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Title:
IDENTIFICATION OF GENES RELATED TO ABIOTIC STRESS TOLERANCE IN JATROPHA CURCAS
Document Type and Number:
WIPO Patent Application WO/2010/058428
Kind Code:
A2
Abstract:
The present invention provides a novel method for identifying genes in Jatropha plants, such as Jatropha curcas L., that confer tolerance to abiotic stress such as salinity, drought, and ion stresses. Such genes allow the development of Jatropha curcas cultivars that display enhanced salinity and stress tolerance, which can be accomplished by genetic modification approaches, directed breeding or selecting for desirable mutations in the identified genes for salinity, drought and ion stress. The present invention also provides methods for generating random mutants in yeast sensitive to salinity, acid or alkaline stress that facilitate the identification of additional novel genes from Jatropha curcas. The present invention also provides novel genes from Jatropha curcas that confer tolerance to salinity, drought and abiotic stress. In a related embodiment, the invention also provides polypeptides that mediate tolerance to salinity, drought and abiotic stress.

Inventors:
SRIRAM, Paramerswaran (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
NALINI, Eswaran (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
BALAJI, Sathram (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
BHAGYAM, Anatharaman (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
TANGIRALA, Sudhakar, Johnson (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
Application Number:
IN2009/000673
Publication Date:
May 27, 2010
Filing Date:
November 20, 2009
Export Citation:
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Assignee:
RELIANCE LIFE SCIENCES PVT. LTD. (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
SRIRAM, Paramerswaran (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
NALINI, Eswaran (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
BALAJI, Sathram (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
BHAGYAM, Anatharaman (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
TANGIRALA, Sudhakar, Johnson (Dhirubhai Ambani Life Sciences Centre, R-282 TTC Area of MIDC,Thane Belapur Road, Rabale,Navi Mumbai 1, Maharashtra, 400 70, IN)
International Classes:
C12N15/82; A01H5/00; C07K14/415
Attorney, Agent or Firm:
PURNIMA, Malkani (Reliance Life Sciences Pvt Ltd, Dhirubhai Ambani Life Sciences CentreR-282, TTC Area of MIDC, Thane Belapur Road,Rabale, Navi Mumbai 1 Maharashtra, 400 70, IN)
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Claims:
CLAIMS

1. A method for identifying a nucleic acid molecule that confers tolerance to an abiotic stress when expressed in Jatropha curcus, wherein the method comprises:

(a) exposing tissue of Jatropha curcas to the abiotic stress; b) constructing a Jatropha curcas cDNA library from the Jatropha tissue;

(c) cloning the cDNA library into a E. coli-yeast expression plasmid, wherein the cDNA is under transcriptional control of an inducible promoter in the plasmid;

(d) transforming E. coli with the plasmid containing the Jatropha curcas tissue cDNA library, and extracting plasmid DNA containing the cDNA from the E. coli;

(e) transforming yeast with the plasmid DNA extracted from the E. coli, and isolating individual transformed yeast clones;

(f) identifying one or more individual transformed yeast clones that do not grow under an abiotic-stress growth condition unless at least one cDNA molecule from the Jatropha curcas tissue cDNA library is expressed upon activation of the inducible promoter; and

(g) isolating and sequencing at least one of the Jatropha curcas tissue cDNA molecules present in at least one of the identified yeast clones.

2. The method of claim 1 , wherein the Jatropha curcas tissue is roots of 3-4 week old Jatropha curcus plantlets.

3. The method of claim 1, wherein the abiotic stress comprises salinity or drought or both.

4. The method of claim 1, wherein the abiotic stress is NaCl salt stress.

5. The method of claim 2, wherein the abiotic stress on the roots is at least 100 mM NaCl for 1.5-3 hours.

6. The method of claim 5, wherein the abiotic stress is 100-150 mM NaCl for 1.5-3 hours, at a relative humidity of 40-50%.

7. The method of claim 1, wherein the inducible promoter is GALl.

8. The method of claim 1, wherein the abiotic stress and the abiotic-stress growth condition are selected from the group consisting of drought, salinity, osmotic pressure, heat, cold, heavy metal, low or high pH, ultra violet light, radiation, and combinations thereof.

9. The method of claim 1, wherein the abiotic-stress growth condition is at least 500 mM of a salt.

10. The method of claim 9, wherein the abiotic-stress growth condition is at least 750 mM NaCl.

11. The method of claim 1 , wherein the abiotic-stress growth condition is at least 750 mM NaCl for 36-72 hours.

12. The method of claim 1 , wherein the abiotic-stress growth condition is low pH.

13. The method of claim 12, wherein the abiotic-stress growth condition is at least 50 mM H2SO4.

14. The method of claim 1, wherein the abiotic-stress growth condition is high pH.

15. The method of claim 14, wherein the abiotic-stress growth condition is at least 25 mM NaOH.

16. An isolated nucleic acid comprising one of the Jatropha curcas root cDNA molecules isolated in step (g) of claim 1.

17. An isolated polypeptide encoded by one of the Jatropha curcas root cDNA molecules isolated in step (g) of claim 1.

18. An isolated polypeptide having an amino acid sequence at least 90% identical to an amino acid sequence selected from any of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 153, 155, 157 and 159.

19. An isolated polypeptide having an amino acid sequence at least 95% identical to an amino acid sequence selected from any of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 153, 155, 157 and 159.

20. An isolated polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 153, 155, 157 and 159.

21. An isolated polypeptide encoded by a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from any SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73-152, 154, 156, 158 and 160-195.

22. An isolated nucleic acid comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from any of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73-152, 154, 156, 158 and 160-195.

23. An isolated nucleic acid comprising a nucleotide sequence selected from any of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73-152, 154, 156, 158 and 160- 195.

24. A vector comprising the isolated nucleic acid of claim 22 or 23.

25. A transgenic plant containing the isolated nucleic acid of claim 22 or 23.

26. A method of selective breeding, wherein the method comprises breeding Jatropha curcas plants, and using the isolated nucleic acid of claim 22 as a probe to identify which of the Jatropha curcas plants overexpress at least one gene that confers tolerance to an abiotic stress.

27. A plant obtained by the method of claim 26.

28. A method for preparing a transgenic Jatropha curcas plant, wherein the method comprises: (i) identifying one or more nucleic acid molecules according to claim 1 ; (ii) overexpressing in individual Jatropha curcas plants one or more nucleic acid molecules identified by the method of claim 1 ; (iii) exposing the individual plants to the abiotic stress of claim 1; and (iv) identifying at least one individual plant that is tolerant of the abiotic stress.

29. A method for identifying a nucleic acid molecule that confers tolerance to abiotic stress when expressed in a plant, wherein the method comprises:

a) exposing plant tissue to the abiotic stress;

(b) constructing a plant cDNA library from the plant tissue after exposing the plant tissue to the abiotic stress;

(c) cloning the cDNA library into a shuttle vector, wherein the plant cDNA is under transcriptional control of an inducible promoter in the vector;

(d) transforming bacteria with the vector containing the cDNA library, and extracting vector DNA containing the plant cDNA from the bacteria.

(e) transforming yeast with the vector DNA extracted from the bacteria, and isolating individual transformed yeast clones;

(f) replica plating individual transformed yeast clones on at least quadruplicate selection plates;

(g) exposing the replica plated clones to at least four separate conditions comprising:

1) a permissive growth condition;

(2) the permissive growth condition and activation of the inducible promoter;

(3) an abiotic-stress growth condition;

(4) the abiotic-stress growth condition and activation of the inducible promoter; (h) identifying transformed yeast clones that grow under conditions (1) and (4), but not condition (3), in step (g).

30. The method of claim 29, wherein the inducible promoter is activated by galactose and repressible by glucose.

31. The method of claim 29, wherein the method further comprises (i) isolating and sequencing at least one of the plant cDNAs present in at least one of the identified transformed yeast clones.

32. A vector comprising at least one of the plant cDNAs isolated in step (i) of claim 31.

33. An isolated nucleic acid isolated in step (i) of claim 31.

34. An isolated polypeptide encoded by one of plant cDNAs isolated in step (i) of claim 31.

35. The method of claim 29, wherein the plant is Jatropha curcas.

36. The method of claim 29, wherein the permissive growth condition is 0 mM NaCl and the abiotic-stress growth condition is at least 750 mM NaCl for 36-72 hours.

37. A method for identifying a nucleic acid molecule that confers tolerance to an abiotic stress when expressed in Jatropha curcus, as claimed above exemplified herein substantially in the examples and figures.

Description:
IDENTIFICATION OF GENES RELATED TO ABIOTIC STRESS TOLERANCE IN JATROPHA CURCAS

CROSS REFERENCE TO RELATED APPLICATIONS

This provisional patent application claims priority to the provisional patent application number 2460/MUM/2008 filed on November 21, 2008, and Application number 2650/MUM/2008 filed on December 19, 2008, which are incorporated herein by entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods for identification of genes in Jatropha, including those conferring tolerance to abiotic stress such as salinity, drought and/or ion stresses. The present invention also provides novel genes conferring abiotic stress tolerance in Jatropha curcas.

