(en)This disclosure relates to the field of secondary metabolite production in plants. More specifically, the disclosure relates to chimeric genes and their use in the regulation of biosynthesis and/or production of secondary metabolites in plants and plant-derived cell cultures.

1.ApplicationNumber: US-201515119681-A
1.PublishNumber: US-2017009249-A1
2.Date Publish: 20170112
3.Inventor: GOOSSENS ALAIN
MERTENS JAN
VAN MOERKERCKE ALEX
POLLIER JACOB
MEMELINK JOHAN

4.Inventor Harmonized: GOOSSENS ALAIN(BE)
MERTENS JAN(BE)
VAN MOERKERCKE ALEX(BE)
POLLIER JACOB(BE)
MEMELINK JOHAN(NL)

5.Country: US
6.Claims:
(en)This disclosure relates to the field of secondary metabolite production in plants. More specifically, the disclosure relates to chimeric genes and their use in the regulation of biosynthesis and/or production of secondary metabolites in plants and plant-derived cell cultures.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2015/053407, filed Feb. 18, 2015, designating the United States of America and published in English as International Patent Publication WO 2015/124620 A1 on Aug. 27, 2015, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 14184392.0, filed Sep. 11, 2014, and to European Patent Application Serial No. 14155634.0, filed Feb. 18, 2014.


TECHNICAL FIELD
This disclosure relates to biology and agriculture and the field of secondary metabolite production in plants. More specifically, this disclosure relates to chimeric genes and their use in the regulation of biosynthesis and/or production of secondary metabolites in plants and plant-derived cell cultures.
STATEMENT ACCORDING TO 37 C.F.R. §1.821(C) OR (E)—SEQUENCE LISTING SUBMITTED AS A TXT FILE
Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.
BACKGROUND
The plant kingdom produces tens of thousands of different small compounds with very complex structures that are often genus or family specific. These molecules, referred to as “secondary metabolites” or “specialized metabolites,” display an immense variety in structures and biological activities that plants have tapped into over the course of evolution, and that is now harnessed by man for industrial and medical applications. These compounds play, for example, a role in the resistance against pests and diseases, attraction of pollinators and interaction with symbiotic microorganisms. Besides the importance for the plant itself, secondary metabolites are of great interest because they determine the quality of food (color, taste, aroma) and ornamental plants (flower color, fragrance). A number of secondary metabolites isolated from plants are commercially available as fine chemicals, for example, drugs, dyes, flavors, fragrances and even pesticides. In addition, various health-improving effects and disease-preventing activities of secondary metabolites have been discovered. Flavonoids and terpenoids, for example, have health-promoting activities as food ingredients, and several alkaloids have pharmacological activities. To illustrate this further, taxol is a highly substituted, polyoxygenated cyclic diterpenoid characterized by the taxane ring system, which presents an excellent anti-tumoral activity against a range of cancers.
Although about 100,000 plant secondary metabolites are already known, only a small percentage of all plants have been studied to some extent for the presence of secondary metabolites. Interest in such metabolites is growing as, e.g., plant sources of new and useful drugs are discovered. Some of these valuable phytochemicals are quite expensive because they are only produced at extremely low levels in plants. In fact, very little is known about the biosynthesis of secondary metabolites in plants. However, some recently elucidated biosynthetic pathways of secondary metabolites are long and complicated, requiring multiple enzymatic steps to produce the desired end product. Most often, the alternative of producing these secondary metabolites through chemical synthesis is complicated due to a large number of asymmetric carbons and, in most cases, chemical synthesis is not economically feasible.
The cellular and genetic programs that steer the production of secondary metabolites can be launched rapidly when plants perceive particular environmental stimuli. The jasmonate phytohormones (JAs) play a prominent and universal role in mediating these responses as they can induce synthetic pathways of molecules of a wide structural variety, encompassing all major secondary metabolites (Zhao et al. 2005; Pauwels et al. 2009). Essential in the “core JA signaling module” in plants is the F-box protein CORONATINE INSENSITIVE 1 (COI1), which is part of a Skp/Cullin/F-box-type E3 ubiquitin ligase complex (SCF COI1 ), to which it provides substrate specificity. The targets of the SCF COI1 complex are the JA ZIM domain (JAZ) family of repressor proteins. JAZ and COI1 proteins directly interact in the presence of the bioactive JA-isoleucine (JA-Ile) conjugate to form a co-receptor complex, which triggers the degradation of the JAZ proteins by the 26S proteasome. The JAZ proteins are further characterized by a conserved C-terminal JAs domain, which is required for the interaction with both COI1 and a broad array of transcription factors (TFs). JA-triggered JAZ degradation releases these TFs, which each modulate expression of specific sets of JA-responsive genes and, thereby, the production of specific sets of secondary metabolites (De Geyter et al. 2012 , Trends Plant Sci. 17:349-359).
In Arabidopsis thaliana , for example, the basic helix-loop-helix (bHLH) factor MYC2 is the best known target of the JAZ proteins. MYC2 has been shown to be both directly and indirectly involved in regulating secondary metabolite induction, more precisely, of phenolic compounds and glucosinolates. The Catharanthus roseus MYC2 homologue regulates the expression of the ORCA TFs by direct binding to the “on/off switch” in the promoter of the ORCA3 gene, and thereby controlling expression of several alkaloid biosynthesis genes. In Nicotiana tabacum , MYC2 proteins up-regulate the ORCA-related NIC2 locus APETALA2/ETHYLENE Response Factor (AP2/ERF) TFs that regulate nicotine biosynthesis as well as the nicotine biosynthesis enzymes themselves. JAZ proteins also directly interact with and thereby repress other TFs with a well-established role in the synthesis of secondary metabolites, such as the bHLH TFs GLABRA3 (GL3), ENHANCER OF GL3 (EGL3) and TRANSPARENT TESTA8 (TT8), and the R2R3-MYB TF PAP1, which together compose transcriptional activator complexes that control anthocyanin biosynthesis and are conserved in the plant kingdom.
Besides direct JAZ interactors, other TFs with a proven role in JA-mediated elicitation of a specific metabolic pathway exist, such as WRKY-type TFs that regulate sesquiterpene biosynthesis in various plants, but the full picture on how the central module exerts control over evolutionary distant metabolic pathways, leading to natural products of a wide structural variety, is still lacking. Although overexpression of several of these transcription factors could stimulate synthesis of some secondary metabolites, no master switches have been found that can mimic the full JA spectrum, neither quantitatively nor qualitatively, or replace JAs in plant engineering programs. Likewise, for many secondary metabolic pathways, such as of triterpenes, no regulatory TFs have been identified yet.
Therefore, a need exists for novel ways, preferably generic ways, to regulate the production of secondary metabolites in plants and plant-derived cell cultures.
BRIEF SUMMARY
A first aspect relates to a chimeric gene comprising the following operably linked sequences:
a) one or more control sequences capable of driving expression of a nucleic acid sequence in a plant cell; b) a nucleic acid sequence encoding a bHLH25-like polypeptide comprising a bHLH domain; and c) optionally, a transcript termination sequence.

In one particular embodiment of the above-described chimeric gene, the nucleic acid sequence of b) is a polynucleotide selected from the group consisting of:
a) a polynucleotide that encodes a bHLH25-like polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 13; b) a polynucleotide that encodes a bHLH25-like polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 14; and c) variants of the polynucleotides according to (a) or (b).

Also envisaged is a vector comprising any of the above-described chimeric genes.
Another aspect relates to a transgenic plant or a cell derived thereof comprising any of the above-described chimeric genes.
Yet another aspect relates to a method for regulating the production of secondary metabolites in a plant or a plant cell, the method comprising modulating expression in a plant or plant cell of a nucleic acid encoding a bHLH25-like polypeptide comprising a bHLH domain. The modulated expression can be effected by introducing and expressing in a plant or plant cell any of the above-described chimeric genes.
In one embodiment of the above-described method, the production of secondary metabolites is increased. In another embodiment of the above-described method, the production of secondary metabolites is decreased.
Also envisaged is a method of producing a plant or a plant cell with a different profile of secondary metabolites relative to a control plant or control plant cell, the method comprising modulating expression in a plant or plant cell of a nucleic acid encoding a bHLH25-like polypeptide comprising a bHLH domain.
In a more particular embodiment of any of the above-described methods, the secondary metabolites are selected from the group consisting of alkaloid compounds, phenylpropanoid compounds, and terpenoid compounds. In a preferred embodiment of any of the above-described methods, the secondary metabolites are saponins. In another preferred embodiment of any of the above-described methods, the secondary metabolites are monoterpenoid indole alkaloids.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 . Saponin biosynthesis in Medicago truncatula.
FIGS. 2A-2D . MtbHLH25 factors are co-regulated with triterpene saponin synthesis genes in M. truncatula . Co-expression analysis of HMGR1 (dashed arrows), CYP93E2 (dotted arrows), MtbHLH25a ( FIG. 2A , full arrow), MtbHLH25b ( FIG. 2B , full arrow), MtbHLH25c ( FIG. 2C , full arrow), MtbHLH25d ( FIG. 2D , full arrow) in various M. truncatula plant tissues under various conditions. Note that MtbHLH25a and MtbHLH25c expression levels elevate under influence of methyl jasmonate. MtbHLH25b is only expressed in green seeds at specific stages of development.
FIG. 3 . MtbHLH25 transcription factors activate promoters of saponin biosynthesis genes in BY-2 protoplast transient expression assays. All activation values (fLUC/fREN) were normalized by dividing with the average control values, hence are expressed relative to the normalized control. Standard errors are designated by error bars (n=8 for panels A, C and D; n=24 for panel B).
FIG. 4 . Quantitative PCR analysis of saponin genes in control (CTR) and MtbHLH25a overexpressing (OE) M. truncatula hairy root lines. Ctr lines Ctr 1, Ctr 2 and Ctr 3 are represented in consecutive order by white bars; MtbHLH25a OE lines OE 1, OE 2 and OE 3 are depicted by bars with different shades of gray. The Y-axis represents fold increase relative to control line 1 (CTR1). Standard errors are designated by error bars (n=3).
FIG. 5 . Increased accumulation of saponins in M. truncatula hairy roots overexpressing MtbHLH25a. Depicted are the average TIC values (n=5; error bars, ±s.e.) of the peaks corresponding to the parent ion of Soyasaponin I (A) and Rha-Gal-GlcA-Soyasapogenol E (B) in three control (CTR) and three MtbHLH25a overexpression (OE) M. truncatula hairy root lines.
FIG. 6 . Pathway leading to the production of monoterpenoid indole alkaloids (MIA) in Catharanthus roseus . MIA are produced from strictosidine, a condensation product of secologanin and tryptamine. 7DLGT: 7-deoxyloganetic acid glucosyl transferase, 7DLH: 7-deoxyloganic acid hydroxylase, DXR: 1-deoxy-5-xylulose-5-phosphate reductase, G3P: glyceraldehyde 3-phosphate, GES: geraniol synthase, G8H: geraniol-8-hydroxylase, GPP: geranyl diphosphate, HDS: (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate synthase, 8HGO: 8-hydroxygeraniol oxidoreductase, IPP: isopentenyl diphosphate, IO: iridoid oxidase, IS: iridoid synthase, LAMT: loganic acid O-methyltransferase, MECS: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate synthase, MEP: 2-C-methyl-D-erythritol 4-phosphate, SLS: secologanin synthase, TDC: tryptophan decarboxylase, STR: strictosidine synthase.
FIG. 7 . Co-expression analysis of CrbHLH25 and CrbHLH18 with G8H and selected seco-iridoid and monoterpenoid indole alkaloid genes in different conditions. Left panel represents selected RNA-Seq data from the Medicinal Plant Genomics Resource (MPGR) (http://medicinalplantgenomics.msu.edu). SEEDL: seedlings, ML: mature leaf, IML: immature leaf. Values were normalized to the seedling reads. The right panel is from RNA-Seq data from the SmartCell consortium and available on the ORCAE database (http://bioinformatics.psb.ugent.be/orcae/overview/Catro). CC_c: mock-treated cell culture, CC_JA: jasmonate-treated cell culture, CC_O2 and CC_O3: ORCA2 and ORCA3 overexpressing cell culture. Values were normalized to the mock-treated cell culture. Blue and yellow denote relative down-regulation and up-regulation, respectively.
FIG. 8 . Transient promoter trans-activation assays in Nicotiana tabacum protoplasts using a promoter:fLUC reporter construct and selected TFs. CrbHLH25 and CrbHLH18, but not ORCA3 transactivate pG8H and p7DLGT, whereas only ORCA3 transactivates pSTR1. Values are fold-changes relative to protoplasts transfected with a pCaMV35S:gusA (gusA) construct. The error bars designate SE of the mean (n=8). Statistical significance was determined by the Student's t-test (*P<0.1, **P<0.01, ***P<0.001).
FIG. 9 . CrbHLH25 overexpression stimulates MIA gene expression and metabolite accumulation. Constitutive overexpression of CrbHLH25 suspension cells of C. roseus . Three independent transformant lines were selected for both pCaMV35S:CrbHLH25 and the pCaMV35S:gusA control. The error bars designate SE of the mean (n=3). (Panel A) Expression analysis in transformed cells by RNA-blot hybridization analysis for selected biosynthesis genes. (Panel B) Accumulation of seco-iridoid and MIA compounds in CrbHLH25 overexpressing cell lines. Control cell lines did not accumulate detectable levels of these compounds. In all cases, statistical significance was determined by the Student's t-test (*P<0.1, **P<0.01, ***P<0.001).
FIG. 10 . Reciprocal transactivation of MIA and TS biosynthesis genes by MtbHLH25 and CrbHLH25. Values are fold-changes relative to protoplasts transfected with a pCaMV35S:gusA (control) construct. The error bars designate SE of the mean (n=8). Statistical significance was determined by the Student's t-test (***P<0.001).



