(en)The present disclosure provides methods, recombinant DNA molecules, recombinant host cells containing the DNA molecules, and transgenic plant cells, plant tissue and plants which contain and express at least one antisense or interference RNA specific for a thiamine biosynthetic coding sequence or a thiamine binding protein or a thiamine-degrading protein, wherein the RNA or thiamine binding protein is expressed under the regulatory control of a transcription regulatory sequence which directs expression in male and/or female reproductive tissue. These transgenic plants are conditionally sterile; i.e., they are fertile only in the presence of exogenous thiamine. Such plants are especially appropriate for use in the seed industry or in the environment, for example, for use in revegetation of contaminated soils or phytoremediation, especially when those transgenic plants also contain and express one or more chimeric genes which confer resistance to contaminants.

1.ApplicationNumber: US-55930104-A
1.PublishNumber: US-7667096-B2
2.Date Publish: 20100223
3.Inventor: MEAGHER RICHARD B.
MCKINNEY ELIZABETH
KIM TEHRYUNG

4.Inventor Harmonized: MEAGHER RICHARD B(US)
MCKINNEY ELIZABETH(US)
KIM TEHRYUNG(KR)

5.Country: US
6.Claims:
(en)The present disclosure provides methods, recombinant DNA molecules, recombinant host cells containing the DNA molecules, and transgenic plant cells, plant tissue and plants which contain and express at least one antisense or interference RNA specific for a thiamine biosynthetic coding sequence or a thiamine binding protein or a thiamine-degrading protein, wherein the RNA or thiamine binding protein is expressed under the regulatory control of a transcription regulatory sequence which directs expression in male and/or female reproductive tissue. These transgenic plants are conditionally sterile; i.e., they are fertile only in the presence of exogenous thiamine. Such plants are especially appropriate for use in the seed industry or in the environment, for example, for use in revegetation of contaminated soils or phytoremediation, especially when those transgenic plants also contain and express one or more chimeric genes which confer resistance to contaminants.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application No. PCT/US 2004/018058, filed Jun. 3, 2004, which application claims benefit of U.S. Provisional Application 60/475,551, filed Jun. 3, 2003.


ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT
This invention was made with funding under Grant No. DEFG796ER2027 from the Department of Energy and Contract No. R01 GM36397-14A1 from the National Institutes of Health. The government has certain rights in this invention.


BACKGROUND OF THE INVENTION
The field of the present invention is plant molecular biology, especially as related to genetically modified plants with conditional male sterility. Specifically, the present invention relates to conditionally male and/or female sterile plants in which sterility is achieved by disrupting the availability of thiamine by high affinity binding proteins expressed in pollen and/or in the developing ovule, by inhibiting functional expression of one or more thiamine biosynthetic proteins or by destroying thiamine in those plant tissues.
Systems of plant sterility are essential tools in the hybrid seed industry, forestry, conservation biology, and phytoremediation. The hybrid seed industry plants millions of acres of in which one of the two elite parent plants in a genetic cross is male sterile as a result of physical or genetic emasculation. Male sterility is the basis for this 400 million dollar per year industry. Foresters are interested in plant sterility, because wood production is dramatically reduced when nitrogen and phosphorus are drained into pollen and megagametophyte production. In addition, genetically engineered trees, shrubs, and grasses are being developed that extract, detoxify, and/or sequester toxic pollutants and for phytomining of precious elements. Conditional male sterility adds value to and limits unauthorized propagation of valuable plants for any purpose. Plant sterility systems are needed if genetically modified organisms (GMOs) are to be released into the natural environment with no release of their germplasm. In this case, complete male-female sterility is desirable so that the organisms cannot reproduce seed by any means.
Numerous strategies have been used to generate male sterility for the hybrid seed industry ranging from manually emasculating plants, altering the levels of essential metabolites in pollen, and generating toxins in developing pollen with two component systems (Perez-Prat and van Lookeren Campagne, 2002). Another approach has been to make the essential vitamin cofactor biotin unavailable in reproductive tissues to render a plant sterile. Applying this harmless vitamin to the plants then restores fertility (Albertsen and Howard, 1999).
There is a need in the art for economical and safe compositions and methods for rendering plants male and/or female sterile, especially where the sterility can be controlled so as to allow the production of viable seeds under controlled conditions.
SUMMARY OF THE INVENTION
The present invention provides DNA constructs comprising tissue specific transcription regulatory sequences which direct expression of an associated sequence in developing pollen and/or ovules and operably linked to the transcription regulatory sequence, a sequence which when expressed, ablates the availability of thiamine in developing pollen or ovules, either by expression of at least one interfering RNA or antisense RNA specific to at least one thiamine biosynthetic enzyme (e.g., AtThi2 or AtThi3) or by the expression of a high affinity thiamine binding protein (e.g., an enzymatically inactive PDC2) such that thiamine is sequestered in the developing pollen and/or ovules or by expression of a thiamine-degrading enzyme (thiaminase). Also within the scope of the present invention are vectors and recombinant host cells comprising the DNA constructs of the present invention. Pollen-specific or pollen- and ovule-specific transcription regulatory sequences, as specifically exemplified herein, include the transcriptional regulatory sequences of the Arabidopsis thaliana Act11, Act12, or Act2 or Lat52p genes. The target for inhibiting expression of a thiamine biosynthetic gene can be AtThi2 or AtThi3. The AtPDC gene can be modified to produce a thiamine-sequestering protein in pollen and/or ovules as described herein. As specifically exemplified, the thiamine-sequestering derivative has coding and amino acid sequences as given in SEQ ID No: 7-8. The sterility resulting from the regulated expression of the constructs of the present invention is conditional; fertility is restored by the application of thiamine to the flowers, for example, in a spray which may optionally further comprise a surfactant such as 0.1% Silwet or Triton X100 (allyloxypolyethyleneglycol methyl ether, OSi Specialties, Inc, Tarrytown, N.Y. or t-octylphenoxypolyethoxyethanol) or in the growth medium.