BACKGROUND ART OF THE INVENTION

The seed and kernel oil from Jatropha curcas Linn, has been proposed as an ideal source for commercial biodiesel. This is due to its high content of extractable seed oil (30-40%, of seed weight), the similarity of its lipid fractions to diesel, the ability of the plant to grow with minimal agricultural inputs and on marginal soils; and its tolerance to drought, salinity and even heavy metals. (Berchmans and Hirata, 2007; Francis et al., 2005; Gϋbitz et al., 1999; Kumar et al., 2007; Heller, 1996; Modi et al., 2007; Srivastava, 2006; Heller, 1996). (Juwarkar et al., 2007; Kumar et al., 2007). The ability to grow biofuel crops such as Jatropha in marginal soils provides multiple economic advantages, such as increasing the acreage under biofuel crop cultivation, non-competition of energy crop plantation with edible crops, lower land accusation costs (F airless, 2007; Fargione et al., 2008; Palmer, 2006; Stein, 2007; Williams, 2007), as well as providing a means for economic substance for marginal farmers. The economics of plant derived biofuel sources can be improved by enhancing oil yield and/or by decreasing cultivation costs.

Despite economic reasons favoring the cultivation of biofuel crops in saline and drought affected soils, most biofuel plantations do not thrive in naturally occurring dry soils (Ismail et al., 2007; Mahajan and Tuteja, 2005; Parida and Das, 2005; Tuteja, 2007). Environmentally inflicted stress to plants, such as salinity, drought and ions limits the productive capacity of agricultural crops (Cheeseman, 1988; Hasegawa et al., 2000; Mahajan and Tuteja, 2005; Parida and Das, 2005; Sanchez et al., 2008; Vij and Tyagi, 2007). Large areas of land have been rendered agriculturally unproductive due to salt accumulation cause by poor irrigation practices (Bohnert et al., 2001; Winicov, 1998). Agricultural engineering can remedy these problems, but is expensive. Hence there is an increasing emphasis on enhancing the intrinsic ability of the plant to survive environmental stress.

It has been known that abiotic stress, such as salinity, dessication, drought and ion stress, affect cellular physiology and elicit a homeostatic response in several plant tissues (Munns, 2005). Saline solutions impose a combination of both ionic and osmotic stress on plants and alter plant physiology that can be distinguished at several levels. Na+ specific damage is associated with root uptake and later accumulation of Na+ in older leaves leading to necrosis, diminishing the average lifetime of leaf tissue (Tester and Davenport, 2003). Other effects of Na+ stress occurs by limiting the uptake of other nutrients via interference of transporters, and interference with intracellular K+ homeostasis (Hasegawa et al., 2000; Tester and Davenport, 2003; Tuteja, 2007).

Different plants display a variation to the salt physiology response that require more careful consideration. For instance, previously reports on model plant systems demonstrate that tobacco appears to show sensitivity to the low osmotic potential of growth solutions containing NaCl, compared to the specific effect of Na+ and Cl- (Murthy and Tester, 1996), while a relation between Na+ accumulation and salt sensitivity in Arabidopsis is not clear (Tester and Davenport, 2003).

Changes in gene expression in response to saline and drought stress have been identified in other plant species, and include: a) cellular adaptation to high Na+ levels in shoots, via vacuolar sequestration, mediated by vacuolar sodium pumps; b) tolerance to osmotic shock mediated by the synthesis and accumulation of small molecular osmoprotectants such as glycinebetain or polyols; c) the ability to tolerate high intracellular concentrations of Na+ ions, by expression and modulation of ribosomal protein synthesis, and d) the control of stress induced damage. (Denby and Gehring, 2005; Fricke et al., 2006; Seki et al., 2004; Sreenivasulu et al., 2007 Tester and Davenport, 2003.)

Salinity and drought tolerance is thought to be a quantitative trait locus (QTL)-associated complex trait, making selective breeding difficult. (Bohnert et al. 2001; Vasil, 2007; Winicov, 1998). Current approaches focus on identification of genes associated with resistance to salinity, drought and other abiotic stress. QTL-associated markers for salinity tolerance have been identified in cereal crops using recombinant inbred lines (Flowers, 2004; Sahi et al., 2006). Identification of abiotic stress-resistance genes permits directed breeding and/or and direct genetic modification to develop varieties with enhanced tolerance (Sahi et al., 2006; Sreenivasulu et al., 2007; Tuteja, 2000).

Plant genes associated with tolerance to abiotic stressors have been described in, for example, U.S. Patent No. 6,376,192; U.S. Patent Application Publications 2003/0162294 and 2006/0137043; and International Patent Application Publications WO2008005619, WO2008006033, WO2008022963, WO2008057642, WO2008061240, WO2008064128, WO2008064222 and WO2008064341. US Patent Application Publication 2003/0162294, describes the screening of environmental stress genes from siliques (fruits, containing mature seeds), such as Arabidopsis, using a silique cDNA library inserted in yeast. This group identified a gene called At-DBF2, which was found to enhance tolerance to cold, heat, salt and drought, in yeast and Arabidopsis cells.

The present invention provides a method for identifying Jatropha, such as Jatropha curcas, genes associated with tolerance to abiotic stressors. For example, the invention provides methods for a molecular genetic screen using yeast (Saccharomyces cerevisae) to identify expressed genes in Jatropha curcas root tissue, which confer resistance to salinity, drought and other abiotic stresses. Using an expression cloning approach that involves the construction of cDNA libraries from Jatropha curcas roots treated with salt, the present invention has efficiently isolated specific genes from Jatropha curcas that are associated with salinity and drought tolerance. Identification of stress-tolerance genes from Jatropha curcas permits targeted breeding programs and/or transgenetic modification of plants. Such activity enables the development of Jatropha curcas plant lines capable of productive growth in poor and marginal soils, with fewer agricultural inputs, yet yielding acceptable oil yields. The novel genes from Jatropha can also be applied for the genetic improvement of other agriculturally important crops.

STATEMENT OF THE INVENTION

The present invention provides the development of a method capable of identifying plant genes involved in salinity, drought, ions and abiotic stress. The invention in particular provides methods for a molecular genetic screen that demonstrates the ability to utilize yeast (Sαcchαromyces cerevisαe) to identify expressed genes from Jαtrophα curcαs tissue,especially root tissue, which confers tolerance to salinity, drought and other abiotic stresses.

Using an expression cloning approach that involves the construction of libraries from Jαtrophα curcαs roots treated with salt, the present invention demonstrates the efficacy of the screen to isolate specific genes from Jαtrophα curcαs L, that are associated with salinity and drought tolerance. The present invention provides genes and gene fragments conferring tolerance to abiotic stressors and related nucleotide and amino acid sequences.

The present invention also provides additional full length sequences to demonstrate the efficacy of the screen to isolate genes from Jαtrophα curcαs.

OBJECTIVES OF THE INVENTION:

It is the objective of the present invention to provide methods for identification of plant genes that confer tolerance to salinity, drought and abiotic stress.

It is the objective of the present invention to identify functional genes of a plant using yeast (Sαcchαromyces cereviseα).

It is the objective of the present invention to provide a method that is able to identify both false-positive as well as false-negative yeast transformants.

It is the objective of the present invention to provide a method that is able to identify known and novel genes under the conditions described in the invention.

It is the objective of the present invention to provide genes identified by the functional screen that can be used for the construction of transgenic plants. It is the objective of the present invention to provide a method for rapid and universal screening system which is able to identify genes that can be extended to any plant genome.

It is the objective of the present invention to provide an efficient method for identification of genes irrespective of the genome information available for the target plant.

It is the objective of the present invention to provide a method for functional genetic screen to prospect genes which are capable of providing enhanced resistance to abiotic stress by screening wild type plants occurring in naturally saline or drought prone eco systems.

It is the objective of the present invention to identify genes that confer tolerance to salinity, drought and abiotic stress using a functional genetic screen using yeast (Saccharomyces cereviseά) in plants.

It is the objective of the present invention to identify genetic functional genes from Jatropha curcas using a yeast (Saccharmomyces cereviseά).

It is the objective of the present invention to identify genes that confer conferring tolerance to salinity, drought and abiotic stress using a functional genetic screen using yeast (Saccharomyces cerevisea) in Jatropha curcas.

It is the objective of the present invention to provide polypeptides encoded by the gene identified that confer tolerance to salinity, drought and abiotic stress.

It is the objective of the present invention to provide methods for producing plants with enhanced tolerance to salinity, drought and abiotic stress based on the identified genes.

It is the objective of the present invention to provide plant cells and plants transformed with the identified genes that confer tolerance to salinity, drought and other abiotic stress.