DETAILED DESCRIPTION
This disclosure provides novel chimeric genes that can be used for the regulation of production of plant-derived secondary metabolites, in particular, to induce or enhance the production and/or secretion of desired secondary metabolites (or intermediates) in plants or cells derived thereof; or otherwise, to repress or decrease the production and/or secretion of undesired secondary metabolites (or intermediates) in plants or cells derived thereof.
DEFINITIONS
This disclosure will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. Where the term “comprising” is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun, e.g., “a,” “an,” or “the,” this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms “first,” “second,” “third,” and the like, in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the disclosure described herein are capable of operation in other sequences than described or illustrated herein.
Unless otherwise defined herein, scientific and technical terms and phrases used in connection with this disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of molecular and cellular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of this disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology , Greene Publishing Associates (1992, and Supplements to 2002).
As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
As used herein, the terms “nucleic acid,” “polynucleotide,” and “polynucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotide molecule may be linear or circular. The polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker or the like. The polynucleotide may comprise single-stranded or double-stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone. A nucleic acid that is up to about 100 nucleotides in length is often also referred to as an oligonucleotide.
Any of the peptides, polypeptides, nucleic acids, etc., disclosed herein may be “isolated” or “purified.” “Isolated” is used herein to indicate that the material referred to is (i) separated from one or more substances with which it exists in nature (e.g., is separated from at least some cellular material, separated from other polypeptides, separated from its natural sequence context), and/or (ii) is produced by a process that involves the hand of man such as recombinant DNA technology, chemical synthesis, etc.; and/or (iii) has a sequence, structure, or chemical composition not found in nature. “Purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 90% by weight, e.g., at least 95% by weight, e.g., at least 99% by weight, of the polynucleotide(s) or polypeptide(s) present (but water, buffers, ions, and other small molecules, especially molecules having a molecular weight of less than 1000 Dalton, can be present).
The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov/).
“Similarity” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. As used herein, “conservative substitution” is the substitution of amino acids with other amino acids whose side chains have similar biochemical properties (e.g., are aliphatic, are aromatic, are positively charged, . . . ) and is well known to the skilled person. Non-conservative substitution is then the substitution of amino acids with other amino acids whose side chains do not have similar biochemical properties (e.g., replacement of a hydrophobic with a polar residue). Conservative substitutions will typically yield sequences that are not identical anymore, but still highly similar. By “conservative substitutions” is intended combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. “Orthologues” and “paralogues” are two different forms of homologues and encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
The terms “motif or “consensus sequence” or “signature” refer to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
Specialist databases exist for the identification of domains or motifs, for example, SMART (Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic et al. (2002), Nucleic Acids Res. 30:242-244), InterPro (Mulder et al. (2003), Nucl. Acids. Res. 31:315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation, in ISMB-94, Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology, R. Altman, D. Brutlag, P. Karp, R. Lathrop, D. Searls, Eds., pp. 53-61, AAA I Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art; such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:443-453) to find the global (i.e., spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J. Mol. Biol. 215:403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics, 2003 Jul. 10, 4:29, MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (T. F. Smith and M. W. Waterman (1981) J. Mol. Biol. 147(1):195-7).
A “deletion” refers to removal of one or more amino acids from a protein.
An “insertion” refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include an activation domain, such as VP16, a (histidine)-6-tag, a glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
A “substitution” refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art; see, for example, Creighton (1984) Proteins, W.H. Freeman and Company (Eds) or Table below.








TABLE 1





Residue
Conservative Substitutions





Ala
Ser


Arg
Lys


Asn
Gln, His


Asp
Glu


Gln
Asn


Cys
Ser


Glu
Asp


Gly
Pro


His
Asn, Gln


Ile
Leu, Val


Leu
Ile, Val


Lys
Arg, Gln


Met
Leu, Ile


Phe
Met, Leu, Tyr


Ser
Thr, Gly


Thr
Ser, Val


Trp
Tyr


Tyr
Trp, Phe


Val
Ile, Leu




































Substitutions that are less conservative than those in Table 1 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions that, in general, are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
As used herein, the term “derivatives” include peptides, oligopeptides, polypeptides that may, compared to the amino acid sequence of the naturally occurring form of the protein or polypeptide of interest (in this case, the bHLH25-like polypeptide), comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides that comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated, etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule that is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally occurring protein. Furthermore, “derivatives” also include fusions of the naturally occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60:523-533, 2003).
The term “chimeric gene” or “chimeric construct,” as used herein, is a recombinant nucleic acid sequence wherein one or more control sequences (at least a promoter) are operably linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the one or more control sequences are able to regulate transcription or expression of the associated nucleic acid coding sequence. The one or more control sequences of the chimeric gene are not normally operably linked to the associated nucleic acid sequence as found in nature.
The terms “regulatory element,” “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and is involved in recognizing and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box, which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers), which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case, it may include a 35-box sequence and/or a 10-box transcriptional regulatory sequence. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
The term “operably linked” as used herein refers to a linkage in which the regulatory sequence is contiguous with the gene of interest to control the gene of interest, as well as regulatory sequences that act in trans or at a distance to control the gene of interest. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if it is expressed as a pre-protein that participates in the transport of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or adapters or linkers inserted in lieu thereof using restriction endonucleases known to one of skill in the art.
The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression,” in particular, means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. The vector may be of any suitable type including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of certain genes of interest. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like, as desired and according to a particular host organism (e.g., plant cell). Typically, a recombinant vector according to this disclosure comprises at least one “chimeric gene” or “expression cassette,” as defined hereinbefore.
DESCRIPTION
This disclosure shows that modulating expression in a plant or cells derived thereof of a nucleic acid encoding a protein of the family of bHLH transcription factors can be used to alter the production of secondary metabolites relative to control plants.
According to a first aspect, the disclosure relates to a chimeric gene, or otherwise expression cassette, comprising the following operably linked sequences: a) one or more control sequences capable of driving expression of a nucleic acid sequence in a plant or plant cell; b) a nucleic acid sequence encoding a bHLH protein comprising a bHLH domain, more specifically encoding a bHLH25-like polypeptide comprising a bHLH domain; and c) optionally, a transcript termination sequence.
The “basic helix-loop-helix (bHLH) proteins” are a superfamily of transcription factors that bind as dimers to specific DNA target sites. The family is defined by the presence of a bHLH domain (as defined further herein), which contains a highly conserved amino acid motif. Outside of the conserved bHLH domain, the proteins exhibit considerable sequence divergence. As used herein, the term “basic helix-loop-helix (bHLH) domain” refers to a highly conserved amino acid motif that defines the group of transcription factors that are known as bHLH proteins. The bHLH domain is well defined in the art and consists of about 60 amino acids that form two functionally distinct segments: a stretch of about 15 predominantly basic amino acids (the basic region) and a section of around 40 amino acids predicted to form two amphipathic α-helices separated by a loop of variable length (the helix-loop-helix region). The basic region forms the main interface where contact with DNA occurs, whereas the two helices promote the formation of homo- or heterodimers between bHLH proteins, a prerequisite for DNA binding to occur. bHLH proteins are found throughout eukaryotic organisms.
Within the scope of this disclosure, a bHLH protein particularly refers to a plant-derived bHLH protein. Plant-derived bHLH proteins are well known in the art (see, e.g., Pires and Dolan 2010 , Mol. Biol. Evol. 27:862-874; Toledo-Ortiz et al. 2003 , Plant Cell 15:1749-1770; and Heim et al. 2003, Mol. Biol. Evol. 20:735-747). Today, plant-derived bHLH proteins are classified into different subfamilies and reference is particularly made to Table 1 on pages 870 to 871 in Pires and Dolan, 2010, Mol. Biol. Evol. 27:862-874, which is incorporated herein by reference. According to a preferred embodiment, the bHLH protein of this disclosure belongs to subfamily IVa.
Examples of plant-derived bHLH nucleotide sequences and encoded proteins belonging to the subfamily IVa include bHLH25 nucleotide sequences of Medicago truncatula , MtbHLH25a (SEQ ID NO: 1), MtbHLH25b (SEQ ID NO: 2), MtbHLH25c (SEQ ID NO: 3), MtbHLH25d (SEQ ID NO: 4), and the MtbHLH25 polypeptides encoded thereby, MtbHLH25a (SEQ ID NO: 5), MtbHLH25b (SEQ ID NO: 6), MtbHLH25c (SEQ ID NO: 7), MtbHLH25d (SEQ ID NO: 8). Other representative members of subfamily IVa bHLH proteins include homologues of the Medicago truncatula bHLH25 polypeptides. For example, the Medicago truncatula bHLH25 polypeptides share homology with a number of bHLH25-like polypeptides that phylogenetically belong to the same subfamily, for example, bHLH25-like polypeptides from Arabidopsis thaliana AtbHLH020 (At2g22770), AtbHLH019 (At2g22760), AtbHLH018 (At2g22750), AtbHLH025 (At4g37850), from Oryza sativa OsbHLH021 (Os12g43620), OsbHLH022 (0s03g46790), OsbHLH020 (Os03g46860), OsbHLH023 (Os10g01530), OsbHLH008 (Os03g51580). Other examples of homologous sequences of the Medicago bHLH25 sequences from other plant species can be found via BLAST searches on public databases such as NCBI (http://blast.ncbi.nlm.nih.gov/), PLAZA (http://bioinformatics.psb.ugent.be/plaza/blast/index) or ORCAE (http://bioinformatics.psb.ugent.be/orcae/) and include, but are not limited to, the homologues from the medicinal plant Catharanthus roseus Caros001862 (CrbHLH25; SEQ ID NO: 15), Caros006385 (CrbHLH18; SEQ ID NO: 16), and Caros017587.
Thus, a “bHLH25-like polypeptide” as used herein collectively refers to a bHLH protein of subfamily IVa. Typically, a bHLH25-like polypeptide has a conserved bHLH domain. Non-limiting examples of bHLH domains as comprised in bHLH25-like polypeptides include bHLH domains defined by SEQ ID NOs: 9-12. Also, a bHLH25-like polypeptide has a bHLH domain comprising an amino acid sequence having at least 50% overall sequence identity and, for instance, at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the amino acid represented by SEQ ID NO: 9.
In particular, the bHLH25-like polypeptides as used herein comprise a bHLH domain comprising at least the following conserved amino acid motif:
(SEQ ID NO: 13) X 1 HX 2 X 3 AERX 4 RRX 5 X 6 LX 7 X 8 X 9 X 10 X 11 ALX 12 AX 13 X 14 PX 15 LX 16 KX 17 DK
wherein X 1 can be D or E; wherein X 2 can be I or V or L; wherein X 3 can be L or M or V or I; wherein X 4 can be K or R or N; wherein X 5 can be R or Q or E; wherein X 6 can be any amino acid, preferably K or Q or E or D; wherein X 7 can be T or S; wherein X 8 can be E or Q; wherein X 9 can be R or S or K or N; wherein X 10 can be F or I or L; wherein X 11 can be I or V or M; wherein X 12 can be S or A; wherein X 13 can be any amino acid, preferably I or V or L or T; wherein X 14 can be V or I or L or P; wherein X 15 can be G or N; wherein X 16 can be K or N or S or R; and wherein X 17 can be any amino acid, preferably M or T. It will be understood that amino acid residues for each X 1 to X 17 represent alternatives.

For example, a consensus motif characteristic for the group of bHLH25-like polypeptides can be defined by the following amino acid sequence:







(SEQ ID NO: 14)


DHIMAERKRREKLTQRFIALSALIPGLKKMDK









Also envisaged are motifs with amino acid sequences having at least 70% overall sequence identity and, for instance, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the amino acid represented by SEQ ID NO: 14.
Other representative examples of motifs comprised in bHLH25-like polypeptides are defined by the following amino acid sequences, as retrieved from PLAZA:







TC01G007250


(SEQ ID NO: 48)


DHIMAERKRREKLSQRFIALSAIVPGLKKMDK





FV0G20600


(SEQ ID NO: 49)


DHIMAERKRREKLSQRFIALSAIVPGLKKMDK





MD02G000440


(SEQ ID NO: 50)


DHIMAERKRREKLSQRFIALSAIXPGLKKMDK





CP00129G00360


(SEQ ID NO: 51)


DHIVAERKRREKLSQRFIALSAIVPGLKKMDK





ZM01G51370


(SEQ ID NO: 52)


DHILAERKRREKLSQRFIALSKIVPGLKKMDK





OS03G51580


(SEQ ID NO: 53)


EHILAERKRREKLSQRFIALSKIVPGLKKMDK





ME09335G00010


(SEQ ID NO: 54)


DHILAERKRREKLSQRFIALSAIVPGLKKMDK





AT2G22770


(SEQ ID NO: 55)


EHVLAERKRRQKLNERLIALSALLPGLKKTDK





AT2G22760


(SEQ ID NO: 56)


EHVLAERKRREKLSEKFIALSALLPGLKKADK





MD02G000420


(SEQ ID NO: 57)


DHVLAERKRREKLSQRFIALSALLPGLKKMDK





FV0G20580


(SEQ ID NO: 58)


DHVLAERKRREKLSQRFIALSALVPGLKKMDK





ME02883G00050


(SEQ ID NO: 59)


DHVLAERKRREKLSQRFISLSAVVPGLKKMDK





RC28179G00220


(SEQ ID NO: 60)