There are numerous hydroxyethylthiazole kinase (HTK) and phosphomethylpyrimidine kinase (PPK) sequences available on the internet site for The National Center for Biotechnology Information, including the following accession numbers: CA765813, U38199, U27350, Oryza sativa ; BU964708, BM524834, BG725189, Glycine max , CA900839, CA900838, CA896676, CA896675, Phaseolus coccineus ; AF193791, Fragaria x ananassa ; AJ251246 , Saccharum officinarum ; X81855, Nicotiana tabacum ; BM 177583, Glycine max ; and BQ618938, Zea mays.
Thiaminase can be expressed under the regulatory control of pollen-specific or pollen- and ovule-specific promoter sequences, with the result that thiamine in the relevant reproductive tissue is degraded and that tissue cannot develop for its intended function.
For the RNAi strategy for conditional plant sterility, it is preferred that there be a very high degree (greater than 95%) of sequence identity between the expressed RNAi nucleotide sequence and the target gene. Preferably, the RNAi construct is derived in sequence from the same plant source and is identical in sequence to the target sequence.
While the AtACT11 and AtACT12 promoters (transcription regulatory sequences) are specifically exemplified herein, the skilled artisan can isolate the corresponding tissue specific promoters from other species and use them in the conditional plant sterility methods of the present invention as well.
The present invention further provides recombinant plant cells, recombinant plant tissue and transgenic plants which contain the DNA constructs of the present invention. Transgenic plants which contain the DNA construct are conditionally male sterile or male-female sterile, i.e.; they are sterile in the absence of exogenously supplied thiamine.
Also within the scope of the present invention are methods for rendering a plant of interest conditionally male and/or female sterile. The method comprises the steps of introducing a vector comprising a DNA construct containing a pollen-specific or pollen- and/or ovule-specific transcriptional regulatory sequence operably linked to a sequence which, when expressed, renders the developing pollen and/or ovules deficient in thiamine. This can be achieved by expression in the developing the pollen and/or ovules of a thiaminase or a protein in the developing pollen which binds thiamine with high affinity or it can be achieved by the expression in developing pollen of an antisense RNA or an interference RNA specific to a sequence which specifies a thiamine biosynthetic enzyme. Supplementation of the transgenic plant during flowering with exogenous thiamine temporarily restores sterility. The methods of the present invention are applicable in forestry, horticulture, agriculture, conservation and phytoremediation, among other areas.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the thiamine biosynthetic pathway.
FIG. 2A illustrates the Arabidopsis thaliana AtThi2 gene structure with the thi2-1 mutant T-DNA insertion. FIG. 2B illustrates expression (from A2pt:AtThi2Ri or A11pt:Thi2Ri or A12pt:Thi2Ri) of an antisense (A) oriented and sense (S) oriented 100 nucleotides of AtThi2 cDNA separated by a GUS spacer in a single transcript. FIG. 2C shows that the RNA product of this engineered construct forms a stem-loop transcript that leads to degradation of native AtThi2 mRNA. (ts, transcriptional start; pA, polyadenylation sites).
FIGS. 3A , 3 B and 3 C provide partial plasmid maps of pACT12pt, pACT11pt and pACT2pt, respectively.
FIGS. 4A-4C provide the Arabidopsis thaliana bifunctional phosphomethylpyrimidine kinase/thiamine phosphate pyrophosphorylase (PPK/TPP) (AtThi2) nucleotide and amino acid sequences, SEQ ID NO:1 and SEQ ID NO:2, respectively.
FIGS. 5A-5B provide the Arabidopsis thaliana hydroxyethylthiazole kinase (HTK) (AtThi3) nucleotide and amino acid sequences, SEQ ID NO:3 and SEQ ID NO:4, respectively.
FIGS. 6A-6B provide the Arabidopsis thaliana pyruvate decarboxylase (AtPDC2) nucleotide and amino acid sequences, SEQ ID NO:5 and 6, respectively. A mutation (PDC E517Q ) useful in the present conditional plant sterility strategy is indicated; the enzymatically inactive, thiamine-binding mutant coding and amino acid sequences are given in SEQ ID NO:7 and SEQ ID NO:8, respectively.
FIG. 7 diagrammatically illustrates the steps for the rapid cloning of RNAi constructs using overlap extension polymerase chain reaction (OE-PCR), as described herein below.
FIG. 8 provides a restriction map of AtThi2. Restriction endonucleases which do not cleave in this region include ApaI, BglII, EcoRI, KpnI, NotI, SacII, SaII, SmaI, SpeI and XhoI. Primer sets useful for PCR manipulations of this gene are also shown.
FIG. 9 provides a restriction map of the AtThi3 gene. Restriction endonucleases which do not cleave in this region include ApaI, BglII, EcoRI, HindIII, KpnI, NotI, PstI, SacI, SacII, SaII, SmaI, SpeI and XhoI. Primer sets useful for PCR manipulations of this gene are also shown.
FIG. 10 provides a restriction map of the AtPDC gene. Restriction endonucleases which do not cleave in this region include BamHI, HindIII, NcoI, NotI, PstI, SacI, SalI, SmaI, SpeI, and XhoI. Primer sets useful for PCR manipulation of this region are also shown.