It is the objective of the present invention to provide a method of identification of genes that can be extended to other stress conditions. SUMMARY OF THE INVENTION

The present invention provides methods for identification of plant genes that confer tolerance to salinity, drought and abiotic stress, using a functional screen in yeast, such as Saccharomyces cerevisea. The present invention provides a method for this purpose that is able to identify both false-positive and false-negative results in yeast transformants, thereby aiding the identification of genes expressed under a given set of conditions. Accordingly, the present invention provides a method for rapid and universal screening system that is able to identify genes that can be extended to any plant genome, irrespective of the genome information available for the target plant.

The present invention also provides genes from Jatropha curcas that confer tolerance to abiotic stressors, such as salinity and drought, and methods for identifying such genes. In a related embodiment, the invention also provides polypeptides that mediate tolerance to abiotic stressors, such as salinity and drought.

The present invention further provides methods for producing plants and plant cells with enhanced tolerance to abiotic stress based on the identified genes, and plants obtained by said methods.

In one aspect, the present invention provides a cDNA library comprising coding sequences. In one embodiment, the present invention provides a cDNA library taken from the Jatropha root.

In one aspect, the present invention provides methods for screening Jatropha sequences that confer tolerance to abiotic stress, such as salinity and drought.

In one aspect, the present invention relates to nucleotide and amino acid sequences referenced in Tables 4-8 and presented in the accompanying sequence listing, including any of SEQ ID NOs: 1 to 195. In related embodiments, the invention is a polypeptide encoded by a nucleotide sequence that is at least 90%, 95%, 98%, or 99% identical to a nucleotide sequence selected from any SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73-152, 154, 156, 158, and 160-195. In another, the invention is an isolated polypeptide having an amino acid sequence at least 90%, 95%, 98%, or 99% identical to an amino acid sequence selected from any of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 153, 155, 157 and 159. In yet further embodiments, the invention is an an isolated nucleic acid comprising a nucleotide sequence that is at least 90%, 95%, 98%, or 99% identical to a nucleotide sequence selected from any of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73-152, 154, 156,158, 160-195.

In certain embodiments, the polynucleic acids referenced in Tables 4-8 and presented in the accompanying sequence listing, including any of SEQ ID NOs: 1 to 195 are obtained and/or expressed by recombinant techniques.

In one aspect, the present invention relates to a method for producing a plant with enhanced tolerance to abiotic stress, such as salinity and drought. In one embodiment, the present invention relates to plant cells, parts or harvestable plants, or any propagation material thereof that are transformed with genes identified as above,

In one aspect, the present invention provides methods for identifying stress tolerant variants of Jatropha. In one aspect, variants of Jatropha can be developed through either genetic transformation methodologies or via directed breeding approaches (Agarwal et al., 2006; Bhatnagar-Mathur et al., 2008; Chen et al., 2007; Jain and Jain, 2000; Qiao et al., 2007; Witcombe et al., 2008; Zhou et al., 2008). For example, the nucleic acids described herein can be used to identify plants that express higher levels of genes confering tolerance.

In one embodiment, the invention is a method for identifying at least one gene that confers tolerance to an abiotic stress in Jatropha curcus, wherein the method comprises:

(a) exposing tissue of Jatropha curcas plantlets to the abiotic stress;

(b) constructing a Jatropha curcas cDNA library from the Jatropha tissue;

(c) cloning the cDNA library into a £-co//-yeast expression plasmid, wherein the cDNA is under transcriptional control of an inducible promoter in the plasmid;

(d) transforming E. coli with the plasmid containing the Jatrophia tissue cDNA library, and extracting plasmid DNA containing the cDNA from the E. coli. (e) transforming yeast with the plasmid DNA extracted from the E. coli, and isolating individual transformed yeast clones;

(f) identifying one or more individual transformed yeast clones that do not grow under an abiotic-stress growth condition unless at least one cDNA molecule from the Jatropha curcas tissue cDNA library is expressed upon activation of the inducible promoter; and

(g) isolating and sequencing at least one of the Jatropha curcas tissue cDNA molecules present in at least one of the identified yeast clones.

Any tissue may be used, whether part of a plant or separated from it. In one embodiment the tissue is roots from 3-4 week old plantlets.

In a related embodiment of the method, the abiotic-stress comprises exposing the roots of Jatropha curcus to 100-150 mM NaCl for 1.5-3 hours, at a relative humidity of 40-50%.

In a related embodiment of the method the inducible promoter is GALl

In a related embodiment of the method the abiotic-stress growth condition comprises exposing the transformed yeast to an abiotic stressor selected from at least 500 mM NaCl, at least 75OmM NaCl; at least 5OmM H 2 SO 4 ; and at least 25mM NaOH.

In a related embodiment of the method, the abiotic stress and the abiotic-stress growth condition are selected from the group consisting of drought, salinity, osmotic pressure, heat, cold, heavy metal, pH, ultra violet light, radiation, and combinations thereof.

In another embodiment, the invention is an isolated nucleic acid comprising a nucleotide sequence that is at least 85, 90, 95, 98, or 99% identical to a nucleotide sequence listed in any of Tables 4-8 and the sequence listing. In a related embodiment, the nucleic acid is isolated from Jatropha curcas. In another related embodiment, the invention is a vector containing such nucleic acid, or a transgenic plant containing the isolated nucleic acid.

In another embodiment, the invention is an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide has an amino acid sequence listed in any of Tables 4-8 and the sequence listing. In a related embodiment, the nucleic acid is isolated from Jatropha curcas. In another related embodiment, the invention is a vector containing such nucleic acid, or a transgenic plant containing the isolated nucleic acid. In another embodiment, the invention is an isolated polypeptide encoded by a nucleotide sequence that is at least 85, 90, 95, 98, or 99% identical to a nucleotide sequence listed in any Tables 4-8 and the sequence listing.

In another embodiment, the invention is an isolated polypeptide comprising an amino acid sequence listed in any of Tables 4-8 and the sequence listing. In another embodiment, the invention comprises a method of selective breeding, wherein the method comprises breeding Jatropha curcas plants, and using the isolated nucleic acid elsewhere described herein as a probe to identify which of the Jatropha curcas plants overexpress at least one gene that confers tolerance to an abiotic stress. In a related embodiment, the invention is a plant obtained by such a method.

In another embodiment, the invention comprises a method for preparing a transgenic Jatropha curcas plant tolerant to abiotic stress, comprising overexpressing in individual Jatropha curcas plants one or more genes or proteins identified herein, exposing the individual plants to an abiotic stress, and identifying at least one individual plant that is tolerant of the abiotic stress.

In another embodiment, the invention comprises a method for identifying genes that confer tolerance to abiotic stress, the method comprising:

(a) exposing plant tissue to the abiotic stress;

(b) constructing a cDNA library from the plant tissue;

(c) cloning the cDNA library into a shuttle vector, wherein the cDNA is under transcriptional control of an inducible promoter in the vector;

(d) transforming bacteria with the vector containing the cDNA library, and extracting vector DNA containing the cDNA from the E. coli.

(e) transforming yeast with the vector DNA extracted from the E. coli, and isolating individual transformed yeast clones;

(c) replica plating individual clones under at least four separate conditions as follows:

(i) permissive growth condition;

(ii) permissive growth condition and activation of the inducible promoter; (iii) abiotic-stress growth condition;

(iv) abiotic-stress growth condition and activation of the inducible promoter;

(d) identifying individual clones that grow under (i) and (iv), but not (iii).

In a related embodiment, the inducible promoter is activated by galactose and repressible by glucose.

Using an expression cloning approach as provided, the same approach can be extrapolated for any other plants for identification of any plant genes that confer tolerance to salinity, drought and abiotic stress.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure Ia: Outline of a process to identify and isolate specific genes involved in abiotic stress responses, using the yeast Saccharomyces cerevisiae.

Figure Ib: Illustration of certain underlying principles of a functional genetic screen in yeast to identify Jatropha genes conferring abiotic stress resistance.

Figure 2: Identification of salt stress conditions for wild-type Saccharomyces cerevisae (BY4741). Sectors in plates marked with a dot are wild-type, while other sectors represent salt hypersensitive yeast mutants obtained through random UV-mutagenesis followed by screening. Wild-type yeast BY4741, shows salt sensitivity from 500 mM NaCl, with complete growth arrest at 2.0M NaCl. In contrast, salt hypersensitive mutants shs-6, shs-B (isolated in the BY4741 background) show growth retardation from 100-250 mM NaCl. Similar amount of inocula were used in all the plates. cDNA libraries, from Figure 3: Agarose gel electrophoresis showing the amplification of Jatropha curcas L. root untreated control as well as roots treated with 150 mM salt for 1.5 to 2.0 hrs. Yield and distribution of cDNA sizes after 22 or 25 cycles of PCR amplification (as described in Table 2) for double-stranded cDNA prepared from various root RNA pools (marked in the legend) are shown. Amplicon size distributions ranged from 0.5 to >5. O kb.