DHILAERKRREKLSQRFIALSALVPGLKKMDK





PT09G08310


(SEQ ID NO: 61)


DHIIAERKRREKLSQRFIALSAVVPGLKKMDK





Caros001862


(SEQ ID NO: 62)


DHIIAERKRREQLSQHFVALSAIVPGLKKMDK





Caros006385


(SEQ ID NO: 63)


DHIIAERKRREILSQRFMALSTLVPGLKKMDK





AT4G37850


(SEQ ID NO: 64)


DHIIAERKRREKLTQRFVALSALVPGLKKMDK





MD00G205570


(SEQ ID NO: 65)


DHIIAERKRREKLTQRFVALSALVPGLKKMDK





AT2G22750


(SEQ ID NO: 66)


DHILAERKRREKLTQRFVALSALIPGLKKMDK





VV07G03050


(SEQ ID NO: 67)


DHVIAERKRRGKLTQRFIALSALVPGLRKMDK





VV00G09620


(SEQ ID NO: 68)


DHVVAERKRREKLTQRFIALSALVPGLRKTDK





Medtr4g066460


(SEQ ID NO: 69)


DHIMAERNRREKLTQSFIALAALVPNLKKMDK





Medtr0246s0020


(SEQ ID NO: 70)


DHIMAERKRREKLSQSFIALAALVPNLKKMDK





GM17G16720


(SEQ ID NO: 71)


DHIMAERKRREKLSQSFIALAALVPGLKKMDK





GM05G23530


(SEQ ID NO: 72)


DHIMAERKRREKLSQSFIALAALVPGLKKMDK





GM11G04680


(SEQ ID NO: 73)


DHIIAERKRREKLSQSLIALAALIPGLKKMDR





GM01G40620


(SEQ ID NO: 74)


DHIIAERKRREKLSQSLIALAALIPGLKKMDK





LJ2G026110


(SEQ ID NO: 75)


DHIIAERRRREKLSQSLIALAALIPGLKKMDK





GM07G03060


(SEQ ID NO: 76)


DHIMAERRRRQELTERFIALSATIPGLNKTDK





GM08G23050


(SEQ ID NO: 77)


DHIMAERRRRQDLTERFIALSATIPGLSKTDK





GM03G25100


(SEQ ID NO: 78)


DHIMAERKRRQDLTERFIALSATIPGLKKTDK





GM07G13500


(SEQ ID NO: 79)


NHIMAERKRRRELTERFIALSATIPGLKKTDK





GM07G03050


(SEQ ID NO: 80)


DHIMTERKRRRELTERFIALSATIPGLKKIDK





Medtr7g080780


(SEQ ID NO: 81)


DHLMAERKRRRELTENIIALSAMIPGLKKMDK





Medtr2g104650


(SEQ ID NO: 82)


DHIMSERNRRQLLTSKIIELSALIPGLKKIDK





GM15G00730


(SEQ ID NO: 83)


SHIMAERKRRQQLTQSFIALSATIPGLNKKDK






















































































































































Thus, in one embodiment, the invention provides a chimeric gene as described above, wherein the nucleic acid sequence of b) is a polynucleotide selected from the group consisting of:
a. a polynucleotide that encodes a bHLH25-like polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 13; b. a polynucleotide that encodes a bHLH25-like polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 14; and c. variants of the polynucleotides according to (a) or (b).