DETAILED DESCRIPTION OF THE INVENTION
As used herein, a male sterile is a plant which does not produce pollen. Seed sterility is where viable seeds are not produced to embryo lethality. Female sterility refers to the inability of the female germline of a plant (ovule and endosperm) to develop, receive pollen or develop once fertilized, and there is no introgression, selfing or outcrossing. Where there is female sterility, pollen from a native plant cannot fertilize the engineered female sterile plant and no fertile offspring are produced.
Systems of plant sterility are important tools in the hybrid seed industry, forestry, and phytoremediation. The hybrid seed industry, for example, plants millions of acres in which one of the two elite parent plants in a genetic cross is male sterile as a result of physical or genetic emasculation. In phytoremediation, genetically engineered plants are being developed that extract, detoxify, and/or sequester toxic pollutants, and their germplasm needs to be tightly controlled. In this case, systems of male and female sterility are needed if plants are to be released permanently into the environment. Control of fertility also limits unauthorized propagation of proprietary material. An especially useful sterility system is one in which sterility is conditional, and in which elite parental lines can still be propagated through fully fertile crosses. The present invention provides a conditional sterility system based on suppression of the pathway for thiamine B1 synthesis, sequestration of thiamine or destruction of thiamine B1 during pollen and/or ovule development such that the plants exhibit thiamine-deficiency based conditional sterility (TDCS). Fertility of the TDCS plants is restored by treatment with excess thiamine, a harmless vitamin. In addition, plant sterility can improve the economics of wood and pulpwood production because phosphorus and nitrogen are not “wasted” in the production of pollen and seed. This is particularly applicable to pine and eucalyptus. Controlled sterility is also applicable to genetically modified turfgrass or bentgrass; to the production of seedless fruit such as watermelon or grapes. These methods can also be applied to the animal forage crops; many forage crops such as alfalfa, fescue and Bermuda grass decline in feed quality when they go to seed. Similarly, the sugar yield from sugar cane is improved if the cane does not go to seed as a result of genetic modification to contain and express a conditional sterility construct of the present invention. A particularly important advantage of the present invention is that it is not labor-intensive.
TDCS can be achieved by altering the expression of three different genes in the model plant Arabidopsis . Two genes, AtThi2 and AtThi3, encoding a bifunctional enzyme (phosphomethylpyrimidine kinase, thiamine phosphate pyrophosphorylase also called thiamine synthase) and a monofunctional enzyme (hydroxyethylthiazole kinase) in the thiamine B1 synthesis pathway, respectively, are targeted for suppression in Arabidopsis reproductive tissue. RNA interference (RNAi) is used to degrade target AtThi2 and AtThi3 RNAs using three distinct actin promoter vector systems: ACT12pt directs pollen specific suppression; ACT11pt directs pollen/ovule specific suppression; and ACT2pt serves as a control by suppressing these genes in all vegetative tissues. In addition, TDCS can be achieved by sequestering thiamine in reproductive tissues by the overexpressing a mutant form of Arabidopsis pyruvate decarboxylase (PDC). Alternatively, or in addition, a thiaminase coding sequence can be expressed under the regulatory control of tissue specific promoters as described therein. The resulting plants with one or more of these transgenes are sterile under normal soil growth conditions, but fully fertile when supplemented with excess thiamine B1.
Thiamine (Vitamin B1) is an essential vitamin in mammals. Plants make their own thiamine, because it is an essential cofactor in metabolism. For example, pyruvate decarboxylase, xylulose transketolase, and acetolactate synthase (Chang and Duggleby, 1997), and other enzymes that convert carboxyl groups to aldehydes or ketones, require thiamine B1 (Bouvier et al., 1998). Thiamine biosynthesis can be ablated or thiamine can be sequestered in reproductive organs and tissues to create conditional auxotrophic sterile mutants (“knockdown lines”) that require thiamine for fertility.
Arabidopsis thiamine (B1) auxotrophic mutants grow well with exogenously added B1 in their growth medium (Li and Redei, 1969; Redei and Li, 1969; Ledoux et al., 1974). Plants appear to use a thiamine (B1) biosynthesis pathway similar to that described in bacteria and yeast, the final steps of which are shown in FIG. 1 (Brown and Williamson, 1987). Pyrimidine pyrophosphate and thiazole monophosphate are combined by the action of thiamine phosphate synthase to make thiamine phosphate. The pyrimidine and thiazole derived components are both made by poorly characterized biochemical pathways (Brown and Williamson, 1987). In the last decade several genes encoding enzymes or regulatory proteins in the thiamine pathway have been characterized in Escherichia coli, Saccharomyces cerevisiae and Schizosaccharomyces pombe.
We have identified genes involved in thiamine B1 synthesis in the Arabidopsis database. Using yeast, S. pombe , and E. coli query sequences, we found several genes encoding homologues to B1 synthesis enzymes. No attempt was made to identify DNA regulatory proteins involved in thiamine synthesis. Examples of the relevant Arabidopsis sequences identified with potential roles in thiamine synthesis or binding are listed in Table 1. This analysis reveals several gene sequence targets in the Arabidopsis genome that are believed essential for thiamine B1 biosynthesis, modification, and degradation. Many of them are single-copy or low-copy genes, which simplifies any strategy for blocking thiamine synthesis or sequestering available thiamine in plant cells.
Only one Arabidopsis gene (AtThi1) implicated in thiamine B1 synthesis (AtThi1) has been partially characterized for function (Machado et al., 1996; Machado et al., 1997; Chabregas et al., 2001). This gene complements E. coli mutations that affect DNA repair, such as uvrA. AtThi1 is also a sequence homologue of the B1 biosynthetic genes of yeast Thi4 and S. pombe Thi2. AtThi1 complements yeast mutants in the essential Thi4 gene ( FIGS. 1 and 2 ), and it appears to complement both yeast cell viability and DNA repair activity as measured for mitochondrial DNA. Using either S. pombe Thi2 or yeast Thi4 protein as the query sequence, we detected a single Arabidopsis Thi1 sequence (NP200288). It has very strong homology over most of its length and 65% identity to the S. pombe Thi2 (Nmt2) protein (Table I, AtThi1). Thus, AtThi1 appears to be a single copy gene. AtThi1 is synthesized in the cytoplasm and then transported into to both the chloroplast and mitochondria by means of a dual N-terminal peptide targeting sequence (Chabregas et al., 2001). Because of this and other information on protein localization of other enzymes in thiamine synthesis, it appears that plant nuclear genes encode thiamine B1 synthesis enzymes. The transcripts are translated on cytoplasmic ribosomes, but thiamine B1 synthesis itself takes place primarily in organellar compartments. AtThi1 is only a secondary target for functional inactivation, because its complex biochemical activities are still poorly defined.
AtThi2 and AtThi3: Yeast Thi6 is a 540 amino acid bifunctional enzyme acting as both a phosphomethylpyrimidine kinase and a hydroxyethylthiazole kinase ( FIG. 1 ). Its N-terminal half is homologous to E. coli ThiE, phosphomethylpyrimidine kinase (Table 1). The C-terminal half of yeast Thi6 is homologous to E. coli Thi4, a hydroxyethylthiazole kinase. Using the yeast Thi6 sequence as a query, we detected two proteins in Arabidopsis , NP — 172707 and NP — 189045, and found homology to the N-terminal and C-terminal halves of the Thi6 query (see Table 1), respectively. We have named these sequences AtThi2 and AtThi3, respectively. AtThi2 and AtThi3 are very different in length (525 and 276 amino acids) and are not homologous to each other. AtThi2 is about the same length as yeast Thi6, but only has homology in its N-terminal half. The question thus becomes, what does the C-terminal half of AtThi2 encode? Using the C-terminal 250 amino acids of AtThi2 as a query against all sequences, we found a thiamine phosphate pyrophosphorylase sequence (thiE, NP — 579063) from Pyrococcus furiosus as the most homologous of many non-plant sequences that are significantly related to this Arabidopsis query (E-value=e-35). In addition, using the yeast thiamine phosphate pyrophosphorylase Thi22 (Goffeau et al., 1996), we found a single Arabidopsis homologue, and it was again the C-terminal, 250 amino acid end of AtThi2 (NP — 173707, Table 1, and see below). Without wishing to be bound by any particular theory, we have concluded that AtThi2 is a different bifunctional enzyme than yeast Thi6. AtThi2 combines an N-terminal phosphomethylpyrimidine kinase with a C-terminal thiamine phosphate pyrophosphorylase (thiamine synthase) ( FIG. 1 ). Similarly, and again without wishing to be bound by theory, we have concluded that AtThi3 is a mono-functional hydroxyethylthiazole kinase, corresponding to the C-terminal portion of the bifunctional yeast Thi6 ( FIG. 1 ).