M: lkb DNA ladder (NEB, USA, Lowest band is 500 bp and highest band corresponds to 10kb).

Lanel : ds cDNA amplified from total RNA prepared from untreated (control) roots.

Lane2: ds cDNA amplified from total RNA prepared from roots treated with 15OmM

NaCI.

Lane3: ds cDNA amplified from poly (A+) RNA prepared from untreated (control) roots.

Lane4: ds cDNA amplified from poly (A+) RNA prepared from roots treated with

15OmM NaCl.

Figure 4: Schematic outline of process and principle of the quadruplicate plate based replica-printing, to screen of yeast transformants to various abiotic stress. In this assay each individual yeast transformant is placed on four selective plates. Comparison of growth of yeast between these plates allows scoring and isolation of transformants expressing genes conferring stress tolerance.

Figure 5: Replica plate screen used to identify and isolated yeast transformants showing the relative growth advantage during salinity and drought stress. Photographs of a representative replica plate screen (as described in Figure 4) demonstrating the ability to identify and isolated yeast transformants showing the relative growth advantage during salinity and drought stress. Yeast transformants (selected for the plasmid-borne URA3 marker), expressing genes derived from Jatropha curcas root libraries, showing tolerance to stress induced by 750 mM NaCl are marked in panel B (black circles). Identical yeast transformants (compare panel D to B) with repressed gene expression shows arrested growth.

Figure 6: Identification of acid and alkali sensitive conditions for wild-type yeast, and isolation of yeast mutants generated though a process of UV mutagenesis followed by forward genetic screening showing sensitivity towards acid and alkali stress.

DETAILED DESCRIPTION OF THE INVENTION Definitions The term "stress" as used herein refers to a condition that decreases growth, prevents growth, prevents a stage of growth (such as production of flowers, seed production, seed germination, production of new shoots), or tends to kill or actually kills an organism.

The term "tolerance" or "tolerant" refers to the ability of a plan to resist stress, such as the ability to grow or survive despite a condition that would decrease growth, prevent growth, or kill a organism that is not tolerant. Tolerance may be observed, for example, as a longer time to death, absence of death, or growth in the presence of the condition. Tolerance also may correspond to a range of protection from a delay to complete inhibition of alteration in cellular metabolism, reduced cell growth and/or cell death caused by the environmental stress conditions defined herein before. A plant identified, isolated, bred or created by methods of the present invention is tolerant of or resistant to abiotic stress in the sense that the plant is capable of growing in a substantially normal manner under environmental conditions where a corresponding wild-type plant shows reduced growth, metabolism, viability, productivity and/or male or female sterility. Methods for determining plant growth or response to stress include, but are not limited to, height measurements, leaf area, plant water relations, ability to flower, ability to generate progeny and yield or any other methodology known to those skilled in the art. The terms tolerance or resistance may be used interchangeably in the present invention.

The term "abiotic stress" as used herein refers to stress that is induced by or associated with non-biological factors, such as salinity, dessication, drought, radiation damage (such as that caused by UV light), heat, cold, pH (low or high), heavy metal, or ion stress. The amount of an abiotic factor that induces stress will vary according to multiple factors including the plant species and variant, soil type, and other co-existing abiotic stressors. For example, drought stress may be exacerabated by heat and salinity, as all three abiotic stressors may reduce the availability of water to plant cells.

The term "salinity" as used herein refers to stress that is induced by an elevated concentration of salt. The term "salt" as used herein refers to any water soluble inorganic salt such as sodium sulfate, magnesium sulfate, calcium sulfate, sodium chloride, magnesium chloride, calcium chloride, potassium chloride, etc., salts of agricultural fertilizers and salts associated with alkaline or acid soil conditions. While low salinity water has EC 5-9 dS m "1 , high salinity includes water with EC 10-28 dS m "1 , and over 28 28 dS m "1 .

The term "drought stress" as used herein refers to stress that is induced by or associated with a deprivation or reduced supply of water, and is a limitation on maximal plant performance imposed by water limitation. Jatropha normally needs at least 500mm of rainfall/yr, but can grow in Cape Verde Islands on 250mm/yr because of the high ambient humidity.

The term "ion stress" as used herein refers to stress caused by excessive concentrations of an ion, or ions in general. Such ions include Fe2+, Ca2+, Li+, OH-, H+, SO 4 2" etc. Excessive amounts of ions may manifest as osmotic stress, drought stress, or salinity stress. Other forms of ion stress include effects on the pH, oxidative potential, or toxicity. Ionic stress includes stress due to anions, cation, or both.

The terms "high pH" and "low pH" are relative terms that vary according to the plant species, soil condition etc. Jatropha curcase normally grows well on well drained soils of pH 6-8.5. Accordingly, a pH of about 6, especially less than 6, and more especially less than 5.3, would constitute "low pH." A pH about 9.0, especially greater than 9.0, and more especially greater than 10.0 would consistite "high pH."

The term "functional gene" as used herein refers to a gene that expresses a protein having substantially the same biological activity as the protein that is expressed from the gene in the natural environment from which it is derived (e.g. in the plant).

A "plantlet" as used herein is young or small plant used as a propagule, such as from division of a plant into several smaller units.

"Replica plating" as used herein refers to technique in which one or more Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate or wells, producing the same spatial pattern of colonies on each.

"Tissue" is a cellular organizational level intermediate between cells and a complete organism. A group of cells that shares a common function may be defined as an "organ." Organs of plants include roots, leaves, stems, flowers, seeds and developmental stages. Tissue may be obtained from an organ. "Root" typically refers to the organ of a plant body that typically lies below the surface of the soil, as part of a plant body that bears no leaves.

"Jatropha curcas" as used herein refers all variants of the species, including Jatropha curcas L. "Jatropha" refers to the genus which encompasses several species. In one embodiment, it is the species Jatropha curcas.

"Grow" or "growth" as used herein refers to refers to an increase in some quantity over time, including height, mass, etc. "Grow" or "growth" also refers to markers of development, including developmental stage, levels of developmentally appropriate compounds, etc.

"Promoter" as used herein refers to a regulatory region of DNA generally located upstream (towards the 5' region of the sense strand) of a gene that allows transcription of the gene. An "inducible promoter" is a promoter that is controlled by an inducer. For example, galactose is an inducer for the GAL promoter, such that galactose induces transcription of any gene under the transcriptional control of the GAL promoter.

"Plant" as used herein refers to any other plant and of any variety not limited to Jatropha curcas.

Method of identifying genes involved in tolerance to abiotic stressors

In one embodiment, the present invention provides a method for identifying plant genes involved in abiotic stress, such as salinity, drought and/or ion stress. For example, the invention provides methods for a molecular genetic screen that uses yeast (Saccharomyces cerevisae) to identify genes expressed in Jatropha curcas root tissue that confer resistance to salinity, drought and other abiotic stresses.

Strategies have been developed to identify genes conferring tolerance to abiotic stressors including mutagenesis-screening followed by positional cloning (Iuchi et al., 2007; Liu et al., 2000; Shi et al., 2002; James et al., 2008; Koh et al., 2007; Langridge et al., 2006; Yu et al., 2007a), activation-tagging (Kant et al., 2007; Langridge et al., 2006; Sreenivasulu et al., 2007), full-length overexpression screens in plants (Fujita et al., 2007), differential gene or protein expression analysis, and functional screening of silique cDNA libraries in yeast (US Patent Application Publications 2003/0162294). While such methods have been successful to isolate certain genes involved in an abiotic stress response in well characterized model plants, such as Arabidopsis, and some economically relevant cereal crops (James et al., 2008; Koh et al., 2007; Langridge et al., 2006; Yu et al., 2007a), directly applying these strategies to non-model plants, such as Jatropha, is challenging.

Unlike many agriculturally-valuable crops, there is little genetic information available for Jatropha curcas, and consequently large-scale expression analysis is not currently possible. Lack of high-density molecular markers, the time associated with breeding and the inability to perform large-scale transformation in Jatropha curcas also prevent application of many genetic techniques.

To overcome these limitations, and to isolate discrete gene sequences from Jatropha curcas that are linked to resistance to various abiotic stresses, the present invention has devised a functional genetic screen. The screen uses yeast, Saccharomyces cerevisae (Cregg et al., 1998, Guthrie and Fink, 1991; Pringle et al., 1997), as a surrogate system to identify and isolate specific genes in Jatropha curcas. Yeast also have been shown to respond to salt stress, which is reflected as retardation in cell growth (Hirasawa et al., 2006). Yeast strains show adaptive resistance (tolerance) to salt stress by intracellular accumulation of osmolytes as well as though membrane modification (Rodriguez-Vargas et al., 2007; Shen et al., 1999). Wild-type yeast strains have been reported to show salt sensitivity from 500 mM (Andreishcheva and Zviagil'skaia, 1999; Gaxiola et al., 1992; Nakayama et al., 2004; Shen et al., 1999), depending on the growth conditions. The conditions that lead to salt stress of wild type yeast BY4741 (yeast obtained from EUROSCARF, Germany) were identified by measurement of relative growth of the yeast under a range of salinity stress. See Examples.