A “variant” as used herein refers to homologs, orthologs and paralogs and include, but are not limited to, homologs, orthologs and paralogs of polynucleotides encoding a bHLH25-like polypeptide. Non-limiting examples include homologs, orthologs and paralogs of SEQ ID NOs: 1-4. Homologs of a protein encompass peptides, oligopeptides and polypeptides having amino acid substitutions, deletions and/or insertions, preferably by a conservative change, relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived; or, in other words, without significant loss of function or activity. Orthologs and paralogs, which are well-known terms by the skilled person, define subcategories of homologs and encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologues and paralogues include phylogenetic methods, sequence similarity and hybridization methods. Percentage similarity and identity can be determined electronically. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://ncbi.nlm.nih.gov/). Preferably, the homologue, orthologue or paralogue has a sequence identity at a protein level of at least 50%, preferably 60%, more preferably 70%, even more preferably 80%, most preferably 90% as measured in a BLASTp. For example, non-limiting examples of functionally homologous sequences include bHLH25-like polypeptides from Arabidopsis thaliana AtbHLH020 (At2g22770), AtbHLH019 (At2g22760), AtbHLH018 (At2g22750), AtbHLH025 (At4g37850), from Oryza sativa OsbHLH021 (Os12g43620), OsbHLH022 (Os03g46790), OsbHLH020 (Os03g46860), OsbHLH023 (Os10g01530), OsbHLH018 (Os03g51580), amongst others.
In a particular embodiment, derivatives (as defined herein) of any of the above encoded polypeptides also from part of this disclosure.
According to one specific embodiment, the chimeric gene of the disclosure is not a chimeric gene that comprises the following operably linked sequences: a) CaMV 35S promoter; b) a nucleic acid sequence encoding Atg22770; and c) nopaline synthase terminator.
Further, it will be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the polypeptides of this disclosure. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides. Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence are a feature of the disclosure. In addition to silent variations, other conservative variations that alter one or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide (i.e., regulating secondary metabolite production, in the context of this disclosure), these conservative variants are, likewise, a feature of the disclosure.
Conservative substitutions or variations, as used herein, are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place and are defined hereinbefore. Deletions and insertions introduced into the sequences are also envisioned by the disclosure. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis or the other methods known in the art. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotides of the disclosure should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function (i.e., enhanced secondary metabolite production, in the context of this disclosure).
Further, the disclosure also envisages a vector comprising any of the above-described chimeric genes.
According to yet another aspect, the disclosure provides a transgenic plant or a cell derived thereof that is transformed with any of the above-described constructs.
The term “plant” as used herein refers to vascular plants (e.g., gymnosperms and angiosperms). A “transgenic plant” refers to a plant comprising a recombinant polynucleotide and/or a recombinant polypeptide according to the disclosure. A transgenic plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, and progeny thereof. A transgenic plant can be obtained by transforming a plant cell with an expression cassette of this disclosure and regenerating such plant cell into a transgenic plant. Such plants can be propagated vegetatively or reproductively. The transforming step may be carried out by any suitable means, including by Agrobacterium -mediated transformation and non- Agrobacterium -mediated transformation, as discussed in detail below. Plants can be regenerated from the transformed cell (or cells) by techniques known to those skilled in the art. Where chimeric plants are produced by the process, plants in which all cells are transformed may be regenerated from chimeric plants having transformed germ cells, as is known in the art. Methods that can be used to transform plant cells or tissue with expression vectors of this disclosure include both Agrobacterium and non- Agrobacterium vectors. Agrobacterium -mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes and is described in detail in G. Gheysen, G. Angenon, and M. Van Montagu (1998), Agrobacterium -mediated plant transformation: a scientifically intriguing story with significant applications, in K. Lindsey (Ed.), Transgenic Plant Research Harwood Academic Publishers, Amsterdam, pp. 1-33; and in H. A. Stafford (2000), Botanical Review 66:99-118. A second group of transformation methods is the non- Agrobacterium -mediated transformation and these methods are known as direct gene transfer methods. An overview is included by P. Barcelo and P. A. Lazzeri (1998), Direct gene transfer: chemical, electrical and physical methods in K. Lindsey (Ed.), Transgenic Plant Research , Harwood Academic Publishers, Amsterdam, pp. 35-55.
Methods include particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated-transformation, etc.
Hairy root cultures can be obtained by transformation with virulent strains of Agrobacterium rhizogenes , and they can produce high contents of secondary metabolites characteristic to the mother plant. Protocols used for establishing of hairy root cultures vary, as well as the susceptibility of plant species to infection by Agrobacterium (Toivounen et al. 1993 , Biotechnol. Prog. 9:12; Vanhala et al. 1995, Plant Cell Rep. 14:236). It is known that the Agrobacterium strain used for transformation has a great influence on root morphology and the degree of secondary metabolite accumulation in hairy root cultures. It is possible by systematic clone selection, e.g., via protoplasts, to find high yielding, stable, and single-cell-derived hairy root clones. This is possible because the hairy root cultures possess a great somaclonal variation. Another possibility of transformation is the use of viral vectors (Turpen 1999 , Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354:665-73.).
Any plant tissue or plant cells capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a construct of this disclosure. The term “organogenesis” means a process by which shoots and roots are developed sequentially from meristematic centers. The term “embryogenesis” means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include protoplasts, leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyls meristem).
A “control plant” as used in this disclosure refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plants for the purpose of identifying a difference in production of secondary metabolite (as described further herein) in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of this disclosure that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line (wild-type) used to generate a transgenic plant herein.
Plants of this disclosure may include, but not limited to, plants or plant cells of agronomically important crops that are or are not intended for animal or human nutrition, such as maize or corn, wheat, barley, oat, Brassica spp. plants such as Brassica napus or Brassica juncea , soybean, bean, alfalfa, pea, rice, sugarcane, beetroot, tobacco, sunflower, quinoa, cotton, Arabidopsis , vegetable plants such as cucumber, leek, carrot, tomato, lettuce, peppers, melon, watermelon, diverse herbs such as oregano, basilicum and mint. It may also be applied to plants that produce valuable compounds, e.g., useful as, for instance, pharmaceuticals, as ajmalicine, vinblastine, vincristine, ajmaline, reserpine, rescinnamine, camptothecine, ellipticine, quinine, and quinidine, taxol, morphine, scopolamine, atropine, cocaine, sanguinarine, codeine, genistein, daidzein, digoxin, calystegins or as food additives such as anthocyanins, vanillin, including, but not limited to, the classes of compounds mentioned above. Examples of such plants include, but not limited to, Papaver spp., Rauwolfia spp., Catharanthus spp., Artemisia spp., Taxus spp., Cinchona spp., Eschscholtzia californica, Camptotheca acuminata, Hyoscyamus spp., Berberis spp., Coptis spp., Datura spp., Atropa spp., Thalictrum spp., Peganum spp., Panax spp., Avena spp., Medicago spp., Quillaja spp., Sapindus spp., Saponaria spp., Betula spp., Digitalis spp., Glycyrrhiza spp., Bupleurum spp., Centella spp., Dracaena spp., Aesculus spp., Yucca spp., amongst others.
The chimeric genes as described above may be expressed in, for example, a plant cell under the control of a promoter that directs constitutive expression or regulated expression. Regulated expression comprises temporally or spatially regulated expression and any other form of inducible or repressible expression. “Temporally” means that the expression is induced at a certain time point, for instance, when a certain growth rate of the plant cell culture is obtained (e.g., the promoter is induced only in the stationary phase or at a certain stage of development). “Spatially” means that the promoter is only active in specific organs, tissues, or cells (e.g., only in roots, leaves, epidermis, guard cells or the like). Other examples of regulated expression comprise promoters whose activity is induced or repressed by adding chemical or physical stimuli to the plant cell. In a preferred embodiment, the expression is under control of environmental, hormonal, chemical, and/or developmental signals. Such promoters for plant cells include promoters that are regulated by (1) heat, (2) light, (3) hormones, such as abscisic acid and methyl jasmonate, (4) wounding or (5) chemicals such as salicylic acid, chitosans or metals. Indeed, it is well known that the expression of secondary metabolites can be boosted by the addition of, for example, specific chemicals, jasmonate and elicitors. In a particular embodiment, the co-expression of several (more than one) polynucleotide sequences, in combination with the induction of secondary metabolite synthesis, is beneficial for an optimal and enhanced production of secondary metabolites. Alternatively, the at least one polynucleotide sequence is placed under the control of a constitutive promoter. A constitutive promoter directs expression in a wide range of cells under a wide range of conditions. Examples of constitutive plant promoters useful for expressing heterologous polypeptides in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues including monocots, the nopaline synthase promoter and the octopine synthase promoter. The expression cassette is usually provided in a DNA or RNA construct, which is typically called an “expression vector,” which is any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages, cosmids, plant viruses and artificial chromosomes. The expression cassette may be provided in a DNA construct, which also has at least one replication system. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. The markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host; or c) provide a visible phenotype through the production of a novel compound in the plant. Exemplary genes that may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable markers are β-glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.
The term “promoter activity” refers to the extent of transcription of a polynucleotide sequence that is operably linked to the promoter whose promoter activity is being measured. The promoter activity may be measured directly by measuring the amount of RNA transcript produced, for example, by Northern blot or indirectly by measuring the product coded for by the RNA transcript, such as when a reporter gene is linked to the promoter.
According to a further aspect of the disclosure, the above-described chimeric genes can be used for modulating the production of secondary metabolites in plants and plant-derived cell cultures. According to a preferred embodiment, this disclosure provides methods for modulating the production of secondary metabolites chosen from the group comprising alkaloid compounds, phenylpropanoid compounds and terpenoid compounds, for which non-limiting examples are provided further herein. In one particular embodiment, this disclosure provides methods for modulating the production of at least one secondary metabolite, which is meant to include related structures of secondary metabolites and intermediates or precursors thereof.
Generally, two basic types of metabolites are synthesized in cells, i.e., those referred to as primary metabolites and those referred to as secondary metabolites (also referred to as specialized metabolites). A primary metabolite is any intermediate in, or product of the primary metabolism in cells. The primary metabolism in cells is the sum of metabolic activities that are common to most, if not all, living cells and are necessary for basal growth and maintenance of the cells. Primary metabolism thus includes pathways for generally modifying and synthesizing certain carbohydrates, amino acids, fats and nucleic acids, with the compounds involved in the pathways being designated primary metabolites. In contrast hereto, secondary metabolites usually do not appear to participate directly in growth and development.
Secondary plant metabolites, as used herein, include, e.g., alkaloid compounds (e.g., terpenoid indole alkaloid, tropane alkaloids, steroidal alkaloids), phenylpropanoid compounds (e.g., quinines, lignans and flavonoids), terpenoid compounds (e.g., monoterpenoids, iridoids, sesquiterpenoids, diterpenoids and triterpenoids). In addition, secondary metabolites include small molecules, such as substituted heterocyclic compounds, which may be monocyclic or polycyclic, fused or bridged. Many plant secondary metabolites have value as pharmaceuticals. Non-limiting examples of plant pharmaceuticals include, e.g., taxol, digoxin, atropine, scopolamine, colchicine, diosgenin, codeine, cocaine, morphine, quinine, shikonin, ajmaline, vinblastine, vincristine, and others.
As used herein, the definition of “alkaloids,” of which more than 15,000 structures have been described already, refers to all nitrogen-containing natural products that are not otherwise classified as amino acid peptides and proteins, amines, cyanogenic glycosides, glucosinolates, antibiotics, phytohormones or primary metabolites (such as purine and pyrimidine bases). Alkaloids can be divided into the following major groups: (i) “true alkaloids,” which contain nitrogen in the heterocycle and originate from amino acids, for which atropine, nicotine and morphine are characteristic examples; (ii) “protoalkaloids,” which contain nitrogen in the side chain and also originate from amino acids, for which mescaline, adrenaline and ephedrine are characteristic examples; (iii) “polyamine alkaloids,” which are derivatives of putrescine, spermidine and spermine; (iv) “peptide and cyclopeptide alkaloids”; (v) “pseudoalkaloids,” which are alkaloid-like compounds that do not originate from amino acids, including terpene-like and steroid-like alkaloids as well as purine-like alkaloids such as caffeine, theobromine and thephylline.
Plants synthesize alkaloids for various defense-related reactions, e.g., actions against pathogens or herbivores. Enzymes and genes have been partly characterized only in groups of nicotine and tropane alkaloids, indole alkaloids and isoquinolidine alkaloids (Suzuki et al. 1999, Plant Mol. Biol. 40:P141).
As used herein, “phenylpropanoids” or “phenylpropanes,” refer to aromatic compounds with a propyl side-chain attached to the aromatic ring, which can be derived directly from phenylalanine. The ring often carries oxygenated substituents (hydroxyl, methoxy and methylenedioxy groups) in the para-position. Natural products in which the side-chain has been shortened or removed can also be derived from typical phenylpropanes. Phenylpropanoids are found throughout the plant kingdom. Most plant phenolics are derived from the phenylpropanoid and phenylpropanoid-acetate pathways and fulfil a very broad range of physiological roles in plants. For example, polymeric lignins reinforce specialized cell wall. Closely related are the lignans that vary from dimers to higher oligomers. Lignans can either help defend against various pathogens or act as antioxidants in flowers, leaves and roots. The flavonoids comprise an astonishingly diverse group of more than 4500 known compounds. Among their subclasses are the anthocyanins (pigments), proanthocyanidins or condensed tannins (feeding deterrents and wood protectants), and isoflavonoids (defensive products and signaling molecules). The coumarins, furanocoumarins, and stilbenes protect against bacterial and fungal pathogens, discourage herbivory, and inhibit seed germination.
As used herein, “terpenoids” or otherwise “isoprenoids” refer to the large and diverse class of naturally occurring organic chemicals of terpenes and can be found in all classes of living organisms. Terpenoids are molecules derived from a five-carbon isoprene unit that are assembled and modified in different ways and have diverse activities. Plant terpenoids are used extensively for their aromatic qualities and contribute to, e.g., the scent of eucalyptus, the flavors of cinnamon, clover and ginger, and the color of yellow flowers. They play a role in traditional herbal remedies and may have antibacterial, antineoplastic, and other pharmaceutical functions. Well-known terpenoids include citral, menthol, camphor, salvinorin A and cannabinoids and are also used to flavor and/or scent a variety of commercial products. The steroids and sterols in animals are biologically produced from terpenoid precursors. They also include pharmaceuticals, e.g., taxol, artemisinin, vinblastine and vincristine.
Terpenoids are classified with reference to the number of isoprene units that comprise the particular terpenoid. For example, a monoterpenoid comprises two isoprene units; a sesquiterpenoid comprises three isoprene units; and a diterpenoid comprises four isoprene units. Polyterpenoids comprise multiple isoprene units. There are many thousands of examples of terpenoids. Artemisinin is a sesquiterpene lactone endoperoxide and is a natural product produced by the plant Artemisia annua . Artemisinin is typically used in combination with anti-malarial therapeutics, for example, lumefantrine, mefloquine, amodiaquine, sulfadoxine, chloroquine, in artemisinin combination therapies (ACT). Endoperoxides like artemisinin, for example, dihydroartemisinin, artemether, and sodium artesunate have been used in the treatment of malaria. Examples of monoterpenoids include linalool, citronellol, menthol, geraniol and terpineol. Linalool and citronellol are used as a scent in soap, detergents, shampoo and lotions. Linalool is also an intermediate in the synthesis of vitamin E. Menthol is isolated from peppermint or other mint oils and is known for its anesthetic properties; it is often included in sore throat medications and oral medications, e.g., for the treatment of bad breath in toothpaste and mouth wash. Geraniol is known for its insect repellent properties and is also used as a scent in perfumes. Terpineol is also used as an ingredient in perfumes and cosmetics and as flavoring. It is apparent that in addition to the pharmaceutical applications of monoterpenoids such as perillyl alcohol, there are additional uses as scents, flavorings, and as insect deterrents.
The synthesis of terpenoids involves a large number of enzymes with different activities. For example, isoprene units are synthesized from monosaturated isoprene units by prenyltransferases into multiples of 2, 3 or 4 isoprene units. These molecules serve as substrates for terpene synthase enzymes, also called terpene cyclase. Plant terpene synthases are known in the art.
A particular class of terpenoids are the saponins. The term “saponins” as used herein are a group of bio-active compounds that consist of an isoprenoidal aglycone, designated “genin” or “sapogenin,” covalently linked to one or more sugar moieties. This combination of polar and non-polar structural elements in their molecules explains their soap-like behavior in aqueous solutions. Most known saponins are plant-derived secondary metabolites, though several saponins are also found in marine animals such as sea cucumbers and starfish. In plants, saponins are generally considered to be part of defense systems due to anti-microbial, fungicidal, allelopathic, insecticidal and moluscicidal, etc., activities. Typically, saponins reside inside the vacuoles of plant cells. Extensive reviews on molecular activities, biosynthesis, evolution, classification, and occurrence of saponins are given by, e.g., Augustin et al. 2011 , Phytochemistry 72:435-57, and Vincken et al. 2007 , Phytochemistry 68:275-97. Thus, the term “sapogenin,” as used herein, refers to an aglycone, or non-saccharide, moiety of the family of natural products known as saponins.
The commonly used nomenclature for saponins distinguishes between triterpenoid saponins (also: triterpene saponins) and steroidal saponins, which is based on the structure and biochemical background of their aglycones. Both sapogenin types are thought to derive from 2,3-oxidosqualene, a central metabolite in sterol biosynthesis. In phytosterol anabolism, 2,3-oxidosqualene is mainly cyclized into cycloartenol. Triterpenoid sapogenins branch off the phytosterol pathway by alternative cyclization of 2,3-oxidosqualene, while steroidal sapogenins are thought to derive from intermediates in the phytosterol pathway downstream of cycloartenol formation (see also FIG. 1 ). A more detailed classification of saponins based on sapogenin structure with 11 main classes and 16 subclasses has been proposed by Vincken et al. 2007, Phytochemistry 68:275-97; particularly from page 276 to page 283, and also FIGS. 1 and 2 ), which is all incorporated herein by reference. In particular, saponins may be selected from the group comprising dammarane-type saponins, tirucallane-type saponins, lupane-type saponins, oleanane-type saponins, taraxasterane-type saponins, ursane-type saponins, hopane-type saponins, cucurbitane-type saponins, cycloartane-type saponins, lanostane-type saponins, and steroid-type saponins. The aglycon backbones, the sapogenins, can be similarly classified and may be selected from the group comprising dammarane-type sapogenins, tirucallane-type sapogenins, lupane-type sapogenins, oleanane-type sapogenins, taraxasterane-type sapogenins, ursane-type sapogenins, hopane-type sapogenins, cucurbitane-type sapogenins, cycloartane-type sapogenins, lanostane-type sapogenins, and steroid-type sapogenins. A well-known example of triterpenoid saponins includes ginsenoside found in ginseng. A well-known example of steroid saponins, also referred to as glycoalkaloids, includes solanine found in potato and tomato.
Triterpenoid sapogenins typically have a tetracyclic or pentacyclic skeleton. As described in the Background section, the sapogenin building blocks themselves may have multiple modifications, e.g., small functional groups, including hydroxyl, keto, aldehyde, and carboxyl moieties, of precursor sapogenin backbones such as β-amyrin, lupeol, and dammarenediol.
The terms “triterpene” and “triterpenoid” are used interchangeably herein.
It is to be understood that the secondary plant metabolites, as used herein, also encompass new-to-nature compounds that are structurally related to the naturally occurring metabolites. These new-to-nature compounds may be currently unextractable compounds by making use of existing extraction procedures or may be novel compounds that can be obtained after genetic engineering of the synthesizing plant or plant cell culture (see further herein).
With “production” of secondary metabolites is meant both intracellular production as well as secretion into the medium. The term “modulates” or “modulation” in relation to production of secondary metabolites refers to an increase or a decrease in production or biosynthesis of secondary metabolites.
Often, an increase of a secondary metabolite is desired but sometimes a decrease of a secondary metabolite is wanted. That decrease can, for example, refer to the decrease of an undesired intermediate product or a toxic end product. With an increase in the production of one or more metabolites, it is understood that the production may be enhanced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% relative to a control plant (or plant cell). Conversely, a decrease in the production of the level of a secondary metabolite may be decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% relative to a control plant (or plant cell).
An “induced production” of a secondary metabolite means that there is no detectable production of secondary metabolite(s) by the control (untransformed) plant (cell) but that detection becomes possible upon carrying out the transformation according to the disclosure.