TABLE 1



Arabidopsis sequence targets to block thiamine B1 biosynthesis








Thi sequence
Ath homolog b





query a /
Accession #
(# seq.)

Length hom.

Organism
Length a.a.
E value
% ID
a.a./query
Comments/Reference



Thi2 (nmt2)
NP_200288
(1)3e−93
65%
266/328
(Manetti et al., 1994) Thi1 Ath

NP_596642
349 a.a.



(Machado et al., 1996;


S. pombe

AtThi1



Machado et al., 1997;






Chabregas et al., 2001)

Thi4 S25321
ARA6, Thi1,
3e−77
50%-
310-100/326
thiamin biosynthesis protein

NP_011660
NP_200288



thi4, thiozole biosyn.

yeast
349 a.a.

Thi2p
No sig.
>0.2

450
Ts activator of Thi B1 genes

NP_009799
homologue

yeast

Thi6
NP_173707
(1)7e−28
37%
225/540
Phosphomethypyrimidine

NP_015110
525 a.a.



kinase. Homology to a.a.

N-terminal
AtThi2



9-233 of query

domain
C-terminus

C-terminal
NP_189045
(1)2e−20
30%
240/540
hydroxyethylthiazole kinase,

domain
276 a.a.



putative, Homology to a.a.

yeast
AtThi3



255-523 of query

ThiE
NP_173707
2e−11
33%
185/211
Phosphomethypyrimidine kinase

NP_312943
525 a.a.


E. coli

AtThi2


C-term

Thi4
NP_189045
9e−43
42%
240/262
hydroxyethylthiazole kinase

NP_416607
276 a.a.


E. coli

AtThi3

Thi22,
NP_173707
(1) e−35
33%
274/572 C-term
See AtThi2 above, Also

NP_015446
525 a.a.



Brassica BTH1 thiamine

yeast.
AtThi2



phosphate pyrophosphorylase

( S. pombe Pho4)
N-terminus

THI80 P35202
NP_563669
(4) 2e−17:
26%
270/319 a.a.
Thiamine pyrophosphokinase

yeast
264 a.a.
4e−8


(TPK) Thiamine kinase, unknown


AtThi5

Thi3 BAA04886
B1 binding
(12) 3e−65:
29-22%
(8)
Yeast: Thiamine positive

& Thi3p
motif
5e−9

550/568 & 609
regulatory factor, Thiamine

NP_010203 yeast




binding motif. Arabidopsis






pyruvate decarboxylase






(Nishimura et al., 1992)

Pyruvate
NP_195752
(12) 4e−78:
33%-31%
560/563
Pyruvate decarboxylase,

decarboxylase

7e−7


oxal-CoA decarboxylase

PO6169 yeast



a Protein sequence from E. coli , S. cerevisiae , or S. pombe used as a query of the Arabidopsis genomic sequences.

b Predicted Arabidopsis protein sequence with homology detected in gDNA database ( Arabidopsis Genome Initiative, 2000). For the purpose of clarity in identification of the Arabidopsis sequences, we will use Ath as a precursor to all Arabidopsis gene names.

c Number of predicted and distinct protein sequences with clear homology (N) followed by the range in E-values.






























































AtThi5: Thiamine pyrophosphate kinase (TPK, thiamine kinase) makes the pyrophosphate modified form of thiamine B1, shown at the bottom right of FIG. 1 . Using the yeast gene THI80 (TPK) as a query, four Arabidopsis sequences with significant sequence homology were detected (Table 1). All four sequences may encode nearly identical proteins with truncations at the N-terminus. These proteins are believed to represent the products of a single gene, that we call AtThi5, with multiple allelic cDNAs. We have not yet confirmed whether all four sequences are in the same chromosomal location (same gene) or if they have significant silent nucleotide substitution differences and represent different genes. Yeast thi80 mutants have less thiamine, but are viable (Nishimura et al., 1991; Nosaka et al., 1993). However, because Thi80 is not an essential gene in yeast, the Arabidopsis homologue(s) has not been chosen as a target for functional inactivation.
AtPDC2: There are alternative or supplementary methods of creating TDCS in addition to blocking the synthesis of thiamine biosynthetic enzymes. Thiamine B1 can be sequestered in reproductive tissue, similar to the strategy using avidin to sequester biotin and thus create biotin-deficiency based male sterility (Albertsen and Howard, 1999). Although there is no precedent for generating sufficient thiamine sequestration capacity with a binding protein to create a deficiency, this concept is straightforward, as described herein. There is a thiamine binding protein activity found in plant seeds (Watanabe et al., 1998; Rapala-Kozik et al., 1999), but the genes and proteins for this activity are not identified. The well-characterized enzyme pyruvate decarboxylase (PDC) contains a strong thiamine B1 binding site. Three-dimensional models are available for PDCs from bacteria, fungi, and plants (Konig et al., 1998; Lu et al., 2000). PDC binds its thiamine B1 cofactor at the interface between two homodimeric subunits. Thiamine binding and subunit assembly appear to require the substrate pyruvate or an analogue. However, we believe that expression of large amounts of active PDC enzyme damages the efficiency of central metabolism. Thus, expression of an altered form of PDC that binds thiamine, but is enzymatically inactive, in plant reproductive tissue results in a sterile phenotype. The thiamine binding site is immediately adjacent to the pyruvate binding site. Mutant analysis of the bacterial enzyme from Zymomonas mobilis has yielded relevant and exciting results. Chang et al., 1999 have characterized several mutant active site mutant enzymes with a lower K m for substrate, most of which exhibit a lower affinity for thiamine. One PDC2 mutant with a single E473Q amino acid change lowers the specific activity to 0.025% of wild-type PDC levels (i.e., a 4000 fold reduction in activity), but appears to have an even tighter binding to thiamine than wild-type enzyme. Wild-type PDC has a k c for thiamine of 1.97 μM, while the release rate of thiamine from mutant enzyme PDC E473Q was too low to be measured. The affinity of PDC E473Q for thiamine could rival that of avidin for biotin. There is a strong sequence identity between the bacterial PDC and AtPDC2 in the region of bacterial residue E 473 . Thus, we can engineer thiamine sequestration based on the tissue specific expression of a catalytically inactive, thiamine binding mutant AtPDC2 (E517Q) to achieve TDCS. Thiamine sequestration based-sterility can stand alone or be used to supplement to genetic means for inactivating thiamine synthesis, for example, using interference RNA or antisense.
When a thiaminase coding sequence is operably linked to a pollen- and/or ovule-specific transcriptional regulatory sequence, the expressed thiaminase degrades thiamine in the relevant developing reproductive tissue. Thiaminase coding sequences are known to the art; see, e.g., Accession No. U17168 ( Paenibacillus/Bacillus thiaminolyticum thiaminase) on the National Center for Biotechnology Information website. The skilled artisan can modify the codons for improved plant gene expression, if necessary. Murray et al. (1989) provides a discussion of codon choice in plants (Murray et al. (1989) Nucl. Acids Res. 17:477-494).
Thiaminases are also produced by other organisms including, but not limited to, Clostridium sporogenes, Naegleria gruberi , carp, lobsters, shrimp, certain clams and the fem bracken Pteridium aquilinum (See U.S. Patent Publication 2004/0013658 for a discussion).
Interference RNA (RNAi) can be used to suppress a gene activity by targeting an mRNA for efficient degradation (Chuang and Meyerowitz, 2000). A single RNA transcript is constructed so that the double stranded mRNA stem of its stem-loop structured RNA product is homologous to part of the target mRNA to be suppressed. This sets up a cycle of efficient target mRNA degradation. Our own laboratory has pioneered a technique to make RNAi constructs very rapidly (one day from PCR to cloning) using overlap extension PCR as described herein below. Using this technique, we have suppressed the levels of actin, profilin, and actin-related protein mRNAs and protein products. We have targeted 100 to 200 bp of the 3′ untranslated regions (3′UTR) and/or 500 bp from the coding regions from these genes. 200 nt 3′UTR sequences from AtThi2 and AtThi3 were PCR amplified by this method to make an RNA product that folds into a stem-loop structure with a 200 bp dsRNA stem. An example of a construct expressing an RNAi to suppress AtThi2 expression is shown in FIG. 2 . An inverted repeat-polymerase chain reaction (IR-PCR) technique is used to create the RNAi constructs in a short time. This technique circumvents the complex multistep cloning protocols generally needed to assemble RNAi constructs.
The ACT11pt vector is used to express an antisense (A) orientation and a sense (S) orientation from AtThi2 mRNA separated by a GUS spacer in a single transcript. The RNA product of this gene forms a stem-loop transcript that leads to the degradation of native AtThi2 mRNA. (ts, transcriptional start; pA, polyadenylation sites). The Act 11 promoter determines preferential expression of an associated sequence in pollen, ovules and in developing embryos, and it is also expressed in the leaves and stem of the inflorescence.
Pollen and ovule tissue-specific expression with the actin promoters: The tissue specific expression patterns of the specifically exemplified three promoter vectors is shown in Table 2 (for vector maps see FIGS. 3A-3C ). The RNAi constructs are cloned into the ACT 11pt and ACT12pt vector derived from the Arabidopsis ACT11 and ACT12 actin gene promoters and pBI121, respectively (see FIGS. 3A , 3 B). The homologous ACT11 and ACT12 terminators, respectively, have been added to update these promoter cassette vectors from their original versions (Huang et al., 1996; Huang et al., 1997). ACT11 is one of five reproductive actin genes. ACT11 is expressed very strongly in ovule, embryo, seed, silique, and pollen. We have already used ACT11pt-related constructs to inactivate ACT11 gene expression with an ACT11-RNAi construct. These ACT11/RNAi plants have a partially sterile phenotype. The use of the ACT11 promoter/terminator vector constructs was more successful at lowering ACT11 protein levels and producing phenotypes than were CaMV 35S promoted RNAi constructs. The ACT11-Thi2-RNAi or Thi3-RNAi constructs inactivate thiamine B1 biosynthesis in ovule, embryo, seed, silique, and pollen, producing a conditionally sterile phenotype.
TABLE 2 Vectors for reproductive and vegetative tissue-specific expression. Major tissue-specific Vector expression Origin ACT11pt Most reproductive tissues- Arabidopsis ACT11 embryo, ovule, seed, actin gene silique, mature pollen ACT12pt Mature pollen Arabidopsis ACT12 actin gene ACT2pt All vegetative tissues- Arabidopsis ACT2 leaves, roots, sepals, actin gene petals
ACT12 is the most tightly regulated of the Arabidopsis actin genes. It is expressed almost exclusively late in pollen development (Huang et al., 1996). Thi2- and Thi3-RNAi constructs expressed from the ACT12pt vector prevent the growth of mature pollen and block fertilization. Another suitable pollen-specific promoter is the Lat52p (Preuss et al., 1994). The constitutive ACT2 actin promoter cassette ACT2pt is used as a control to express the RNAi constructs in all vegetative tissues to make plants that do not grow at all without added thiamine.