To study the effect of salinity stress on Jatropha curcas, we treated the roots of young 3- 4 week old Jatropha curcas plants to various levels of NaCl salt stress. Some prior art studies have used LiCl to mimic the effects of salinity stress because cells are more sensitive to LiCl as compared to NaCl. For example, 1OmM LiCl inhibits yeast growth similarly to that obtained with ~200mM NaCl. In other words, cells exhibit approximately 20-fold greater sensitivity to Li+ over Na+. LiCl reduces the effect of drought responses (more precisely, osmotic effects) in the screen, due to less salt concentration. In one embodiment, however, the present approach uses NaCl because it (a) mimics both salt and drought stressors to which Jatropha is exposed; (b) mimics the physiological effect of NaCl, since LiCl is more toxic to cell growth; and (c) is less expensive than LiCl.

The present invention has identified different conditions of salt stress ideal for the construction of Jatropha curcas root tissue cDNA libraries. See Examples.

For instance, a cDNA library was constructed from normal and salt-stressed Jatropha roots, cloned into a shuttle vector, amplified in bacteria, and then shifted into yeast. The shuttle vector provided a catabolite-regulated GALl inducible promoter (Flick and Johnston 1990; Lohr et al., 1995; Stargell and Struhl 1996),. which allowed for conditional expression of Jatropha genes in the yeast cell (Silar and Thiele, 1991).

To screen yeast transformants, a replica printing based screening process was devised to identify and isolate yeast transformants expressing heterologous gene sequences derived from Jatropha curcas root cDNA libraries. To improve performance of the screen, a more exhaustive approach was devised, which is described herein. Additional controls were introduced in the screen to allow identification of both false-positive and false- negative yeast transformants, as illustrated in Figure 4. In the Examples below, the method of the invention has identified 345 candidate yeast clones from 20,000 yeast transformants. These clones will enhance the capability of wild-type yeast cells to tolerate unfavorable conditions imposed by salinity and drought stress.

Genes identified by the screening method of the invention have several industrial uses.

These include:

(i) screening Jatropha and other plants for mutant variants that exhibit increased tolerance to abiotic stress;

(ii) acting as a marker for breeding and selection of plants with increased tolerance;

(iii) transforming into plants to increase tolerance;

(iv) identification of abiotic stress tolerance genes in other plant species. The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention

EXAMPLES

Example 1: Design of the functional screen using yeast Saccharomyces cerevisea

The screen used a yeast, Saccharomyces cerevisae (Cregg et al., 1998, Guthrie and Fink, 1991; Pringle et al., 1997), as a surrogate system to identify and isolate specific genes in Jatropha curcas. An overview of a screening process developed to identify and isolate abiotic stress response genes from Jatropha is presented in Figure 1 a.

In one embodiment, the screen system of the present invention uses an inducible promoter, and specifically the catabolite-regulated GALl promoter (Flick and Johnston 1990; Lohr et al., 1995; Stargell and Struhl 1996), to conditionally express a Jatropha gene in the yeast cell when cloned in a shuttle yeast vector system (Silar and Thiele, 1991). The assay system is based upon the ability to select individual yeast transformants harboring plasmids containing a selectable yeast marker, and the ability to select yeast transformants that survive exposure to stress (Figure Ib). The ability to score the relative survival of a yeast cell exposed to salinity, drought or other abiotic stress, when compared to a yeast cells harboring the same gene in a repressed state when subject to the similar stress conditions, allows identification of tolerant transformants.

Data is presented herein from experiments involving expressed Jatropha cDNA fragments derived from root tissue, which were tested for their ability to rescue yeast growth when expressed in yeast with the GALl -regulated promoter when exposed to salinity and drought stress. Example 2: Identification of salt stress condition in Saccharomyces cervisea yeast, and treatment of 3-4 week in vitro germinated seedlings of Jatropha curcas L in NaCl to induce Jatropha genes involved in salinity stress.

To study the effect of salinity stress on Jatropha curcas, we treated roots of young 3-4 week old Jatropha curcas plants to various levels of salt stress. The present invention has identified different conditions of salt stress that allow for efficient subsequent construction of Jatropha curcas cDNA libraries. The present invention has identified that exposure of Jatropha roots to 100-150 mM NaCl for 1.5-3 hours, at a relative humidity of 40-50%, sufficed to observe a salt stress response. These conditions were used for the subsequent construction of Jatropha curcas cDNA libraries.

For salt treatment, Jatropha curcas seeds (Reliance breed plant R-044, RLS, DALC, Rabale) were removed from the seed coats, surface sterilized with 70% ethanol, followed by 1-5% hypochloride solution, prior to being placed in MS-Agar media bottles, as described previously in Deofe and Johnson (2008) and maintaining, at 23-25 0 C at 50- 60% RH, under long day conditions as described previously (Deore and Johnson, 2007). Young 3-4 week old in vitro germinated Jatropha curcas (grown as described above) were removed from the media, and randomly separated into groups of 15-20 plantlets. Groups of these plants were either placed in sterile water or into 150 mM NaCl solution for 1.5-2.0 hours at 50%-60% ambient humidity. This protocol was adopted to identify early stress responsive genes as distinct from delayed onset stress genes. In addition, 1.5-2.0 hrs was observed to be sufficient to observe visible leaf wilting of the experimental plants (indicating stress), and longer salt stress treatment could potentially lead to poor RNA isolation due to tissue necrosis associated with salt stress. Post- treatment Jatropha root tissue from plants that were untreated or treated were dissected frozen in liquid nitrogen, prior to being stored at -8O 0 C.

To identify and define conditions that lead to salt stress to wild-type yeast BY4741 (genotype detailed in Table 1) (yeast strain obtained from EUROSCARF, Germany), we measured the relative growth of yeast in YPD media (containing 2% peptone, 1% yeast extract, 2% dextrose, solidified with 1.5% Agar, Hi-Media, Mumbai, India) under a range of salinity stress, from 0.0 mM NaCl to 2.0 M NaCl. Through screening wild-type yeast though these condition, we identified that wild-type yeast BY4741 exhibited salt stress from 500 mM with total arrest of growth at 1.5 M NaCl (Figure 2).

Table 1 : Strain details genotype of Saccharomyces cerevisea yeast used in the functional screening process.

Organism Accession Number Strain Genotype

Saccharomyces YOOOOO BY4741 MATa;his3Δl;leu2Δ0; cerevisea metl5Δ0; ura3Δ0

Example 3: Construction of Jatropha curcas cDNA libraries from untreated and salt treated root tissue into yeast expression system driven by GAL-regulated expression system

Jatropha curcas root samples control treated or challenged with 150 mM NaCl (as discussed above) were used to generate pools of cDNA. Total RNA for cDNA synthesis was extracted from Jatropha curcas root samples using Qiagen RNA Miniprep Kit (Qiagen, Germany). Briefly, for total RNA extraction, root tissue samples were homogenized to a fine powder in liquid nitrogen. Total RNA was then extracted as described in the Plant mini RNA prep kit (Qiagen, Germany).

Subsequently the quantity/yield of total RNA was estimated spectrophotometrically at 230, 260 and 280 nm (Nanodrop). To prepare poly (A+) mRNA pools suitable for cDNA synthesis, lOμg total RNA was treated with RNAase-free DNAaseI (Sigma- Aldich, St Louis, USA) for 15-20 min at 37 0 C, and mRNA fraction was enriched using oligo-d(T) beads (Oligotex, Qiagen, Germany).

First strand cDNA pools were synthesized from normalized amounts of RNA derived from either untreated root tissue or from tissue challenged with salt stress, using PowerScript reverse transcriptase (Takara, CloneTech) as described in the Super SMART cDNA synthesis Kit, 1998). Following first strand synthesis, double stranded DNA was generated though PCR amplification using the conditions described in Table 2 (as detailed in Super SMART cDNA synthesis Kit, 1998).

Table 2: PCR cycling conditions and primer information used for double stranded cDNA generation and colony PCR analysis, as described in SMART cDNA Library Construction Kit (1998).

After cDNA synthesis, cDNA pools were then size separated using NucleoSpin columns (BD clontech, USA). The yield and quality of the amplicons were monitored with agarose gel analysis. A schematic diagram elaborating a construction of Jatropha root cDNA libraries is outlined in Figure Ia. A profile of amplification patterns (as observed by agarose gel electrophoresis) for the Jatropha curcas root cDNA libraries, obtained after 22 and 25 cycle of amplifications (as described in the Super SMART cDNA synthesis Kit, 1998), is shown in Figure 3. To express the cDNA libraries in yeast, the cDNA library amplicons were cloned into a yeast expression vector, pYES 2.1 TOPO TA (Invitrogen, Carlsbrad, USA). The pYES 2.1 TOPO TA, a E coli-Yeast shuttle vector (Silar and Thiele, 1991), can be propagated in E. coli with the bacterial selection marker for Amp r ; while the transformants in the yeast BY4741 strain background are selected for the URA3 marker.