An “enhanced production” of a secondary metabolite means that there already exists a detectable amount of secondary metabolite(s) by the control (untransformed) plant cell but that detection becomes possible upon carrying out the transformation according to the disclosure and that an increase of secondary metabolite(s) can be measured by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% compared to basal secretion by the control (untransformed) plant (cell).
Preferably, the organism (such as a plant or plant cell line) transformed with a polynucleotide of this disclosure is induced before it produces secondary metabolites. The wording “inducing the production” means that, for example, the cell or tissue culture, such as a plant cell or tissue culture, is stimulated by the addition of an external factor. External factors include the application of heat, the application of cold, the addition of acids, bases, metal ions, fungal membrane proteins, sugars and the like. One approach that has been given interesting results for better production of plant secondary metabolites is elicitation. Elicitors are compounds capable of inducing defense responses in plants. These are usually not found in intact plants but their biosynthesis is induced after wounding or stress conditions. Commonly used elicitors are jasmonates, mainly jasmonic acid and its methyl ester, methyl jasmonate (MeJA). Jasmonates are linoleic acid derivatives of the plasma membrane and display a wide distribution in the plant kingdom. They were originally classified as growth inhibitors or promoters of senescence, but now it has become apparent that they have pleiotropic effects on plant growth and development. Jasmonates appear to regulate cell division, cell elongation and cell expansion and thereby stimulate organ or tissue formation. They are also involved in the signal transduction cascades that are activated by stress situations such as wounding, osmotic stress, desiccation and pathogen attack. MeJA is known to induce the accumulation of numerous defense-related secondary metabolites (e.g., phenolics, alkaloids, triterpenoids and sesquiterpenoids) through the induction of genes coding for the enzymes involved in the biosynthesis of these compounds in plants. Jasmonates can modulate gene expression from the (post)transcriptional to the (post)translational level, both in a positive way and a negative way. Genes that are up-regulated are, e.g., defense- and stress-related genes (PR proteins and enzymes involved with the synthesis of phytoalexins and other secondary metabolites, whereas the activity of housekeeping proteins and genes involved with photosynthetic carbon assimilation are down-regulated. For example, the biosynthesis of phytoalexins and other secondary products in plants can also be boosted up by signal molecules derived from micro-organisms or plants (such as peptides, oligosaccharides, glycopeptides, salicylic acid and lipophilic substances) as well as by various abiotic elicitors like UV-light, heavy metals (Cu, VOSO4, Cd) and ethylene. The effect of any elicitor is dependent on a number of factors, such as the specificity of an elicitor, elicitor concentration, the duration of the treatment and growth stage of the culture.
Generally, secondary metabolites can be measured intracellularly or in the extracellular space by methods known in the art. Such methods comprise analysis by thin-layer chromatography, high-pressure liquid chromatography, capillary electrophoresis, gas chromatography combined with mass spectrometric detection, radioimmuno-assay (RIA) and enzyme immuno-assay (ELISA). For example, taxol and taxanes can be measured as described in Ketchum et al. 2007 , Plant Cell Rep. 26:1025-1033; tobacco alkaloid content can be analyzed by GC-MS (Millet et al. 2009 , J. Pharm. Biomed. Anal. 49:1166-1171); Medicago flavonoid and triterpene saponin content can be analyzed by reverse-phase UPLC/ICR/FT-MS (see also Example section).
When the chimeric genes of this disclosure are used to modulate the production of secondary metabolites in plants or plant cells, the disclosure can be practiced with any plant variety for which cells of the plant can be transformed with an expression cassette of the current disclosure and for which transformed cells can be cultured in vitro. Suspension culture, callus culture, hairy root culture, shoot culture or other conventional plant cell culture methods may be used (as described in: Drugs of Natural Origin , G. Samuelsson, 1999, ISBN 9186274813). By “plant cells,” it is understood to mean any cell that is derived from a plant and can be subsequently propagated as callus, plant cells in suspension, organized tissue and organs (e.g., hairy roots). Tissue cultures derived from the plant tissue of interest can be established. Methods for establishing and maintaining plant tissue cultures are well known in the art (see, e.g., R. N. Trigiano and D. J. Gray (1999), “Plant Tissue Culture Concepts and Laboratory Exercises,” ISBN: 0-8493-2029-1; E. B. Herman (2000), “Regeneration and Micropropagation: Techniques, Systems and Media 1997-1999 ,” Agricell Report ). Typically, the plant material is surface-sterilized prior to introducing it to the culture medium. Any conventional sterilization technique, such as chlorinated bleach treatment, can be used. In addition, antimicrobial agents may be included in the growth medium. Under appropriate conditions, plant tissue cells form callus tissue, which may be grown either as solid tissue on solidified medium or as a cell suspension in a liquid medium.
A number of suitable culture media for callus induction and subsequent growth on aqueous or solidified media are known. Exemplary media include standard growth media, many of which are commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt (SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, and other variations and supplements well known to those of skill in the art (see, e.g., Plant Cell Culture , Dixon, ed. IRL Press, Ltd. Oxford (1985), and George et al., Plant Culture Media , Vol 1, Formulations and Uses Exegetics Ltd. Wilts, UK, (1987)). For the growth of conifer cells, particularly suitable media include 1/2 MS, 1/2 L.P., DCR, Woody Plant Medium (WPM), Gamborg's B5 and its modifications, DV (Durzan and Ventimiglia, in Vitro Cell Dev. Biol. 30:219-227 (1994)), SH, and White's medium.
In yet another aspect, the disclosure envisages a method for regulating the production of secondary metabolites in a plant or a plant cell relative to a control plant or control plant cell, the method comprising modulating expression in a plant or plant cell of a nucleic acid encoding a bHLH25-like polypeptide, wherein the bHLH25-like polypeptide comprises a bHLH domain. In specific embodiments, modulating can be increasing or decreasing the expression of a nucleic acid encoding a bHLH25-like polypeptide.
The term “modulation” means, in relation to expression or gene expression, a process in which the expression level is changed by the gene expression in comparison to the control plant, wherein the expression level may be increased or decreased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. For the purposes of this disclosure, the original unmodulated expression may also be absence of any expression. The term “modulating the activity” shall mean any change of the expression of the nucleic acid sequences or encoded proteins, which leads to increased production of secondary metabolites by the plants. The expression can increase from zero (absence of, or immeasurable expression) to a certain amount, or can decrease from a certain amount to immeasurably small amounts or zero.
The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level. For the purposes of this disclosure, the original wild-type expression level might also be zero, i.e., absence of expression or immeasurable expression. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers, which are described hereinbefore.
Reference herein to “decreased expression” or “reduction or substantial elimination” of expression is taken to mean a decrease in endogenous gene expression and/or polypeptide levels and/or polypeptide activity relative to control plants. The reduction or substantial elimination is in increasing order of preference, at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control plants. Methods for decreasing expression of genes or gene products are well documented in the art and include, for example, RNA-mediated silencing of gene expression (down-regulation). Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J. 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682). Further, artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation.
According to a preferred embodiment, the modulated expression is effected by introducing and expressing in a plant or plant cell a chimeric gene of the disclosure that comprises a nucleic acid encoding a bHLH25-like polypeptide, as described hereinbefore.
Further, this disclosure also relates to a method for the production or for stimulating the biosynthesis of secondary metabolites in a plant or a plant cell comprising the steps of transforming the plant or plant cell with a chimeric gene of this disclosure and allowing the plant or the plant cell to grow.
In a particular embodiment, the disclosure herein can be combined with other known methods to enhance the production and/or the secretion of secondary metabolites in plant cell cultures such as (1) by improvement of the plant cell culture conditions, (2) by the transformation of the plant cells with a transcription factor capable of up-regulating genes involved in the pathway of secondary metabolite formation, (3) by the addition of specific elicitors to the plant cell culture, and 4) by the induction of organogenesis, amongst other methods.
The chimeric genes of this disclosure can be used in all types of plants to boost the plant's own secondary metabolite production or in plants that are transformed with a combination of genetic material that can lead to the generation of novel metabolic pathways (for example, through the interaction with metabolic pathways resident in the host organism or, alternatively, silent metabolic pathways can be unmasked) and eventually lead to the production of novel classes of compounds. This novel or reconstituted metabolic pathway can have utility in the commercial production of novel, valuable compounds.
The following examples are intended to promote a further understanding of this disclosure. While this disclosure is described herein with reference to illustrated embodiments, it should be understood that the disclosure is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, this disclosure is limited only by the claims attached herein.
EXAMPLES
Example 1
Selection of Novel Transcription Factors that are Co-Regulated with Known Saponin Biosynthesis Genes in Medicago truncatula
To identify new regulators of saponin biosynthesis, co-expression analyses of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMGR1) and the cytochrome P450 monooxygenase CYP93E2 were performed using the Medicago truncatula Gene Expression Atlas (MtGEA) Web Server (Benedito et al. 2008, Plant J. 55:504-513). The HMGR1 enzyme catalyzes the rate-limiting step in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all triterpenes. The CYP93E2 enzyme hydroxylates C-24 on the β-amyrin backbone, which is the first specific step for the biosynthesis of the soyasaponins (for an overview of saponin biosynthesis in M. truncatula , see FIG. 1 ). Co-expression was checked in various tissues, with a main focus on the roots as production site for saponins. Four transcription factors TFs, all belonging to clade IVa of the basic helix-loop-helix (bHLH) family of TFs to which also Arabidopsis thaliana bHLH25 (At4g37850) belongs (Heim et al., 2003, Mol. Biol. Evol. 20:735-747), were significantly co-expressed ( FIG. 2 ). The complete open reading frames (ORFs) were obtained by blasting the Affymetrix probe set sequences against the M. truncatula genome v4.0 ( Medicago truncatula genome project) and were denominated as follows: MtbHLH25a (Medtr7g080780.1), MtbHLH25b (Medtr2g104650.1), MtbHLH25c (Medtr4g066460.1) and MtbHLH25d (Medtr0246s0020.1) (Table 2).
Example 2
Chimeric Constructs of M. truncatula bHLH25 Homologues for Functional Analyses
Complete ORFs of MtbHLH25a, b, c and d were PCR-amplified with primer sets P1+P2, P3+P4, P5+P6 and P7+P8, respectively (Table 3). As a template for MtbHLH25b, cDNA made from RNA extracted from green M. truncatula seeds was used. For the other three genes, cDNA made from RNA from M. truncatula hairy root material that was treated with methyl jasmonate was utilized. Subsequently, all MtbHLH25 genes were cloned into the entry vector pDONR221 using the GATEWAY® recombination system (Invitrogen Life Technologies).
The capacity of the MtbHLH25 factors to transactivate promoters of saponin biosynthesis genes was assessed in an automated transient expression assay in Nicotiana tabacum (tobacco) BY-2 protoplasts (see Example 3) (De Sutter et al. 2005, Plant J. 44:1065-1076). Therefore, the MtbHLH25 ORFs were transferred from the entry clones into the p2GW7 high copy vector in which the genes are subjected to regulation by the cauliflower mosaic virus (CaMV) 35S promotor (Karimi et al. 2002, Trends Plant Sci. 5:193-195). Promoter sequences of M. truncatula genes involved in saponin biosynthesis were constituted by selecting the first 1000 base pairs upstream of the corresponding ORFs, which were identified in the M. trunctatula genome v 3.5. This was done for HMGR1, β-amyrin synthase (BAS), cytochrome P450 monooxygenase CYP93E2 and two UDP-dependent glycosyl transferases (i.e., UGT73F3 and UGT73K) (Table 2). The promoter sequences were PCR-amplified using the following primer sets, respectively: P9+P10, P11+P12, P13+P14, P15+P16, P17+P18 (Table 3). All PCR fragments were recombined into the GATEWAY® entry vector pDONR221 (Invitrogen Life Technologies) from which they were transferred into the pGWL7 destination vector (De Sutter et al. 2005, Plant J. 44:1065-1076). Hence, these reporter constructs consist of a fusion of the promoter with the firefly luciferase ORF (fLUC).
In order to over-express the MtbHLH25 factors in M. truncatula hairy roots as described (Pollier et al., 2011, J. Nat. Prod. 74:1462-1476) (see Examples 4 and 5), the ORFs were inserted in the pK7WGD2 destination vector, in which they become expressed from a 35S promoter (Karimi et al. 2002, Trends Plant Sci. 5:193-195). This plasmid was then transformed into the Agrobacterium rhizogenes strain LBA 9402/12 to transfect M. truncatula hairy roots (ecotype Jemalong J5).
Example 3
Functional Analysis of MtbHLH25 Homologues by Transient Expression Assays
BY-2 tobacco protoplasts were transfected as described by De Sutter et al. 2005, Plant J. 44:1065-1076. A dual firefly/Renilla luciferase assay was carried out in which the fLUC readout is a measure for the promoter activation level and fREN a measure for transfection efficiency. As control, a non-functional β-glucuronidase (GUS) gene expressed from the 35S promoter was used. It was observed that MtbHLH25a mediates a strong activation of the saponin biosynthesis gene promoters pHMGR1, pCYP93E2, pUGT73F3 and pUGT73K15 ( FIG. 3 ). The other MtbHLH25s mediate activation of the same promoters, but with various strengths. For pBAS, a minor activation was observed with all MtbHLH25 factors.
Example 4
Overexpression of MtbHLH25 Homologues in Transgenic M. truncatula Hairy Roots
Transgenic M. truncatula hairy roots were generated as described (Pollier et al. 2011, J. Nat. Prod. 74:1462-1476) and grown for 10 days in 5 mL Murashige and Skoog basal salt mixture including vitamins (Duchefa) medium supplied with 1% sucrose. Successively, 20 mL medium was added after which the roots were incubated for an additional 2 weeks. The hairy roots were harvested, rinsed with water and ground in liquid nitrogen into a fine powder. 10 mg of root material was used for RNA extraction and cDNA generation.
Three MtbHLH25a OE lines and three control (CTR, i.e., overexpressing GUS from a 35S promoter) lines were analyzed by quantitative PCR (qPCR) to assess the transcript levels of saponin biosynthesis genes. Primers were designed with Beacon Designer. As reference genes, a 40S ribosomal protein S8 (40S) and translation elongation factor 1α (ELF1 α) were used. Overexpression of MtbHLH25a was shown to up-regulate a variety of saponin biosynthesis genes, including HMGR1, HMGR2, HMGR4, HMGR6, BAS, CYP93E2, UGT73F3 and UGT73K1 ( FIG. 4 ).
Example 5
Enhanced Production of Saponins in MtbHLH25a OE Lines
To assess the effects of overexpression of MtbHLH25a on the metabolite level, five cultures of each of the three MtbHLH25a OE lines and the three control lines were analyzed by liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC-ESI-FT-ICR-MS, Pollier et al., 2011 , J. Nat. Prod. 74:1462-1476). The resulting LC-MS chromatograms were processed and analyzed using the XCMS software package (Smith et al. 2006 , Anal. Chem. 78:779-787) and yielded a total of 6,945 m/z peaks. A Student's t-test with Welch correction (α=10 −5 ) identified 296 m/z peaks, corresponding to at least 19 metabolites, that were significantly different between the control and MtbHLH25a OE lines. Tentative identification of these 19 significantly different metabolites revealed that most of them were saponins with a soyasapogenol aglycone, and the intensity of the corresponding peaks indicated these metabolites were present in much higher levels in the MtbHLH25a OE lines as compared to the control lines ( FIG. 5 ).
Example 6
Selection of bHLH25 Homologues from Catharanthus roseus that are Co-Regulated with Known Seco-Iridoid Biosynthesis Genes and Functional Analysis by Transient Expression Assays
Catharanthus roseus produces over 150 monoterpenoid indole alkaloid (MIA) compounds, which are derived from the central MIA compound strictosidine, a condensation product of the monoterpenoid (more specifically seco-iridoid, the term that is used hereinafter) compound secologanin and the indole compound L-tryptamine ( FIG. 6 ) (Courdavault et al. 2014 , Curr. Opin. Plant Biol. 19C:43-50; van der Heijden et al. (2004), Curr. Med. Chem. 11:607-628). Secologanin is exclusively produced from MEP-derived geranyl diphosphate (GPP), involving nine biosynthesis genes, of which the seven genes upstream of loganic acid methyltransferase (LAMT) are highly co-expressed ( FIGS. 6 and 7 ) (Miettinen et al. (2014), Nat. Commun. 5:3606). Previously, the APETALA2 (AP2)-domain TF ORCA3 was identified as the JA-inducible regulatory TF that modulates the JA-induced expression of the genes of the indole branch of the pathway and several steps downstream of strictosidine (J. van der Fits, et al. (2000), Science 289:295-297). Overexpression of ORCA3 in C. roseus cell suspension cultures also increased the expression of LAMT and secologanin synthase 1 (SLS1), but not of the seco-iridoid genes upstream of LAMT ( FIG. 6 ). The latter finding also indicated that the seco-iridoid branch of the pathway is limiting for MIA production in C. roseus cell suspension cultures (van der Fits et al. (2000), Science 289:295-297).
To identify regulators of the seco-iridoid pathway branch, recently generated RNA-Seq data (Van Moerkercke et al. (2013), Plant Cell Physiol. 54:673-685) was mined for TFs that were highly co-expressed with the seco-iridoid genes upstream of LAMT. Among them, two bHLH TFs, both belonging to clade IVa of the family; thus, homologues of M. truncatula bHLH25a were identified. Both clade IVa bHLH TFs, called CrbHLH18 and CrbHLH25, were cloned analogous to the procedure used for the M. truncatula bHLH25a TF (primer sets P21+P22, P19+P20, respectively, see Table 3) and were found capable of transactivating the promoters of the geraniol-8-hydroxylase (G8H) and the 7-deoxyloganetic acid glucosyltransferase (7DLGT) genes in the Nicotiana tabacum protoplast-based screen ( FIG. 8 ). The clade IVa bHLH TFs could not transactivate the promoter of the strictosidine synthase 1 (STR1) gene, delimiting their action range to the seco-iridoid branch of the MIA pathway and distinguishing it from that of ORCA3 ( FIG. 8 ).
Example 7
Enhanced Production of Seco-Iridoids and Monoterpenoid Indole Alkaloids in CrbHLH25 Overexpressing C. roseus Cell Suspension Lines
To assess the effect of overexpression of the clade IVa bHLH TFs on MIA biosynthesis in plants, stably transformed C. roseus cell cultures were generated. Therefore, C. roseus cell line MP183L was transformed by co-bombardment of a TF plasmid or the empty overexpression plasmid as a control with a plasmid carrying a hygromycin resistance gene (as described in van der Fits et al. (1997), Plant Mol. Biol. 33:943-946). Individual calli resistant to 50 mg/l hygromycin-B were converted to transgenic cell suspensions maintained weekly in LS-13 medium supplemented with hygromycin. Cell lines were screened for high expression levels of the co-transformed TF using RNA blot hybridization. For RNA blot analysis, total RNA was extracted from frozen cells by hot phenol/chloroform extraction followed by overnight precipitation with 2 M lithium chloride and two washes with 70% v/v ethanol, and resuspended in water. Ten μg RNA samples were subjected to electrophoresis on 1.5% w/v agarose/l % v/v formaldehyde gels and blotted onto Genescreen nylon membranes (Perkin-Elmer Life Sciences, http://perkinelmer.com). Probes were 32P-labeled by random priming. (Pre-)hybridization and subsequent washings of blots were performed as previously described (J. Memelink, K. M. M. Swords, L. A. Staehelin, J. H. C. Hoge, in Plant Molecular Biology Manual , S. B. Gelvin, R. A. Schilperoort, Eds. (Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994), pp. F1-F23) with minor modifications. For metabolite profiling, C. roseus cells from three biological repeats of each selected independent transgenic line were harvested 5 days after transfer, frozen in liquid nitrogen and freeze-dried. One-hundred mg dried cell mass was extracted in two steps with 10 ml methanol and centrifuged. The dried supernatant was resuspended in 1 M phosphoric acid and filtered. Extracts were analyzed using an Agilent Technologies 1100 HPLC. For the seco-iridoids, tryptophan and tryptamine, an Agilent Technologies Zorbax eclipse XDB-C18 4.6×250 mm 5-μm column was used. MIA were analyzed using a Phenomenex Luna C18(2) 4.6×150 mm 5-μm Axia packed column. The Phenomenex column used a flow rate of 1.5 ml/minute and a 25 μl injection volume using the solvents: A, 0.1% trifluoroacetic acid (TFA) in H 2 O, and B, acetonitrile with 0.1% TFA. The Agilent Technologies column used a flow rate of 1.5 ml/minutes and a 50 μl injection volume using the solvents: A, 5 mM disodiumhydrophosphate and B, acetonitrile. Seco-iridoids were detected using 220, 254, 280, 306 and 320 nm wavelengths. MIA were analyzed using 220, 240 and 280 nm wavelengths.
In contrast to differentiated C. roseus plant tissues, the C. roseus cell suspension line MP183L does not accumulate any secologanin, strictosidine or MIAs without exogenous supply of JA and loganin, illustrating that the seco-iridoid pathway is limiting in this cell line (van der Fits et al. (2000), Science 289:295-297). This was corroborated by analysis of recent RNA-Seq data that confirmed the low transcript accumulation of MEP and seco-iridoid genes in these suspension cells compared to that of the ORCA3-regulated genes, such as LAMT, SLS1 and STR1 ( FIG. 7 ). In contrast, the CrbHLH25 overexpressing cells showed a high up-regulation of the MEP and seco-iridoid genes, but not of the known ORCA3-regulated genes ( FIG. 9 , Panel A). The effect on the transcript level in the suspension cells was accompanied by a strong effect on the metabolite level. Loganic acid, secologanin and strictosidine, as well as MIAs such as ajmalicine, serpentine and tabersonine, accumulated to levels previously unreported in untreated control C. roseus suspension cells ( FIG. 9 , Panel B). Control cell lines did not accumulate detectable levels of these compounds.
Example 8
Reciprocal Transactivation of MIA and Saponin Biosynthesis Genes by MtbHLH25a and CrbHLH25
The outcome of the above screens and functional studies in M. truncatula and C. roseus suggest that clade IVa bHLH factors play a conserved role in the regulation of plant terpenoid biosynthesis. To further assess this hypothesis, transient transactivation assays in tobacco protoplasts were performed in which the bHLH TFs and promoters were switched from both species. Evaluation of the effect of CrbHLH25 overexpression on the pCYP93E2 or pHMGR1 reporter genes demonstrated that CrbHLH25 can transactivate M. truncatula saponin gene promoters ( FIG. 10 ). Conversely, transactivation of the G8H promoter by MtbHLH25 indicated that the clade IVa bHLH TFs from both species act reciprocally and thus fulfill a conserved role in diverse plant species ( FIG. 10 ).
C. roseus and M. truncatula belong to the Apocynaceae and Fabaceae, respectively. Both are dicot plant families, but are representative of the two different clades within that group, the Asterids and Rosids, respectively. Hence, the results show that clade IVa bHLH TFs in two distantly related dicot species exert a similar function in regulating terpenoid biosynthesis. These TFs are capable of acting on different classes of terpenoids and act as generic metabolic engineering tools to boost production of high-value bioactive molecules to exploitable levels in plant species.
Tables