The Thi-RNAi constructs are transformed or cotransformed into Arabidopsis via vacuum infiltration of each regulated RNAi construct subcloned into a Agrobacterium T-DNA plasmid (Bariola et al., 1999). Thi2-RNAi is subcloned into pCambia1300 with a hygromycin drug marker for plant selection (provided by Ray Wu, Cornell University, Ithaca, N.Y.). pCAMBIA 1300 and numerous other vectors for cloning and stable introduction of transgenes into plants are available from CAMBIA (Black Mountain, ACT, Australia). Where pBIN10 is used, selection is for kanamycin resistance. The Thi3-RNAi construct is subcloned into the pBIN19 vector with a kanamycin drug marker for plant selection (Bevan, 1984). With such transformations, progeny show between 0.1 and 2% of the seed to be transformed based on Hyg or Kan drug selection, and no non-transformed seeds escaped selection and grow. Plants doubly transformed with mixtures of Agrobacterium strains containing independent KanR and HygR plasmids are co-transformed at a rate of about 60%. When two different Agrobacterium populations carrying different T-DNAs are mixed and vacuum infiltrated together, their T-DNA transgenes are efficiently co-transformed into the same plants. Co-transformation saves three months over transforming the two genes in two successive separate rounds of transformation. The T1 generation of vacuum infiltrated transformed seed from the single and double Thi gene transformations are plated on media containing MS salts, the appropriate drugs for selection, and thiamine. Plants with one or both drug markers, expressing Thi2-RNAi, Thi3-RNAi or both Thi2-RNAi and Thi3-RNAi constructs, are characterized further for TDCS phenotypes.
The molecular model for Thi-RNAi suppression in these experiments is that the AtThi2 and AtThi3 mRNAs are degraded in reproductive tissues. RNA degradation results from the dsRNA structure of the transcript initiating a cycle of target mRNA degradation into small 23-24 nt RNA fragments, as described for several example cases (Hamilton and Baulcombe, 1999). AtThi2 and AtThi3 activities are functionally inactivated by this RNAi approach in a tissue specific fashion. One reason we are producing doubly suppressed lines for AtThi2 and AtThi3 is that the efficiency of blocking the thiamine biosynthesis is then be the multiple of the two phenotypes. In other words, the suppressed phenotype is stronger if two genes are inactivated instead of just one. In addition, AtThi2 encodes a bifunctional enzyme, further strengthening the suppression of thiamine synthesis. If each of the three enzymes are suppressed to 10% of normal levels then the thiamine pathway is blocked to 0.1% of normal levels (i.e., f=(0.1) 3 −0.001).
With respect to the tissue specificity of RNA interference, there is very little information as to RNAi activity being restricted to a single organ or tissue. We are not aware of examples of RNAi purposefully directed at a tissue or organ. Virus-induced RNA silencing can be naturally restricted to the veins or leaves of plants (Voinnet et al., 1999). In contrast, there is more evidence for the systemic nature of RNA-directed cosuppression from a number of sources (Citovsky and Zambryski, 2000; Fagard and Vaucheret, 2000). Grafted transgenic plants often transmitted co-suppression phenotypes to other parts of the plant. However, most of the systemic behavior reported is due to RNA virus movement and expression throughout the plant (Voinnet et al., 2000). However, these experiments are biased in nature because they were directed at exploring co-suppression and some of its systemic properties. The experiments described herein are believed to be the first using tissue-specific promoters to express interference RNAs in order to inactivate target RNAs in a tissue-specific manner. These experiments are counterintuitive because of prejudice in the art that PTGS is always systemic.
We PCR amplify cDNA sequence (AtPDC2) for one of the Arabidopsis AtPDC2 sequences but modify it to contain appropriate cloning sites, a mutation one codon (see FIGS. 6A-6B ), with and without an epitope tag. There are five Arabidopsis sequences with reasonable 4044% identity overall with the well characterized bacterial Zymomonas sequence. We focus on the highly expressed AtPDC2 sequence (see FIGS. 6A-6B ). Twenty four of the 27 resides surrounding the AtPDC2 target residue E517 are identical between the plant and bacterial sequences. We PCR amplify the Arabidopsis AtPDC2 cDNA from an Arabidopsis library using a two fragment overlap extension strategy mutating the codon for E517 to encode Q517. This cloning strategy creates the mutant cloned sequence PDC E517Q . First, the Arabidopsis AtPCD2 gene is modified to mutate GAG codon 517 encoding Glu to the new codon sequence CM encoding Gln. Second, the PDC E517Q protein product is C-terminally tagged with an HA epitope. The HA tagging allows one step purification of the protein to facilitate preparing AtPDC2-specific antibody. The resulting sequence is called PDC E517Q . See also SEQ ID NO:7 and SEQ ID NO:8. This cDNA is cloned into the ACT11pt and ACT12pt expression vectors described above and transformed into Arabidopsis selecting for a linked hygromycin resistance markers. Maps of the first vectors to be used are shown in FIGS. 3A-3C . We screen plants from these two promoter systems for a dominant male-female sterility and male sterility phenotypes, respectively. Again as a simple control, the PDC E517Q encoding sequence is expressed from an actin ACT2pt promoter vector to make a plant whose vegetative growth is dependent upon added thiamine. The thiamine requiring phenotype depends less on the tissue/organ specificity of gene expression, so vegetative expression of the thiamine-sequestering PDC is an option for conditional plant sterility.
AtThi2, AtThi3, and AtPDC2 are soluble enzymes that are sequence homologues of bacterial sequences. Their mRNAs are translated in the cytoplasm and are specifically targeted to the prokaryotic environments (e.g., chloroplast and mitochondria). Therefore, they are efficiently expressed as native proteins in E. coli . A PCR amplified cDNA sequence is cloned which encodes Arabidopsis AtThi2 and AtThi3 without their organellar target peptides of 20 and 21 amino acids, that are removed during organellar transport in plants. A ATPDC2 cDNA is amplified from Arabidopsis total plant cDNA. The three sequences are given in FIGS. 4A-4C , 5 A- 5 B and 6 A- 6 B. Commercially available pBluescript and pET expression vectors are used. Appropriate bacterial stop codons (for LacZ), Shine-Delgarno sequences and cloning sites are added during PCR as we have explained in several previous publications in which we have described the expression of plant sequences in E. coli (Kandasamy et al., 1999; McKinney et al., 2001; McKinney et al., 2002). Synthetic multiple antigenic peptides (MAPs) with homology to the mature N-terminal and C-terminal 30 amino acid residues of AtThi2, AtThi3, and ATPDC2 are prepared. The MAP peptides are used as immunogens in mice to make polyclonal and monoclonal antisera to these proteins following the protocol published recently for three soluble enzymes (Li et al., 2001). Also by this established protocol the crude protein extracts from E. coli with and without the expressed cDNAs are used to characterize polyclonal sera and screen out monoclonal antibodies. Thus, AtThi2, AtThi3, and ATPDC2 proteins do not need to be purified for these assays. These antibodies are used in assays of AtThi2, AtThi3, and AtPDC protein levels in RNAi suppressed plants.