Cloning of gene of interest downstream of the GALl promoter allows regulated gene expression of the library in yeast (Invitrogen, Carlsbard, USA). After cloning the Jatropha root cDNA libraries into pYES 2.1 TOPO TA, the vectors were used to transform chemically competent E. coli TOP 1OF' (Invitrogen, Carlsbad, USA), which were revived in SOC, plated on LB plates supplemented with 100 μg/ml ampicillin, and grown overnight at 37 0 C (as recommended by the manufacturer).

Unlike previously reported cDNA library synthesis methodologies that yielded 3' end biased partial cDNA, the SMART cDNA synthesis system yielded a large fraction of full-length cDNAs (Chenchik et al., 1994; Chenchik et al., 1998). Transformation of Jatropha cDNA library pools cloned in pYES2.1 TOPO TA yielded -48,000 c.f.u's, in each pool, representing un-amplified libraries. Through pilot-scale sequencing, the library was found to be representative and approximately 45-50% of the library inserts were determined to contain full-length cDNA sequences (data not shown). The average insert size in the library was approximately 800bp-lkb.

While plant genomes display large size variations, the number of genes expressed during cellular processes are more conserved. Based on the information available for model plant Arabidopsis and rice, approximately 3000-5000 genes out of a transcriptome of -27,000 genes is expressed in root tissue (Albert et al., 2005; Fizames et al., 2004; Ko and Han, 2004; Poroyko et al., 2005; Schrader et al., 2004). Recent analysis indicates a modest genome size for Jatropha curcas, with an estimated genome size of -450MB that provides an approximation of transcriptome complexity (Carvalho et al., 2008; Gregory, 2002). Based on an expected 3000-5000 genes, and the high fraction of complete cDNA, a library of 48,000 clones is expected to have >90% probability of obtaining a given expressed gene. 20,000 individual clones were screened, representing a -66% probability of obtaining a given gene. Example 4: Amplification and transformation of wild type yeast strain BY4741 with Jatropha root cDNA expression libraries.

Each library pool was plated completely into LB antibiotic plates, (supplemented with 100 μg/ml ampicilin) and grown overnight at 37 0 C. E. coli containing the cDNA library were recovered from the plates and transferred to LB antibiotic media (supplemented with 100 μg/ml ampicilin), grown overnight and plasmid DNA was extracted using Plasmid Midi preparation Kit (Qiagen, Germany). Plasmid transformation of yeast {Saccharromyces cerevisae) was accomplished using PEG-lithium acetate based transformation protocols (Becker and Lundblad, 2001; Gietz and Schiestl, 2007; Gietz and Woods, 2006), while the plasmid selection in yeast was based on the URA3 marker borne on the yeast expression plasmid pYES2.1 TOPO TA (Invitrogen, Carlsbad, USA). Amplified plasmids were incubated with wild-type yeast strain BY4741. Following heat-shock at 15 min at 42 0 C, the yeast cells for were revived in YPD media and plated on synthetic minimal medium plates lacking uracil (see Table 3 for media details), and placed at 23-25 0 C for 48-96 hours. Transformants were selected for their ability to grow on ura- plates, by virtue of the plasmid borne URAS marker (Gietz and Schiestl, 2007; Goldstein et al., 1999; Silar and Thiele, 1991). Typically, we obtained 10 3 -10 5 transformants/μg plasmid DNA, and these were screened using conditions as described in the section below.

Stock com ositions used in s nthetic media

Table 3: Composition of synthetic selection media and stocks used in the functional screen

pH adjusted to 7.2

Example 5: Development of replica printing based screening methodology for the identification of yeast transformants expressing genes conferring resistance to salinity and drought stress.

The screening scheme was based on replica-printing yeast transformants picked onto 96- well microtitre plates followed by testing the growth of yeast using quadruplicate-plate screening of yeast transformants (Figure 4).

Under these screening conditions, each individual yeast transformant (arrayed from 96- well plates) was tested for its ability to grow under the described four conditions: (A) Describes control conditions where GAL mediated gene expression is repressed and stress is not provided to the yeast cells. Under these conditions the cells are expected to grow on synthetic selection plates with glucose as the carbon source. Experimental conditions are defined by (B), where the gene expression is activated/induced using galactose as the carbon source, and the cells are subjected to stress, (here 75OmM NaCl). In these conditions only the yeast transformants that are capable of survival under stress (acquired due to expression of heterologous cDNA) are able to grow (Figure 4). The additional plates allow identification and elimination of transformants displaying cell- lethality caused by expression of heterologous cDNA as false-negatives. In condition (C), yeast transformants are grown in synthetic selection plates containing galactose, but without subjecting then to stress conditions. If the expression of any heterologous gene is detrimental to cell grown, it can be identified, thus eliminating recovery of false- negative transformants in the screen. To enable identification of false-positives and facilitate isolation of true-positive clones, the yeast transformants were subject to yet another condition. Transformants in type (D) conditions are grown in synthetic selection media with glucose as the carbon (i.e., without galactose) source, but treated under stress conditions. In these plates all transformants are expected to show retarded growth, due to the stress conditions. By comparing relative growth of marker array of transformants between above conditions it will be possible to screen against a diverse array of abiotic stresses in S. cerevisae. A schematic illustration of the output expected from the replica print based screen is presented in Figure 4.

To demonstrate proof-of-concept, S. cerevisae (BY4741) transformants containing Jatropha curcas root cDNA library clones in GALl regulated yeast expression system were screened for salinity and drought resistance. 20,000 individual yeast transformants were picked and inoculated into sterile 96 well U-bottom microtitre plates (Nunc, USA) containing synthetic selection media, after which the individual yeast transformants were replica printed on to quadriplicate selection plates containing either 0 mM NaCl or 750 mM NaCl (as described in Table 3) using a 96-pin replicator (Nunc, USA) (Figure 4). Comparison of the relative growth of yeast transformants under different conditions enabled the identification and isolation of genes that show consistent tolerance to stress imposed by salt and drought. A representative data set showing results obtained from this screen is presented in Figure 5. These results demonstrate proof-of-principle that the screen is capable of isolating leads genes involved in salinity and drought tolerance, including by using visible growth as a means of screening. The results also demonstrate the advantages of using an inducible gene promoter (GALl, in this case) that can allow the evaluation of the same yeast transformant for its relative ability to survive (and grow) when exposed to stress, depending on if the gene (derived from Jatropha curcas) is repressed (when grown on glucose) or expressed (when grown on galactose).

Example 6: Isolation of gene sequences from salinity and drought tolerant yeast transformants though plasmid rescue and plasmid amplification for determination of gene sequence

Isolated yeast transformants displaying the ability to tolerate salinity and drought stress imposed at 750 mM NaCl were grown on synthetic selection media containing 2% dextrose without salt (Table 3) for 36-72 hours, after which the yeast were lysed with 10 U/μl lyticase (Sigma-Aldich, St Louis, USA) and 2%-4% SDS (final concentration). Because high concentration of NaCl used here also poses osmotic (drought) stress to cells, the yeast transformants are expected to to be useful in isolating genes that provide a response to salt and/or drought stressors.

The nucleic acid fraction recovered from yeast was purified with two sequential rounds of phenol: chloroform :isoamyl alcohol (25:24:1) extraction, followed by ethanol precipitation of nucleic acids. To analyze the inserts cloned in the pYES2.1 TOPO TA based yeast expression plasmid, the nucleic acid preparations recovered from individual yeast transformants were back-transformed into E . coli TOPlO cells via electroporation (GenePulser II, BioRad, USA)(Lundblad and Zhou, 2001; Marcil and Higgins, 1992), and then revived and plated onto LB antibiotic plates (supplemented with lOOμg/ml ampicillin).

E. coli backtransformants containing yeast expression plasmids were analyzed for the presence of inserts using PCR analysis with the GALl and V5/6XHIS primers as described in the pYES2.1 TOPO TA kit (Invitrogen, Carlsbad, USA). Conditions for colony PCR analysis are provide in Table 2. Subsequently, these E. coli transformants were grown and the plasmid DNA extracted as described in Qiagen plasmid miniprep kit (Qiagen, Germany). Sequencing of the Jatropha cDNA inserts was performed with vector-specific GALl and V5/6XHIS primers with the BigDye Sequencing Kit (ABI, USA), and analyzed on GeneticAnalyzer (ABI, USA), subsequent to clean up with Montage Kit (Millipore, France). Using this process of plasmid rescue, we determined the sequences of 112 clones out of which 33 are full length genes (Table 4) and 79 are partial sequences (Table 5) derived from Jatropha curcas that confer salinity and drought tolerance in when expressed in yeast. DNA sequence information for these clones are presented in the sequence listing.