TABLE 2



Gene and promoter sequences




ID
Sequence



MtbHLH25a
Nucleotide (SEQ ID NO: 1)


ATGGAGGATTCACTGGAAAATTTGATTTCTTA


TATGGAAATGGAAGATGATGTGATCTTGAATC


AAAGTAGCACCACCACATTTGATGAGCAAGAG


TTTCTCAAAGATATCATCCTTGAAGAACCAGA


ATGTATTGAACTCTCTTCTTATCTTTGTTCCA


ATAAAACCAAAGACAATAGTACAACTATAATT


AATGTTGAAGGTGATGCTACTAGCCCCACAAA


TAGTATTTTGTCCTTTGATGAGACAAGTTTAT


TTTGTGGTGATTATGAGAATGTTGAAACAAAC


CACAAAAGTAATAACTCCAACTCAATCAAGTC


TTTGGAAAGATCTTGTGTTAGTTCTCCAGCCA


CATACCTTCTATCTTTTGGTAACTCAAGTATT


GAACCAATCATTGAACCAATGTCACATAAAAC


TAAAAGAAGGACAGATGAATCAAGGGGGGTGA


AGGAAGCAACAAAGAAGGTTAGAAGATCATGT


GAGACAGTACAAGATCATTTGATGGCTGAGAG


GAAAAGGAGAAGGGAATTAACTGAGAATATCA


TAGCACTTTCAGCCATGATACCTGGCTTGAAA


AAGATGGACAAGTGTTATGTACTTAGCGAAGC


TGTGAATTACACAAAACAGCTTCAAAAGCGCA


TTAAAGAATTGGAGAATCAAAACAAAGATAGC


AAACCAAATCCAGCAATATTCAAGTGGAAATC


TCAAGTTTCATCAAATAAAAAGAAGTCCTCAG


AATCACTGCTCGAGGTTGAAGCTAGAGTCAAA


GAAAAGGAAGTACTCATCAGAATTCATTGTGA


GAAGCAAAAAGACATAGTGCTCAAAATACATG


AATTGCTTGAAAAGTTCAATATCACTATAACA


AGTAGTAGCATGTTACCATTTGGTGATTCTAT


TCTTGTAATCAACATTTGTGCTCAGATGGATG


AAGAAGACAGCATGACCATGGATGACCTTGTG


GAAAATCTGAGAAAATATCTATTGGAAACTCA


TGAGAGTTACTTGTGA


Protein (SEQ ID NO: 5)


MEDSLENLISYMEMEDDVILNQSSTTTFDEQE


FLKDIILEEPECIELSSYLCSNKTKDNSTTII


NVEGDATSPINSILSFDETSLFCGDYENVETN


HKSNNSNSIKSLERSCVSSPATYLLSFGNSSI


EPIIEPMSHKTKRRTDESRGVKEATKKVRRSC


ETVQDHLMAERKRRRELTENIIALSAMIPGLK


KMDKCYVLSEAVNYTKQLQKRIKELENQNKDS


KPNPAIFKWKSQVSSNKKKSSESLLEVEARVK


EKEVLIRIHCEKQKDIVLKIHELLEKFNITIT


SSSMLPFGDSILVINICAQMDEEDSMTMDDLV


ENLRKYLLETHESYL



MtbHLH25b
Nucleotide (SEQ ID NO: 2)


ATGGAGGAGAATCCATGGGGCAATTGGTCTTA


TGATTTGGAAATGGAAGAACATTTGTGTCACA


CAAACAACACATTTGACGAAGAGTTCCTCAGA


GATATCCTGTATCAGATTCCACAAGATCAATT


CAATGTTCCTATTGCCACAACTGACCTAGTAA


ACAACTCATCCATCAATGTGTCACAACATGCT


GAAGAAATGCCAACCAACTCATTATCAATACC


AACAACTGAACAACATCATGATTCTTTGCCTT


TGTCATCATCAACAGCTAACCAAGGGTCGAAT


TCGAAGAAGCCTCGAAACACTTCGGATACACT


AGATCACATAATGTCAGAGAGAAATAGGAGAC


AACTACTTACAAGTAAGATCATAGAACTTTCG


GCCTTGATACCTGGATTGAAGAAGATTGATAA


GGTTCATGTGGTAACGGAAGCTATCAATTACA


TGAAACAACTTGAAGAACGTTTGAAAGAGCTA


GAAGAAGACATTAAGAAGAAAGATGCAGGATC


ATTGAGCACCATAACAAGATCTCGTGTTTTAA


TTGACAAAGACATTGCAATCGGTGAAATGAAC


ACTGAAGAATGTTACGGGAGAAATGAATCACT


TCTAGAGGTTGAAGCTAGGATTCTAGAGAAGG


AAGTTTTAATCAAGATTTATTGTGGAATGCAA


GAAGGGATTGTGGTCAATATAATGTCCCAGCT


TCAACTTCTTCATCTGTCCATAACAAGTATCA


ATGTCTTGCCATTTGGAAATACTCTTGACATC


ACCATTATTGCCAAGATGGGTGACAAATACAA


CTTGACAATAAAGGACCTAGTGAAAAAACTAA


GAGTAGTGGCTACGTTGCAGGTATCTCATAAT


GTGCAATTTCATATCTAA


Protein (SEQ ID NO: 6)


MEENPWGNWSYDLEMEEHLCHTNNTFDEEFLR


DILYQIPQDQFNVPIATTDLVNNSSINVSQHA


EEMPTNSLSIPTTEQHHDSLPLSSSTANQGSN


SKKPRNTSDTLDHIMSERNRRQLLTSKIIELS


ALIPGLKKIDKVHVVTEAINYMKQLEERLKEL


EEDIKKKDAGSLSTITRSRVLIDKDIAIGEMN


TEECYGRNESLLEVEARILEKEVLIKIYCGMQ


EGIVVNIMSQLQLLHLSITSINVLPFGNTLDI


TIIAKMGDKYNLTIKDLVKKLRVVATLQVSHN


VQFHI



MtbHLH25c
Nucleotide (SEQ ID NO: 3)


ATGGAGGAAATCAACAACTCAGCTATGAAAGT


ATCATCATCAATCAGCAGCTGGTTATCTGATT


TGGAAATGGACGAATACAATATATTTGCTGAG


GAATGCAACCTTAATTTCCTTGATGCTGATGT


GGGAGGGTTTCTTTCAAATGACATATCTAATG


TATTTCAAGAACAAAACAAACAACAATGTTTA


TCTTTGGGGTCCACTTTTCATGAAACAATTGA


TAATAGTGACAAAAACAATGAATCTCTTTCTC


CATCTTTTCAGTTTCAAGTTCCATCTTTTGAC


AACCCCCCAAATTCATCCCCTACTAACTCAAA


AGAGAATATTGAAACAATACCATTGTCTCCAA


CCGATTTGGAAAATATGAATCACTCAACAGAA


ACCTCAAAAGGGTCATTGGAAAATAAAAAGTT


GGAAACAAAAACCTCAAAAAGCAAAAGGCCAC


GTGCTCATGGTAGAGATCACATCATGGCTGAG


AGAAATCGAAGAGAGAAACTCACCCAAAGCTT


CATTGCTCTTGCAGCTCTTGTTCCTAACCTTA


AGAAGATGGATAAACTATCTGTACTAATTGAC


ACTATCAAATACATGAAAGAGCTTAAAAATCG


TTTGGAAGATGTGGAAGAACAAAACAAGAAAA


CAAAAAAAAAATCATCGACCAAACCATGCCTA


TGCAGCGATGAAGATTCGTCATCATGTGAGGA


TAACATTGAATGTGTTGTTGGTTCACCATTTC


AAGTGGAAGCAAGAGTGTTAGGAAAACAAGTG


CTGATTCGGATCCAATGCAAGGAGCATAAGGG


GCTTCTGGTTAAAATTATGGTCGAAATTCAAA


AATTTCAACTATTTGTTGTCAATAACAGTGTC


TTACCCTTTGGAGATTCTACGCTCGACATTAC


CATCATTGCTCAGTTGGGTGAAGGGTACAACT


TGAGCATAAAGGAACTTGTGAAGAACGTACGC


ATGGCATTATTGAAGTTTACGTCATCATAA


Protein (SEQ ID NO: 7)