The thiamine B1 deficient phenotypes in RNAi-Thi2, RNAi-Thi3, and PDC E473Q plant lines are characterized as follows. The tissue specificity of the ACT11 promoter directs AtThi-RNAi and PDC E473Q gene expression to etiolated hypocotyls and reproductive tissues, which is lethal to seedling growth and mature plant reproduction, respectively. As described above the AtThi2-RNAi construct is linked to a KanR marker and the AtThi3-RNAi construct to a HygR marker. Thus, three classes of plants, KanR, HygR, and HygR+KanR, are characterized as potentially suppressed for AtThi2, AtThi3 and AtThi2+AtThi3, respectively. In order to allow RNAi suppressed plants and PDC E473Q plants with the strongest phenotypes to grow and reproduce, the vacuum infiltrated seed with T1 generation transformed plants are germinated on medium supplemented with thiamine (Li and Redei, 1969). Twenty RNAi plant lines for each of the three drug resistance phenotypes are grown through seed maturation on soil, while being watered with thiamine (Redei, 1969). Ten plants with the drug marker linked to PDC E517Q are examined. As a positive control, we also germinate KanR seed carrying the act7-2 mutation. The act7-2 mutant has no detectable phenotype, because its T-DNA insertion lies downstream from the ACT7 gene and before the next gene in Arabidopsis . The first inflorescence branch from each Thi suppressed and act7-2 plant is isolated in an Aracon tube and is not treated with thiamine. The remaining inflorescences are sprayed with thiamine. The unsprayed inflorescence branches are scored initially for numbers of siliques and mature seeds as compared to the number on sprayed adjacent inflorescence branches.
Thirty single transformed lines for each of the three genes (e.g., Thi2-RNAi, Thi3-RNAi, PDC E517Q ) and thirty doubly transformed lines blocked for thiamine biosynthesis (i.e., Thi2-RNAi and Thi3-RNAi) are characterized further at the molecular level. Plant extracts from young siliques taken from the T2 generation are assayed for AtThi2, AtThi3, and PDC E517Q protein levels are determined on Western blots using the above described antibodies or the commercial HA antibody. Like the strong expression of the ACT11 promoter in siliques, these tissues also show a significant reduction in Thi protein expression or increase in PDC E517Q protein expression. The actual stage in plant growth and tissue that first shows a phenotype is noted. Without wishing to be bound by theory, it is believed that the transgenic plant forms sepals, petals, carpels, and anthers, but fails to form embryos or mature pollen. The plant may begin to form embryos, but those embryos die during development.
RNAi was expressed to knock down HTK or TPP/PPK in vegetative organs and tissues produced almost no phenotype; these plants were essentially the same as control plants. By contrast, RNA interference expressed to decrease HTK or TPP/PPK in reproductive organs and tissues produced strong sterility phenotypes. An A2pt:Thi3Ri-1 HTK resulted in a phenotype in which the plants were fertile and 80-100% of normal size. These plants exhibited a slight reduction in initial growth rates but only moderate long-term dwarfing. The adult plants appeared almost normal. The A2 (Actin 2) promoter directs expression in vegetative tissues. Examination of plant tissues genetically modified with an A2pt:GUS construct indicated that expression occurred in seedlings, leaves, roots, petal and sepals.
An A11pt:Thi3-RiRi-1 HTK construct resulted in plants that were partially or fully sterile. The A11 (actin 11) promoter directs expression in female and male organs and tissues of the plant. This was confirmed using an A11:GUS fusion construct. Expression of GUS was observed in ovule, embryo, endosperm, and mature pollen. Female-male specificity was observed. All the A11pt:Thi3Ri-1 plants are partially or fully sterile. About 20% of the T 1 lines make few or no siliques. The RNAi targeted only about 70 nucleotides of the much larger Thi3 transcript. From those partially sterile liens that produce a few siliques, most of the seeds that are produced are sterile (aborted or dead). An A11pt:Thi3Ri-1 TPP/PPK construct resulted in plants that were partially or completely sterile despite the elaboration of large numbers of flowers. Whereas wild-type seeds rarely include nonviable seeds, 20 to 100% of the seeds produced from this construct are inviable (seeds are dark brown and shriveled).
An A12pt:Thi2Ri-1 TPP/PPK construct resulted in a fully male sterile phenotype. The A12 (actin 12) promoter directs expression in late in pollen development. Expression was examined using an A12pt:GUS fusion construct; activity was observed in the inflorescence of the genetically modified Arabidopsis.
Three lines already characterized as fully sterile in a parent plant and known to be suppressed for the Thi target genes are selected for a more quantitative examine examination of sterility in a population. One hundred T3 generation RNAi or PDC E517Q expressing seedlings germinated with thiamine are grown to maturity on soil lacking added thiamine. When the average height of the first two inflorescences stems in the population reaches about 12 in., each plant is scored for numbers of developing siliques and seeds. This process takes about four to five weeks. Then half the plants are sprayed with thiamine and the sprayed, and unsprayed plants are scored again two weeks later for siliques and seeds. Wild-type plants are scored at the same two times as positive controls.
Based on homology to E. coli , yeast, and S. pombe sequences, we have identified two Arabidopsis targets, AtThi2 and AtThi3, to suppress thiamine biosynthesis and one protein product PDC E473Q to sequester thiamine. Together the two Thi genes determine three essential enzymatic steps in thiamine synthesis. AtThi2 and AtThi3 are both undoubtedly essential to thiamine biosynthesis. The genes are inactivated individually and together by an RNAi strategy using a reproductive tissue-specific actin promoter system. Each is shown to be an essential gene for the development of siliques and seeds. Arabidopsis AtPDC2 genes were identified by homology to bacterial and yeast pyruvate decarboxylase sequences and form a small gene family in Arabidopsis . In bacteria and yeast, the mutant form of the enzyme PDC E473Q has lost 99% of its enzyme activity but has greatly enhanced binding capacity for thiamine. This strong binding should sequester any thiamine present in these cells, including any that is transported in from adjacent tissues. Thiamine-deficient plants are shown to have a male-female sterile or male-sterile TDCS phenotypes depending upon the promoter used. The TDCS phenotypes are rescued by direct application of thiamine to the plants or their soil. In the future, this system is applied to TDCS trees, shrubs, and grasses to enhance there use in phytoremediation of toxic elements and organics such as our previously described mercury and arsenic resistant plants (Meagher, 2000; Meagher et al., 2000; Bizily et al., 2002; Dhankher et al., 2002). This flexible system of TDCS is also easily applied to forestry for more efficient wood or fiber production and to the hybrid seed industry.
Targeted gene suppression in plants can be achieved through the induction of RNA interference (RNAi), also known as post-transcriptional gene silencing. This is accomplished through in vivo production of an RNA species containing a double stranded region composed of sequence homologous to a segment of the mRNA to be targeted. Production of this dsRNA leads to the induction of RNAi and subsequence degradation of the corresponding mRNA.
The Overlap Extension-PCR (OE-PCR) procedure can be used to generate a DNA molecule containing two copies of the target sequence in inverted orientation of one another, as shown in FIG. 7 . The transcript produced from this cloned DNA molecule forms the requisite double-stranded structure needed to trigger RNAi; thus, transformation of plants with such a construct leads to a loss of function phenotype for the targeted gene [Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990].
The OE-PCR procedure requires three DNA fragments: the linker fragment, a target sequence fragment with homology to the 5′ end of the linker, and a second target sequence fragment which is identical to the first except that it has homology to the 3′ end of the linker. Each of these fragments is produced in a separate PCR, and all three are then combined in an OE-PCR to generate the final product (see FIG. 7 ), which is treated with appropriate restriction enzymes and cloned into an expression vector.
The linker fragment consists of a 1 kb internal segment of the GUS gene, which is amplified with the following primers:






GUS Sense:


5′-CCG ACG AAA ACG GCA AGA AAA AGC
(SEQ ID NO:9)

AGT-3′



GUS Antisense:

5′-CCA GAA GTT CTT TTT CCA GTA CCT-
(SEQ ID NO:10)

3′














The target sequence is desirably 100 bp or more in length and consists of sequence unique to the gene to be suppressed. The sequence is amplified in two separate reactions, using different primer sets for each reaction, as shown in FIG. 7 . Thus, four primers are required: two sense strand primers and two antisense strand primers. Two fragments having identical internal sequence (the target sequence) are produced, but they differ at their ends such that each fragment overlaps a different end of the linker and contains unique restriction sites for use in cloning.
The two sense strand primers S1 and S2 contain at their 3′ ends approximately 25 nt of homology to the upstream end of the target sequence, and this region is identical in both primers. Immediately 5′ to this region is 20 nt of homology to one end of the GUS linker. In this region the S1 oligonucleotide is identical to the antisense strand of the upstream end of the linker, and the S2 oligonucleotide is identical to the sense strand of the downstream end of the linker.






S1:


5′TTT CTT GCC GTT TTC GTC GG + 25nt
(SEQ ID NO:11)

target “A”-3′

GUS homology



S2:

5′-ACT GGA AAA AGA ACT TCT GG + 25nt
(SEQ ID NO:12)

target “A”-3′















The antisense strand primers A1 and A2 both have at their 3′ ends an identical 25 nt region of homology to the downstream end of the target sequence. Immediately 5′ to this segment are unique restriction sites (different ones in each primer) that can be used in directional cloning of the final product. Each oligo then has at its 5′ end a unique “clamp” sequence of 21 nt. These unique sequences serve as priming sites for “clamp” primers used to amplify the full length OE-PCT product at the end of the procedure. The “clamp” primers are identical to the “clamps” in each oligo shown below. The primer Clamp-sense is the underlined sequence in A1 below, and Clamp-antisense is the underlined sequence in A2. Amplification of the final product using the clamp primers helps to reduce the background generated in OE-PCT, as explained below.