Example 7: Sequence annotation for identifying novel salinity and drought tolerant genes from Jatropha curcas L.

To understand and assign function classes to the sequence information determined in this screen, the vector sequence and poly(A+) regions were manually removed and the resulting nucleotide sequence analyzed using the BLAST algorithm (Altschul et al., 1990; Altschul et al., 1997; Gish and States, 1993) against the NCBI (http://www.ncbi.nlm.nih.gov/blast/) database, and TIGR plant transcript assemblies (http://tigrblast.tigr.org/euk-blast/plantta_blast.cgi)

BLASTX at NCBI was used to search for hits to non-redundant protein sequences, utilizing a standard genetic coding. Search parameters included (i) automatic adjustment of parameters for short input sequences (ii) an expectation threshold of 10 (expected number of chances in a random model); (iii) word size of 3 (the length of the seed that initiates an alignment); (iv) BLOSUM 62 matrix; (v) Match/Mismatch Scores of +1 and -2, respectively; (vi) Gap Costs of 11 for existence, 1 for extension; (vii) no compositional adjustments; (viii) filters and masking filter on for regions of low complexity; (ix) no discontiguous word options template length;and (x) template type set to "coding."

Sequences were also searched using the BLASTN and TBLASTX on the Wu-BLAST server at TIGR, against the following plant-specific EST collections.

1) Viridiplantae (Green Plants) [252, ESTs: 11006058]

2) Coniferopsida (Conifers) [10, ESTs: 663755]

3) Eudicotyledons (Dicots) [167, ESTs: 5481286]

4) Liliopsida (Monocots) [43, ESTs: 4280352] 5) Other Angiosperms [ 5, ESTs: 47444]

6) Others [27, ESTs: 53322]

Search parameters were (i) BLOSUM62 matrix; (ii) default filter mask; (iii) expectation threshold of 10; (iv) default cutoff for reporting (v) default on analysis of top or bottom strand (vi) default limit of 20 short descriptions (vii) default word length for blastn (viii) and echofilter off (ix) ignore hypotheticals off.

Full length sequences of 33 genes derived from Jatropha curcas root cDNA, cloned in yeast expression vector pYES2.1 TOPO TA (Invitrogen, Calrlsbad, USA) recovered from yeast transformants showing enhanced tolerance to salinity and drought stress. Annotation of best match and degree for has been presented in Tables 4 and 5. Table 4 also presents details on the degree of match (E value and % identity) and the protein sequences for the full length ORFs.

Tables 4 and 5 indicate possible gene/protein function for isolated inserts as predicted based on nucleotide sequence homology to non-Jatropha genes. Searches of sequences against database information indicate the presence of homology hits corresponding to sequences from other plant species that have functional implications in tolerance to salinity, drought and related stresses. This indicates a possible conservation in some of the genetic pathways as well as the genes regulating salinity and drought stress in plants (Seki et al., 2003; Sreenivasulu et al., 2007; Tuteja, 2007; Vashisht and Tuteja, 2006). Several genes isolated using this screening process, however, correspond to previously uncharacterized but hypothetical proteins, or unknown or novel genes Accordingly, these genes are entirely novel determinants of tolerance to salinity and drough abiotic stress.

A summary of Jatropha curcas L gene sequences recovered from yeast that confer tolerance to salt and drought is presented in Tables 4 & 5. In this analysis, out of 112 sequences with assignable hit to the sequence databases, 46 (~41% of the sequences) showed matches, (greater than accepted threshold as elaborated by Altshul et al., 1990) to other known plant genes, 66 sequences (~59 % of the sequences) could not be assigned any function ( Table 4 & 5 ) based on a match other the ability to confer tolerance to the abiotic stresses of salinity and drought, as demonstrated here. The present invention has identified genes in the disclosed screening process that confer dominant resistance to salt and drought stress when expressed in yeast cells. Sequencing of the inserts, followed by sequence annotation, suggests similarity of some of the inserts to other plant gene members, of which a few have been implied previously to be involved in salinity or drought stress. Tables 4 & 5 presents insert clone names, as well as grouping of clones based on "hits" in the databases. "Sequence annotation" refers to "hits" corresponding to genes present in non-Jatropha, plants or yeast. For clones receiving "hits" corresponding to genes in other plants or yeast that have been shown previously to confer resistance to salt, drought or related stress. The fact that the inventors have discovered numerous novel Jatropha homologs of previously identified non-Jatropha genes involved in confering tolerance to salt and drought stress indicates that the methods disclosed herein work unexpectedly well to identify genes related to abiotic stress in Jatropha curcas.

Not withstanding homology "hits" as pertaining to certain clones, the present invention has uncovered for the first time that the clones/genes presented in Figure 6 are involved in salinity or drought tolerance in Jatropha curcas in particular. Of the 33 clones identified so far, inventors have identified one AP2/EREBP domain encoding gene from Jatropha curcas that has been associated with a drought response, although exact mechanism of action is currently unknown for this gene. Tang et al. Plant Molecular biology, Vol. 63, No.3, pp.419-428 (Feb 2007).

These sequences represent novel determinants of salinity and drought stress tolerance (listing nucleotide and amino acid sequences together).

Table 4: Full length sequences of 33 genes derived from Jatropha curcas root cDNA, cloned in yeast expression vector pYES2.1 TOPO TA (Invitrogen, Calrlsbad, USA) recovered from yeast transformants showing enhanced tolerance to salinity and drought stress. Column 1 lists the Reliance Life Science clone identification number. Column 2 lists the accession number of the sequence deposited in GenBank. Columns 3 and 4 list the sequence identifiers of the nucleotide and amino acid sequences, respectively. Column 5 lists the number of amino acids in the predicted protein. Columns 6-9 describe the results of a BLASTX search of GenBank data base: the best match of each sequence (col. 6); references for the best match (col. 7); the significance of the match, as an E value (col. 8); and the % identity over the region of alignment (col. 9).

Table 5: List of 79 partial gene sequences conferring salinity and drought tolerance obtained from the functional genetic screen

Column 1 lists the Reliance Life Science clone identification number. Column 2 lists the accession number of the sequence deposited in GenBank. Columns 3 lists the sequence identifiers of the nucleotide sequences, and column 5 lists the best match of each sequence, obtained from a BLASTX search of GenBank.

Using the above-described initial screening process (Figure Ia, 5), we solated, analyzed and sequenced inserts from 112 yeast expression plasmids confering tolerance to salinity and/or drought.

As discussed above, upon analysis it was found that -41% of the identified Jatropha clones comprised sequences that shared similarity to non-Jatropha plant gene families or other organisms. Reported functions for some of the non-Jatrophic plant families imply involvement of the herein identified Jatropha genes in conferring dominant salt stress tolerance. For example, previous investigation indicate that metallothionines (Hirasawa et al., 2006; Jin et al., 2006; Quan et al., 2008), thioredoxins and the cellular redox machinery (Hong et al., 2004; Scharf et al., 1998; Serrato et al., 2004), and factors involved in the regulation of protein translation (Langland et al., 1996; Tester and Davenport, 2003; Williams et al., 2003) are associated with stress resistance in non- Jatropha plant or microorganism systems.

Amongst these, one example is a sequence ortholog corresponding to allene oxidase cyclase. Sequences of allene cyclase oxidase, isolated from mangrove have been shown in another previous investigation to confer salinity stress tolerance in multiple organisms (Yamada et al., 2002). A summary of DNA sequence annotation isolated in the screen and their implications to confer salinity and drought stress resistance has been compiled in Tables 4 and 5 Approximately 59% of the sequences identified do not have a yet assigned function based on their best match (other than tolerance to salinity and/or drought), but encode hypothetical proteins conserved across plants, or sequences that do not show significant matches to existing sequences. Although the exact mechanisms of action have yet to be determined for certain clones, it is expected that all clones identified by the disclosed method confer tolerance to abiotic stress in Jatropha curcas, or are otherwise involved in abiotic stress in such plants. Clones disclosed herein will be useful in identifying or preparing Jatropha plants that confer tolerance to abiotic stress, such as salinity, drought and/or ion stresses.

For example, the disclosed classes of sequences (Tables 4 and 5) represent a set of novel genes that could provide leads to yet undiscovered stress tolerance biochemical or signaling pathways operational in Jatropha curcas, through further characterization of gene function. Moreover, the disclosed clones are likely to be relevant to abiotic stress in other plants and/or organisms, particulary regarding the disclosed Jatropha clones with no known homology to previously identified genes.

Genes sequences of the present invention are useful to perform directed or selection breeding to generate elite plant cultivars in Jatropha and other species, using the methods disclosed in Sreenivasalu et al.(2007).