MEEINNSAMKVSSSISSWLSDLEMDEYNIFAE


ECNLNFLDADVGGFLSNDISNVFQEQNKQQCL


SLGSTFHETIDNSDKNNESLSPSFQFQVPSFD


NPPNSSPTNSKENIETIPLSPTDLENMNHSTE


TSKGSLENKKLETKTSKSKRPRAHGRDHIMAE


RNRREKLTQSFIALAALVPNLKKMDKLSVLID


TIKYMKELKNRLEDVEEQNKKTKKKSSTKPCL


CSDEDSSSCEDNIECVVGSPFQVEARVLGKQV


LIRIQCKEHKGLLVKIMVEIQKFQLFVVNNSV


LPFGDSTLDITIIAQLGEGYNLSIKELVKNVR


MALLKFTSS



MtbHLH25d
Nucleotide (SEQ ID NO: 4)


ATGGAGGAAATCAACAACACACCAATGAACGT


ATCAGAAGAAACCAGCAAATGGCTATCTGATT


TGGAAATGGATGAGTATAATTTATTTCCTGAA


GAATGCAACCTAAACTTCCTTGATGCTGATGA


GGAAGAGTTTCTTCCACAAGAACAAACCCAAC


AACAATGTTTGAGTTCAGAGTCCAATTCCACA


ACTTTCACCAACTCATTCACTGATGAAACAAA


TTTTGACTCTTTTGACTTTGATTTTGAAATTG


AGAGACCAACCATGGAGCTGAACACAATCTTT


AGTGACAACAGCATCATTGAAACCATTTCACC


AAAACTTTCTCCATCATCATCTAACTCATCTT


TGCACTCTCAGATTTTGTCTTTTGACAACCTC


CCAAATTCACCTGCTACCAACACCCCTCAATT


TTGTGGACTCACCCCTACCTTAATCTCAAAGT


CAAAACAAAACAAAACAGTGTTAGTGTCTCCA


CCCCAAATAAGAAACATTCATGTCTCAACTCA


AAATCCTATAGGGTTATCCAAAAATCAAAACT


TTGCAACAAAAACCTCTCAAACCAAAAGGTCT


CGAGCCAACGCTGATGATCATATCATGGCTGA


GAGAAAGCGAAGAGAGAAACTTAGCCAAAGCT


TCATTGCTCTTGCAGCTCTTGTTCCTAACTTG


AAAAAGATGGACAAGGCTTCTGTATTAGCTGA


GTCTATAATCTACGTGAAAGAGCTTAAAGAGC


GTTTGGAAGTTTTGGAAGAACAAAACAAGAAA


ACAAAGGTAGAGTCCGTGGTTGTTCTGAAGAA


ACCAGACCATAGTATCGATGATGATGATGATG


ATGATGATAACTCATCATGTGATGAGAGTATT


GAAGGTGCTACTGATTCATCCGTACAAGTGCA


AGCAAGAGTGTCAGGGAAAGAGATGCTGATTC


GGATTCACTGCGAGAAGCACAAGGGAATTCTG


GTGAAAGTCATGGCTGAGATTCAAAGCTTTCA


ATTGTTTGCTGTGAATAGTAGTGTCTTACCCT


TTGGGGATTCCATTGACATTACTATCATTGCT


GAGATGGGTGAAAGGTACAACTTGAGCATAAA


GGAACTTGTCAAGAACCTACACATGGCAGCAT


TGAAGTTTATGTCATCAAAAATCACAGACTGA


Protein (SEQ ID NO: 8)


MEEINNTPMNVSEETSKWLSDLEMDEYNLFPE


ECNLNFLDADEEEFLPQEQTQQQCLSSESNST


TFTNSFTDETNFDSFDFDFEIERPTMELNTIF


SDNSIIETISPKLSPSSSNSSLHSQILSFDNL


PNSPATNTPQFCGLTPTLISKSKQNKTVLVSP


PQIRNIHVSTQNPIGLSKNQNFATKTSQTKRS


RANADDHIMAERKRREKLSQSFIALAALVPNL


KKMDKASVLAESIIYVKELKERLEVLEEQNKK


TKVESVVVLKKPDHSIDDDDDDDDNSSCDESI


EGATDSSVQVQARVSGKEMLIRIHCEKHKGIL


VKVMAEIQSFQLFAVNSSVLPFGDSIDITIIA


EMGERYNLSIKELVKNLHMAALKFMSSKITD



bHLH domain
TVQDHLMAERKRRRELTENIIALSAMIPGLKK

of
MDKCYVLSEAVNYTKQLQKRIKEL

MtbHLH25a
(SEQ ID NO: 9)



bHLH domain
DTLDHIMSERNRRQLLTSKIIELSALIPGLKK

of
IDKVHVVTEATNYMKQLEERLKEL

MtbHLH25b
(SEQ ID NO: 10)



bHLH domain
HGRDHIMAERNRREKLTQSFIALAALVPNLKK

of
MDKLSVLIDTIKYMKELKNRLEDV

MtbHLH25c
(SEQ ID NO: 11)



bHLH domain
NADDHIMAERKRREKLSQSFIALAALVPNLKK

of
MDKASVLAESIIYVKELKERLEVL

MtbHLH25d
(SEQ ID NO: 12)



CrbHLH25
Nucleotide (SEQ ID NO: 15)


ATGACAATGATGATGACGATGGATAATTCAGT


CAATTCATGGTTCTCTGATCTGGGAATGGAGG


ATCCTTTCTCCAGCGATCAATATGACATCACG


GACTTTCTGAATGAGGATTTCGCTGCACTTGG


AGAGGACTTGCAAGCATTCACTCCAACAGCCG


AATCTGATTCATCCAATAACTTCATTAATATT


CCAACTAGCAATTCATCAAACACCTTATGTGC


ATTGGCTACGGAACTTCCTTCGGTTGTGGCCG


AAATTCCAACCACCATCACTGCCACTACCACT


ACTAAAAAACGAAAATCCAATTCGTCGACAAA


TCAAAATGTGCCGAATGCTCGTAGAGCCGCTC


GTACTCCCATCGTTCTCACATTTGGGAATACA


CGGCAGAAACCAATCCTAATAAACACAGCTTA


AGCCCTGATATTAATGATGATTCATTAATATC


AACTGAGAATTTGACCTCCCAAGGAAATCTTG


AAGAGGCAGTAGCAGCTGCCAAAAGTACAAAA


CTAAACAAGAAAACTGGTGGCCGCGTTAGGCC


TGCATCCCAAACCTATGATCACATAATTGCTG


AAAGAAAGCGACGTGAGCAGCTCAGCCAGCAT


TTTGTCGCACTTTCTGCCATTGTTCCTGGCCT


TAAGAAGATGGATAAAACTTCTGTACTTGGAG


ATGCGATTACCTACTTAAAACATATGCAAGAG


CGAGTAAAATCACTAGAAGAACAAACAACAAA


ACAAACAATGGAATCAGTGGTGCTAGTGAAGA


AATCACAAGTGTTAGTTGAAGATGAAGGTTCT


TCAGATGAGATTGATCAAGATCAGTCCTCGTC


ACAGCTCCCTGAAATTGAAGCCAAAGTTTGTG


ACAAAACCATTTTACTCAGAGTTCACTGCGAA


AAGAACAAAAGGGTCCTTATTAATATACTCTC


TCAACTTGAAACACTCAATCTTGTTGTTACTA


ACACCAGCGTTTCAGCTTTTGGAAGTTTGGCT


CTTGATATTACTATCATCGTTGAGATGGAGAA


AGAATCAAGCATAAACATGAAAGAACTTATTC


AAACTCTTCGGTCAGCTGTCATGCGTGCAAAT


TTAGAAGATTGA


Protein (SEQ ID NO: 17)


MTMMMTMDNSVNSWFSDLGMEDPFSSDQYDIT


DFLNEDFAALGEDLQAFTPTAESDSSNNFINI


PTSNSSNTLCALATELPSVVAEIPTTITATTT


TKKRKSNSSTNQNVPNARRAARTPIVLTFGNT


TAETNPNKHSLSPQDINDDSLISTENLTSQGN


LEEAVAAAKSTKLNKKTGGRVRPASTYDHIIA


ERKRREQLSQHFVALSAIVPGLKKMDKTSVLG


DAITYLKHMQERVKSLEEQTTKQTMESVVLVK


KSQVLVEDEGSSDEIDQDQSSSQLPEIEAKVC


DKTILLRVHCEKNKRVLINILSQLETLNLVVT


NTSVSAFGSLALDITIIVEMEKESSINMKELI


QTLRSAVMRANLED



CrbHLH18
Nucleotide (SEQ ID NO: 16)


ATGATGACGATGATGATGGATAATTCAGCAAC


GTCCCCTAATTGCTTCTCTGAGCTTCAGCAGG


GAATGGAGGATCCTACTAACTTCCTCATTAGC


AATGATGATAAATGTGATGTAATGGAGTTTTT


GGATGAAGATATATGTGCAGTTCTTGGTCAAG


ACTACTTCCAAATTTCCTCTTTCTCCCCAAAT


GACTACTCATTTTCTCCAAACTTGGATCCAAA


TTCCACTTTATATCCATCTTCTTCTTCAACTC


CTACGGATATTCATGACCAATCTCCACCATTT


ATGCTTAATGATGATATTGATGAAATAATGAA


TAGACGACCAGCCAAACAGCTGAAGAGCACTA


GTAATAATAATCAAAATAACCAAAATCCGTCG


ACTATTCATGATAGCTTTGATGCTCAAATGTC


TACTCCCTACCTTCTTACTTTTGGGAATCCAA


ATTCACCTGAAATTATTAATCCACCTCATCAT


CAACAACACCATCAACCTAATGCAACATTAAA


CTTAAACCCCTCTGATGAAGATGTTCAAGTAT


CCGAGGTTTTCAACTCCCAAAGTTCATCATAT


GGAAATCTTATTGAAGAGGAGGCAGCAGCACC


CAAAAGTTCAAAACCAACGTCCAAGAAAAGTG


GAGGCCGTGTAAGGCCGGCTTCTCAAACTTAT


GATCACATTATAGCTGAAAGAAAGAGAAGGGA


GATCCTCAGCCAGCGCTTTATGGCTCTTTCTA


CTCTAGTTCCCGGTCTCAAGAAGATGGATAAA


ACATCAGTACTTGGAGATGCAATTAAGTACTT


AAAATATCTCCAAGAAAGAGTTCAGATTCTTG


AGGATCAAGCAGCCAAACAAACTATGGAATCG


GTGGTGATGGTGAAGAAATCACATGTCTTCAT


CCAAGAAGAAGAAGATGATGAAGAAGGATCTT


CAGATGATCAGATCACCAGCGATGGCGGAAGC


TCAGAAGAACACCCATTACCTGAAATTGAGGT


TAAAGTTTGCAATAAAACACTTCTTCTGAGAA


TTCACTGCGAGAAGCAAAAAGGGGTGCTTATT


AAGTTACTTAATGAGATTGAAAGGCTCAATCT


TGGCGTTACCAACATTAACGTTGCACCATTTG


GAAGCTTGGCTCTTGACATTACCATTATTGCT


GAGATGGAGAAAGAGTACAATATGACAACGGT


ACAAGTGATTAAAAATCTTCGGTCAGTTCTTC


TTAACAGTCCACCAATGGCAGACTGA


Protein (SEQ ID NO: 18)


MMTMMMDNSATSPNCFSELQQGMEDPTNFLIS


NDDKCDVMEFLDEDICAVLGQDYFQISSFSPN


DYSFSPNLDPNSTLYPSSSSTPTDIHDQSPPF


MLNDDIDEIMNRRPAKQLKSTSNNNQNNQNPS


TIHDSFDAQMSTPYLLTFGNPNSPEIINPPHH


QQHHQPNATLNLNPSDEDVQVSEVFNSQSSSY


GNLIEEEAAAPKSSKPTSKKSGGRVRPASQTY


DHIIAERKRREILSQRFMALSTLVPGLKKMDK


TSVLGDAIKYLKYLQERVQILEDQAAKQTMES


VVMVKKSHVFIQEEEDDEEGSSDDQITSDGGS


SEEHPLPEIEVKVCNKTLLLRIHCEKQKGVLI


KLLNEIERLNLGVTNINVAPFGSLALDITIIA


EMEKEYNMTTVQVIKNLRSVLLNSPPMAD



bHLH domain
(SEQ ID NO: 19)

of CrbHLH25
QTYDHIIAERKRREQLSQHFVALSAIVPGLKK


MDKTSVLGDAITYLKHMQERVKSL



bHLH domain
(SEQ ID NO: 20)

of CrbHLH18
QTYDHIIAERKRREILSQRFMALSTLVPGLKK


MDKTSVLGDAIKYLKYLQERVQIL



pHMGR1
(SEQ ID NO: 21)