A1:
5′- TGA TAG TGA TAG TGA TAG TGA (SEQ ID NO:13) +


restriction sites + 25nt target “C′”-3′




Clamp 1 (underlined)







A2:
5′- AGC GTT AGC GTT AGC GTT AGC (SEQ ID NO:14) +


restriction sites + 25nt target “C′”-3′




Clamp 2 (underlined)
























The GUS linker fragment is amplified from pBI121 using the primers GUS-sense and GUS-antisense. The 50 μL reaction contains 200 ng of pBI121, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 4 pmol of each primer, and 2 units of Taq DNA polymerase in 1×PCR buffer. The reaction is run through 1 cycle of 94° for 3 min and 45 cycles of 94° for 45 sec, 55° for 50 sec, 72° for 1 min, followed by a final extension at 72° for 5 min. The reaction product is purified with the Qiagen PCR purification kit (Valencia, Calif.) and eluted in 50 μL of water.
We have observed that gel purification of any of the three fragments tends to foul the OE-PCR. Therefore in lieu of gel purification, small amounts of primer and a large number of cycles are used to reduced carry-over of GUS primers. Carry-over of large amounts of these primers into the OE-PCR promotes formation of an additional smaller product which results from amplification of the OE product of the GUS linker and one or the other target fragment.
The target sequence fragments are amplified from a plasmid cDNA library in two separate reactions; one using primers S1 and A 1, and another using primers S2 and A2 (see FIG. 7 ). Conditions are identical for both reactions and are as follows: 1 μg cDNA library, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 16.25 pmol of each primer, and 2 units of Taq DNA polymerase in a 50 μL total volume of 1×PCR buffer. The reactions are run through 1 cycle of 94° for 3 min and 30 cycles of 94° for 50 sec, 55° for 50 sec, 72° for 50 sec, followed by a final extension at 72° for 3 min. The products are purified using the Qiagen PCR purification kit and eluted in 50 μL of water.
The three purified PCR products are combined in a 1:1:1 ratio (approximately 20 ng of each) in the following OE-PCR reaction: 1.5 mM MgCl 2 , 0.2 mM each dNTP, and 2 units of Taq DNA polymerase in 50 μL total volume of 1×PCR buffer. Thermal cycling consists of one cycle of 94° for 2 min and 8 cycles of 94° for 50 sec, 55° for 50 sec, 72° for 1 min, followed by a final extension at 72° for 5 min. See FIG. 7 .
The final full length OE product is amplified with primers Clamp-sense and Clamp-antisense using 1 μL of the OE-PCR as template under the following conditions: 1.5 mM MgCl 2 , 0.2 mM each dNTP, 16.25 pmol of each primer, and 2 units of Taq DNA polymerase in 50 μL total volume of 1×PCR buffer. The reaction is run through 1 cycle of 94° for 2 min and 20 cycles of 94° for 1 min, 56° for 1 min, 720 for 1 min 30 sec, followed by a final extension at 72° for 5 min. The full-length product is then gel purified and cloned into an appropriate vector where it can be transcribed into the stem-loop RNA shown in FIG. 7 .
Techniques and agents for introducing and selecting for the presence of heterologous DNA in plant cells and/or tissue are well-known. Genetic markers allowing for the selection of heterologous DNA in plant cells are well-known, e.g., genes carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamycin, or bleomycin. The marker allows for selection of successfully transformed plant cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene. In most cases the heterologous DNA which is inserted into plant cells contains a gene which encodes a selectable marker such as an antibiotic resistance marker, but this is not mandatory. An exemplary drug resistance marker is the gene whose expression results in kanamycin resistance, i.e., the chimeric gene containing nopaline synthetase promoter, Tn5 neomycin phosphotransferase II and nopaline synthetase 3′ non-translated region described by Rogers et al., Methods for Plant Molecular Biology , A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988).
Techniques for genetically engineering plant cells and/or tissue with an expression cassette comprising an inducible promoter or chimeric promoter fused to a heterologous coding sequence and a transcription termination sequence are to be introduced into the plant cell or tissue by Agrobacterium -mediated transformation, electroporation, microinjection, particle bombardment or other techniques known to the art. The expression cassette advantageously further contains a marker allowing selection of the heterologous DNA in the plant cell, e.g., a gene carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin.
The choice of vector in which the DNA of interest is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication, protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules. The vector desirably includes a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In addition, preferred embodiments that include a prokaryotic replicon also include a gene whose expression confers a selective advantage, such as a drug resistance, to the bacterial host cell when introduced into those transformed cells. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline, among other selective agents. The neomycin phosphotransferase gene has the advantage that it is expressed in eukaryotic as well as prokaryotic cells.
Those vectors that include a prokaryotic replicon also typically include convenient restriction sites for insertion of a recombinant DNA molecule of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, Calif.) and pPL, pK and K223 available from Pharmacia (Piscataway, N.J.), and pBLUESCRIPT and pBS available from Stratagene (La Jolla, Calif.). A vector of the present invention may also be a Lambda phage vector including those Lambda vectors described in Molecular Cloning: A Laboratory Manual, Second Edition , Maniatis et al., eds., Cold Spring Harbor Press (1989) and the Lambda ZAP vectors available from Stratagene (La Jolla, Calif.). Other exemplary vectors include pCMU [Nilsson et al. (1989) Cell 58:707]. Other appropriate vectors may also be synthesized, according to known methods; for example, vectors pCMU/K b and pCMUII used in various applications herein are modifications of pCMUIV (Nilson et al., supra).
Typical expression vectors capable of expressing a recombinant nucleic acid sequence in plant cells and capable of directing stable integration within the host plant cell include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al. (1987) Meth. in Enzymol. 153:253-277, and several other expression vector systems known to function in plants. See for example, Verma et al., No. WO87/00551; Cocking and Davey (1987) Science 236:1259-1262.
A transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens -mediated DNA transfer, Agrobacterium rhizogenes -mediated DNA transfer, both preferably with a disarmed T-DNA vector, electroporation, direct DNA transfer, liposomes, diffusion, microinjection, virus vectors, calcium phosphate, and particle bombardment (See Davey et al. (1989) Plant Mol. Biol. 13:275; Walden and Schell (1990) Eur. J. Biochem. 192:563; Joersbo and Burnstedt (1991) Physiol Plant. 81:256; Potrykus (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:205; Gasser and Fraley (1989) Science 244:1293; Leemans (1993) Bio/Technology 11:522; Beck et al. (1993) Bio/Technology 11:1524; Koziel et al. (1993) Bio/Technology 11:194; and Vasil et al. (1993) Bio/Technology. 11:1533.). Techniques are well-known to the art for the introduction of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues.
Many of the procedures useful for practicing the present invention, whether or not described herein in detail, are well known to those skilled in the art of plant molecular biology. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning , Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning , Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation , University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology ; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization , IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods , Vols. 1-4, Plenum Press, New York, Kaufman (1987) in Genetic Engineering Principles and Methods , J. K. Setlow, ed., Plenum Press, NY, pp. 155-198; Fitchen et al. (1993) Annu. Rev. Microbiol. 47:739-764; Tolstoshev et al. (1993) in Genomic Research in Molecular Medicine and Virology , Academic Press. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals as cited herein.
All references and patent documents cited herein are incorporated in their entireties to the extent that there is no inconsistency with the present disclosure.
Where features or aspects of the invention are described in terms of Markush groups or other groupings of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
The examples provided herein are for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention.
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