Functional genes of the present invention are also useful to genetically engineer Jatropha and other economically important plants with these sequences or their homologs for salt and drought tolerance. First, the overexpressed candidate genes recovered in this screen are optimized in existing plant expression systems to generate and evaluate transgenic plants (generated via Agrobacterium tumefaciens mediated or biolistic transformation) to ensure optimal levels of expression and regulation, such as by switching promoters. The resulting genes are then used to genetically engineer the target plant.

Example 8. Identification of genes conferring resistance to other abiotic stressors.

In the present invention, due to the fact the actual screen itself is performed in yeast (Saccharomyces cerevisae), the plant-life cycle is not limiting to the screen. Variations in the ability to tolerate salinity differ among plants that can often be ascribed to variation in their gentotype variations. Across diverse taxonomical groups, plants show distinct differences in their ability to tolerate and grow in saline ecosystems. Halophytes, for example, survive in saline environments by accumulating salt to levels in leaf vacuoles that are lethal to glycophytes. The present invention helps to gain insights in the genetic difference as well as uncover novel genes by comparing the genetic basis this variation. The described functional genetic screen may be used to prospect for novel genes, which are capable of providing enhanced resistance to abiotic stress by screening genomes of wild-plants occurring in naturally saline or drought prone ecosystems. Such populations of wild plants would be expected to have different expression patterns, or different genotypes, that confer resistance to the stresssor. In the present invention, minor modification to the above-discussed functional screening methodology may be used to isolate plant genes that confer tolerance to a diverse array of possible abiotic stresses. Some possible stress condition that may be screen with this assay include: a) pH stress (due to acidic or basic conditions), b) oxidative stresses, (simulated by use of media containing H 2 O 2 ), c) unfavorable temperature (by incubating media in heat or cold condition), d) heavy metal, as well as e) DNA damage/radiation (by exposure to UV, chemicals that induce DNA breakage). For example, ion stress conditions were determined for wild-type yeast BY4741 under acidic and alkaline conditions. Yeast was sensitive to acid from 5OmM H 2 SO 4 , and 50-75mM alkali NaOH (Figure 6). To improve the signal-to-noise ratio of the screen. BY4741 was mutated to be hypersensitive to the stressor being screened The yeast strain BY4741 was grown on YPD medium. A little inoculum of the strain was diluted in sterile distilled water and 300μl was plated on plates containing YPD medium. These plates were exposed to UV radiation. The UV dosage was determined using Stratagene crosslinker (From 50μjoules X 100 Energy to 500μjoules X 100 Energy). These plates were stored in the dark for two days at room temperature. Colonies which grew after two days were picked and replica printed on plates containing YPD and YPD + 50OmM NaCl and kept at room temperature. Mutant colonies which grew on YPD and did not grow on YPD + 50OmM NaCl (hypersensitive salt mutants) were picked and the screen was repeated. The mutant colonies were streaked on YPD plates containing salt series (5OmM to 1.5M) along with wild type. Finally freezer stocks were prepared of the colonies showing hypersensitivity to salt. This entire screen was repeated to obtain hypersensitive acid mutants (5OmM Sulphuric acid) and hypersensitive alkali mutants (25mM NaOH).

By mutating ~10 12 yeast, followed by screening yeast mutants hypersensitive to salt, alkali-salt and acidic conditions were identified. Yeast mutant strains (derived from BY4741) with salt hypersensitive (shs) (Figure 2), acid sensitive (has) and alkali-salt hypersensitive (alks) phenotypes, isolated in with the process of random mutagenesis- selection, are shown in Figure 6. Transformation of Jatropha root cDNA libraries into these isolated yeast mutant backgrounds, and screening as described, enables isolation of additional genes involved in complementing these mutations as well as identification of more genes involved in and/or regulating these processes.

Functional screen for obtaining genes conferring acid tolerance

Methodology

An acid hypersensitive mutant S. cerevisiae (has 1) was generated as a derivative of the wild type S. cerevisiae (BY4741) after UV mutagenesis. This was then transformed with the Jatropha curcas root cDNA library clones and screened for tolerance to acid stress. Around 20,000 individual yeast transforrnants were picked and inoculated into sterile 96 well U-bottom microtitre plates (Nunc, USA) containing synthetic selection media, subsequent to which the individual yeast transformants were replica printed on to quadruplicate selection plates containing either OmM H 2 SO 4 or 5OmM H 2 SO 4 and either glucose or galactose(as described for salinity in figure 4) using a 96-pin replicator (Nunc, USA). Upon comparison of relative growth of yeast transformants we isolated yeast transformants that show consistent tolerance to stress imposed by acid/ Results

To understand and assign functional classes to the sequence information determined in this screen, we performed computational searches against sequence databases at NCBI (http://www.ncbi.nlm.nih.gov/blast/) and TIGR plant transcript assemblies (http://tigrblast.tigr.org/euk-blast/plantta blast.cgi) using the BLAST algorithm (Altschul et al., 1990; Altschul et al., 1997; Gish and States, 1993). Searches suggests the presence of hits to plant specific sequences from other related plant species that have functional implications in tolerance to salinity, drought and related stresses. This indicates a possible conservation in some of the genetic pathways as well as the genes regulating abiotic stress in plants (Seki et al., 2003; Sreenivasulu et al., 2007; Tuteja, 2007; Vashisht and Tuteja, 2006), while several of the genes isolated in from this screening process corresponded to yet uncharacterized but conserved hypothetical protein, or unknown or novel genes. A summary of Jatropha curcas L gene sequences (full length and partials) recovered from yeast, and conferring tolerance to acid is presented in Tables 6 and 7 respectively. Out of 19 sequences with assignable hit to the sequence databases, 9 (47%) showed matches to other known plant genes, while for 10 sequences (53% of the sequences) significant matches to existing sequences in the sequence database was not apparent. These sequences represent novel determinants of acid stress tolerance. Out of the 19 sequences obtained, 2 were full length genes (Table 6) and the remaining 17 were partials (Table 7).

Functional screen for obtaining genes conferring alkali tolerance Methodology

An alkali hypersensitive mutant (alks 1) of S. cerevisiae (BY4741) was generated by UV mutagenesis, and then transformed with the Jatropha curcas root cDNA library clones as before (Flick and Johnston 1990) Around 20,000 individual yeast transformants were picked and inoculated into sterile 96 well U-bottom microtitre plates (Nunc, USA) containing synthetic selection media, subsequent to which the individual yeast transformants were replica printed on to quadruplicate selection plates containing either OmM NaOH or 25mM NaOH, with glucose or galactose(as described for salinity in figure 4) using a 96-pin replicator (Nunc, USA). Upon comparison of relative growth of yeast transformants, we isolated yeast transformants that show consistent tolerance to stress imposed by alkali. Results

To understand and assign function classes to the sequence information determined in this screen, we performed computational searches against sequence databases at NCBI (http://www.ncbi.nlm.nih.gov/blast/) and TIGR plant transcript assemblies (http://tigrblast.tigr.org/euk-blast/plantta_blast.cgi) using the BLAST algorithm (Altschul et al., 1990; Altschul et al., 1997; Gish and States, 1993). Searches of the sequences against database, suggests the presence of hits to plant specific sequences from other related plant species that have functional implications in tolerance to salinity, drought and related stresses. This indicates a possible conservation in some of the genetic pathways as well as the genes regulating abiotic stress in plants (Seki et al., 2003; Sreenivasulu et al., 2007; Tuteja, 2007; Vashisht and Tuteja, 2006), while several of the genes isolated in from this screening process corresponded to yet uncharacterized but conserved hypothetical protein, or unknown or novel genes. A summary of Jatropha curcas L gene sequences (full length and partials) recovered from yeast, and conferring tolerance to alkali has been presented in Tables 6 and 8 respectively. In this analysis out of 21 sequences with assignable hit to the sequence databases, 10 (47%) showed matches, (greater than accepted threshold as elaborated by Altshul et al., 1990) to other known plant genes, while for 11 sequences (53% of the sequences) significant matches to existing sequences in the sequence database was not apparent. These sequences represent novel determinants of alkali stress tolerance. Out of the 21 sequences obtained, 2 were full length genes (Table 6) and the remaining 19 were partials (Table 8).

Table 6: List of 4 full length genes obtained from functional genetic screen showing enhanced tolerance to Acid and /or alkali stress.

Column 1 lists the Reliance Life Science clone identification number. Column 2 lists the accession number of the sequence deposited in GenBank. Columns 3 and 4 list the sequence identifiers of the nucleotide and amino acid sequences, respectively. Column 5 lists the number of base pairs in the clone. Columns 6-8 describe the results of a BLASTX search of GenBank data base: the best match of each sequence (col. 6); the E value, measuring the significance of the match (col. 7); and the % identity over the region of alignment (col. 8).

GQ 906362 and GQ 906363 refer to genes conferring acid tolerance and GQ906364 & GQ906365 refer to genes conferring alkali tolerance.

Table 7: List of 17 partial gene sequences conferring acid tolerance obtained from the functional genetic screen

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Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.