ATTGGTAGGATCAATTGTTGATTGTCTTGGTT


TATGTAAAATGTTAAAATGTGACAATTAAAAA


GAAACGGAGGTAGTACTTCTATCATATTTAAT


GCTATTACTTCTAAATTCGTTTTCAATTATTT


ATGTTTGAATCCTCCGAGTGTCAATTTTTGTT


GATTTGTTTATAGTGGGATTGGTCCTTTGGAT


TAGTCGGTTCTTGAATCAAATACTAAATTTTC


AAACAAAAATTTAGATTTTACTTTTCTATAAC


CATTTTATTATCATAGAAAAAAAAATTAAATC


TCCGAAAAATTATCATGAACAAGTTTTCATGA


GGGACAATTTAGAAAATGCAATTATGGCTAAA


CTATGTTTCAAGTCCCTTAAATTGTTCATTTT


GTTCACTTAAATTACAAACATTGAGTTATTCA


AATAAAAATTACAAACATTGAGTAAGAAAACA


AAATTAAACACGTTTTTGACTTTAGATTCGAT


TCATATTAAACACATTTGAGTCAAAAACAAAA


TTAAACACGTTTTTTTTGCAGCACAAGTGATG


TATAAAACTAATTAGTTTTTATTTCAGCAACA


AAAAATGTACTTATAGTTTGATTAGTTTGTGC


AAATAAGAAATATATATAGTATTTTAGAAACG


TCAAGTAAAGGCTTCTTCTAGTGATGTGTAAT


GTTGGCATCTCTCCTCCATAAAATATGTTATC


TTGGAGTAAAAGTTAGGTACTGATTACAATAT


TCTTCCCTCGTTGTTCACGAGTTCCTTACGAT


CTAGCTATCAAGCTACTGTTACTTGACTTCTT


AAAATATTGGCATATACCTAATTCCTCACTCA


CTCCCTGAGTACTCTTTATCTATAAATAACAT


TCAAAATAAATCTTGGCATTCATTTTCAAGTT


AAGTTTCAATCATCCCCTAACCTGAACTCCTT


TTTCTCCTTTCTTTTTCCATTTTCGGATTTCA


AATCCTTCACTTAATAACCAAGAGAAAGAGAG


AGAGAGAG



pBAS
(SEQ ID NO: 22)


TAATTAATTAATTTGTAGTAGTCCTCGTTTGT


GACCACGCTTCCCTCGTGTTTCATTTATTTTA


TCCTGCTCTCCACGCTATACTCGCTACATATA


TAATAATAATCAATGAATGTATATTGTTAAAT


TCCATCCTGTTCACCAATAATGATTGAATTCA


AGAATTAATTTTAAATATATATTCTAAAATAA


AAGGAAACTGCAAAAAATAAACGATGAATCAT


ATGAGACAAAAATGAAGAGATAACACTTATAC


TGCTTAATTATTATCATTGTAAAGATAAATTG


AGAAGAATACATGACGGCAACTGCAACTCAAT


GGAAGAACACTTACAAGATGTATATGAAAACA


CCTACTTCAAAAATGTACATTAAAGCGAAAAA


GGCATTGTAGTTAGGAGTCACGAATATTTTAT


ATTTTTCAAAGCGAAAAAGGATCGATCCATTT


ATTTATATATTATTAATATTAAAATGAATATA


TGTTTTTGTTGGTATATTAAAGCGGTGGTCAT


TGTTTAATAAGAAAAGCTGTGCACATATGTGA


AAAGAAAATGACAAAATCAGTATGAGTGGTCT


TATTTAGGAAGTTATGATGGTCATGTTAACAA


ATGCTTTAAGAATATTAGTAAAAAGTAATAAC


GTCACTTTTGTATTGACAATAATGATTTTTAA


ATTTAAAAAAAAAAAAAAGAAAAAATTAAGTA


CAGCAATTTGAAATACGATTTTGTTAACATAA


TCATGTAATAAATAATATTATTGCAAAAATGA


ATAGTTGTAATTAGGTGTCACGCTATAAAATA


AAATAAACAAAAAGGTGGGTTTGGTTCGAAAA


GTAAGAAATAGTTTTAAATTAGATTGAGAGAG


TTGTTGTGGATATCTACTATAAGTAGCGGCAA


TATGAATGAGTCTTTCCTCATATCAATCAAAT


TAAGGAGGTCTTGCTAGCTTCCATATATAACT


CATCACTAAAACTTCTAATAATTGAAAAAAAG


TAATTAAG



pCYP93E2
(SEQ ID NO: 23)


AAAATAATATGTCATTGTAGACATGTTTTCTT


TTTGCCTACTACCATAAGAATCTTTTCCTAGT


AGGCGTCCAAAATATTTAGATTTAAATATTAT


AATTATTATTTTAGATTTAAATGTATTTATTA


TTACTTTTAATCGAATAGTTTAGAGGCGAGAA


GAAACACACAATAAATATTAAGAAGTGGAAAA


TTCAAAATTAGAACCCGAGATCAAAAACTTGC


GGTTTTCAACCCGGGATCATACTAACAAGTGT


CATAAATATTTTGTTTAAAGAATACTCTCTGG


TAACATTTATAAGCAAAAAAAACTTTTTAGGT


ACATTGAATAACTAATATATATGACATATAAA


TGTGACCAGATACATTGATTATTCAATGAATT


TAAAATGCTGATTTTTACTTACAAATTTGATC


GGAGGGAGTACATAAAAAGAAAATTTATATTA


GAAATATAAATCAAACATACGTCGAAGTCATA


TATACATCATTTTTTATGCAAAAGTTGCAATT


CCTAACAATGTTCTCACGATATTTGTTAGCCT


TTCTCAATTAAAAATGTATTTTTAAAATAATT


TGCGAGTGTGTAAATTAACATTGGGATTATAC


AAGTATTCTCAACCATTTATTCATCAAGACAA


AACAAAATCTTAACAATTTACCATAAATAAAC


ATAAAAAACACGTATCAATCACACACCGTTAG


CTGACAAACAACGTCAGCAATCATGAAATTAT


TTTATGTACCACGAGGGACACATAAGCAACAT


TCTTATTATAATATTCGACCACGAGGATCGCA


AAAATTAATAACTTAACTTCACTCAACAGTAC


CATTCAAAATCATACTAGACCAAGTCAAATTT


GTTCTTCTATTTATGTTCCATCATAGTTCAAG


AATTTAGATTCATCTTAAGCATAGACGTTGAG


TTGTGTTTGGTATTTTGCATTGACAACATTGT


CCTTTTTTGTCACACCAATTACCAAATCAAGT


ACTCAACC



pUGT73F3
(SEQ ID NO: 24)


TTATCGTAATATATGATCAATATTCTCTATTA


TGAGTTACAAAAAAAAGAAAAAAAATCAATTT


TATTTCGCCAACACGTTTTGTAAATAAGATTT


AGTTATTAAAGTTATAAAGACTAGCGAAAAGA


CGGGTACTCACGTGCCCGTCTTTCCGCTCTTT


TTATTGCGCTAATGTTTGAAAATTATGAATGA


TGTTTTTTTTATGAAATTTTGATTTTGATTAA


AACTAAAAAATATATAAATGTTTGTTTCTTTC


AACTTATAAAAATCAACTAACTATGATTATCT


ATTTCAACAACACCCACAATAATACAATATAA


CAAAAAATATAATCTAAATTCCTTTTTAATGA


CAGCAACAACAATTTTATTATAGAAAAAGTTG


TTTAAGGATATAATTGGAATGATGAAAAAGTA


AGTATATTCATATCGTGTTTGTTACATTTGTT


TTAATATTCAAATTTATTCTTATCTAATTGTT


ACATGTATTATTTGTATTAAAAAATTTTGGAA


AGAAAAATATAACCAAAAAATACTGAAAGGGA


TAGTTCTCTTTATATATAGTATAGATAAAAAA


TATTTCTTTTTCAAAATAAAAAACTCATTTAA


TTATTAATATAAAATCATATGTATCTGATACA


TTTAAATTTTCGAGTGTAGCATGTAGTTGTCA


TTTGGGAATGCACAACAAATTGTATCTGATAC


AATTTTTTCTTTTGAAGGATGAAGTTACAATC


TTTAATTTAGTATTAATAGTCGGCATTCAAAA


CTAAGATGGAAATCTCGCGGAAAAGATTCCTC


TCCTCGTGGGACACGAGCACATAGATCATAGA


TAAACTAATAACAAAAACAACATATAATATTA


AAGAAGTAAATTATTTATGTTAAACCAAAGTA


TGGTTGAAATTGAATATATAGAGATGCACTCA


CAGTGACAAACACAAACATAGAAACGTGAATA


GAATAGAATAGAATAGAGAGAATAATTTGATC


CATAAGCA



pUGT73K1
(SEQ ID NO: 25)


TTTTATCTATGGAGTGACTTTATGAAGACATC


CACCAAATTATCTTTCGATGCAACTTTCTCAG


TAATAATTTTCTTAAGTCTGATCTACAACAAA


CCATATCATACATGGTGCTTCCTACGACACTT


GCATATGGTGTACCCTCCATCTTCTCATCCTC


AAATTGAGGATAATGCATAGTCAAAAGTTTTG


TGTGATGATCCAAAGGAATCTCAGTGGTCATA


GCACTACTCTTTATGAGTTGTCCCACCACTTT


CTTTAGGTAAACATATTGAGATAAGAAAAATT


CATCCTTGTCCTAGTTTTTCCGGTATTTTTTT


CAAGTATACTCTTAGCAACAACAAAAAATTTC


ATCTCAAGCTAGGGGACTAAGTTGTCCCTTGT


ATAAAAAACGAGACACTAATTTGGTATTATCT


CTTGTCAGGAACTAATTTGTGTGATGAAACTC


AGATACACTAAATGTTTCATTATACGTAGTCT


TCGTGAGCATAGCTCAGTTGACAAGGACAATA


CATAATATCGCAAGGTTTGAGTTCAAATCTCA


GACACAAAAAAAAAAATGTAAAAAATTATATG


TACGAATTTGTGCTTCAATATATTAAATATTG


TTAAACTTTATCATCTGAATTGTACTTATAAA


AATGTCCACTCTAGGATCAAATATAGAAAATA


ATAAGATTCAATCATTTACAAAAAATATTTTA


AAAATTATTCACGCAGTTCACGAGTACAGTTT


TTATCAATATAAACAATGAACAATAAACTAAT


AAAATATGCAGCCCGTGTATCTCGCTATTGCA


AGTATATATATTTTAATGAATCATTTCGTTTT


CTTTGGACCGTCTTTAATTTAGATAAATAATA


GTGACAAATAATACATTCATAATTCATCAATC


AACCCCTTTTATAAACACTTCTAAGTTGTAAC


AGATTTAGAACACAGAGCACTAAACCAAGAAG


AAGAAAAAAGAGAAGTAGAAGAATCACATAAG


CTAAAAAA


























































































































































































































































































































































































































































































































TABLE 3



Primer sequences







Primer
SEQ ID
Sequence 5′ to 3′




P1
26
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAATGGAGGATTCACTGGAAAATTTG




P2
27
GGGGACCACTTTGTACAAGAAAGCTGGG




TATCMCAAGTAACTCTCATGAG




P3
28
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAATGGAGGAGAATCCATGGG




P4
29
GGGGACCACTTTGTACAAGAAAGCTGGG




TATCMGATATGAAATTGCACATTATG




P5
30
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAATGGAGGAAATCAACAACTC




P6
31
GGGGACCACTTTGTACAAGAAAGCTGGG




TATTATGATGACGTAAACTTCAATAATG




P7
32
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAATGGAGGAAATCAACAACAC




P8
33
GGGGACCACTTTGTACAAGAAAGCTGGG




TATCAGTCTGTGATTTTTGATGAC




P9
34
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAATTGGTAGGATCAATTGTTG




P10
35
GGGGACCACTTTGTACAAGAAAGCTGGG




TACTCTCTCTCTCTCTTTCTCTTGG




P11
36
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTATAATTAATTAATTTGTAGTAGTC




P12
37
GGGGACCACTTTGTACAAGAAAGCTGGG




TACTTAATTACTTTTTTTCAATTATTAG




P13
38
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTAAAAATAATATGTCATTGTAGACATG




P14
39
GGGGACCACTTTGTACAAGAAAGCTGGG




TAGGTTGAGTACTTGATTTGG




P15
40
GGGGACAAGTTTGTACAAAAAAGCAGGC




TTATTATCGTAATATATGATCAATATTC




P16
41
GGGGACCACTTTGTACAAGAAAGCTGGG




TATGCTTATGGATCAAATTATTCTC




P17
42
GGGGACAAGTITGTACAAAAAAGCAGGC




TTATITTATCTATGGAGTGACTTTATG




P18
43
GGGGACCACTTTGTACAAGAAAGCTGGG




TATTTTTTAGCTTATGTGATTCTTC




P19
44
GGGGACAAGTTTGTACAAAAAAGCAGGC




TGTATGACAATGATGATGACGATG




P20
45
GGGGACCACTTTGTACAAGAAAGCTGGG




TATCAATCTTCTAAATTTGCACGC




P21
46
GGGGACAAGTTTGTACAAAAAAGCAGGC




TGTATGATGACGATGATGATGGATAA




P22
47
GGGGACCACTTTGTACAAGAAAGCTGGG




TATCAGTCTGCCATTGGTGGACTG