WO2010095150A2

WO2010095150A2 - Nucleic acids of jatropha curcas and its applications

Info

Publication number: WO2010095150A2
Application number: PCT/IN2010/000096
Authority: WO
Inventors: Nalini Eswaran; Sriram Parameswaran; Satharam Balaji; Bhagyam Anatharaman; Tangirala Sudhakar Johnson
Original assignee: Reliance Life Sciences Pvt. Ltd.
Priority date: 2009-02-20
Filing date: 2010-02-18
Publication date: 2010-08-26
Also published as: WO2010095150A3

Abstract

The present invention provides expressed sequence tags (ESTs) and contigs (comprising the sequence of two or more ESTs) of Jatropha curcas and its applications in isolating full length genes, identifying genes involved in different biotic and abiotic stresses, and determining gene function.

Description

NUCLEIC ACIDS OF JATROPHA CURCAS AND ITS APPLICATIONS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to Indian Provisional Patent Application No. 389/MUM/2009 filed February 20, 2009, the entire content of which is incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

[A copy of the Sequence Listing in CDROM containing file name Jatropha EST.txt of file size 8.47 Kb and created on Feb 15, 2010 is presented herein and the 987 sequences are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to expressed sequence tags (ESTs) and cDNAs of Jatropha curcas and their application to isolating full length genes, identifying genes involved in different biotic and abiotic stresses, and determining gene function.

BACKGROUND ART

The seed and kernel oil from Jatropha curcas Linn, has received great attention as a source for commercial biodiesel. This is primarily due the similarity of its lipid fractions to diesel (Modi et al., 200.7;.. Sriyastava.et.al., 2006),. the ability.of the. plant to , grow, with minimal agricultural inputs on poor and marginal soils (e.g., under conditions of drought, or high salinity), and that it does not compete with agricultural crops (Berchmans and Hirata, 2007; Francis et al., 2005; Gubitz et al., 1999; Kumar et al., 2007; Heller, 1996). Generation of primary genome information is an essential requisite for execution of research involving genetic improvement and/or analysis of Jatropha at a molecular level. Jatropha curcas has been gaining importance as a biodesel source. The seed and kernel oil from Jatropha curcas Linn, has been proposed as an ideal source for commercial biodiesel. This is primarily due the similarity of its lipid fractions to diesel (Modi et al., 2007; Srivastava et al., 2006); coupled with ability of the plant to grow with minimal agricultural inputs (Berchmans and Hirata, 2007; Francis et al., 2005; Gϋbitz et al., 1999; Kumar et al., 2007; Heller, 1996). Generation of primary genome information is an essential requisite for execution of research involving genetic improvement and/or analysis of Jatropha at a molecular level. Research from such perspective often also provides a leadership advantage.

Jatropha, a drought-resistant, photo-insensitive perennial plant belonging to the family Euphorbiaceae is attracting increasing attention as an important source of biodiesel (Kochhar et al, 2005). J curcas seeds contains 30-35% edible oil which can be easily converted to bio-diesel that meets the American and European standards (Achten et al, 2007). J curcas seeds can also be used for manufacturing other useful products such as candles, high quality soaps and cosmetics as well as for healing several skin disorders. Because of its above mentioned industrial and medicinal uses, initial investments towards commercial scale plantations of this plant are in process. This euphorbia which is a "drought resistant" plant grows on wasteland and could easily be cultivated ' by ^ι low income farmers.- It is grown as a shrub and thought that it could benefit energy provision to remote areas. In this respect, J. curcas is considered a strategic crop for countries such as Brazil or India (Carvalho et al, 2008).

Till date there hasn't been any literature on the genomic aspects of Jatropha. . Recently Jatropha has gained importance- due to its -application in biofuels Jatropha curcas, also known as physic hut, is unique among biofuels. Although oil can be extracted from over 80 known plant species, Jatropha is currently the first choice for biodiesel. Efforts are going on in obtaining transgenic Jatropha deficient in Curcin (toxin), improved oil biosynthesis, salinity tolerance etc. Identification of genes in Jatropha responsible for oil biosynthesis is a major field of research which has gained importance world- wide. While J curcas germplasm is being harvested all over the world with the purpose of crop improvement, little is known about its genome. Till date there has been no Expressed sequence tags (EST) database created for Jatropha curcas which will give an insight into its genome. With regards to the present scenario where lack of genome information is a key limitation to research, the present invention herein provides EST database of Jatropha curcas which consists of ESTs obtained from the root tissue.

Expressed sequence tags (ESTs) are short (100-1000 nucleotide bases in length), randomly selected single-pass sequence reads derived from cDNA libraries. EST sequencing projects are underway for numerous organisms, generating a vast amount of publicly available sequence data from plant species. Such data is useful for understanding various aspects of the transcriptomes of plant species. There are numerous EST databases available from different plants created with different purposes in mind. The aim of the existing EST database were: i. to provide accurate gene annotation and a dedicated platform for storage, processing and retrieval of sequence information (Maheswari et al, 2005). ii. to uncover conserved and potentially novel genes such as glutamate receptor like genes to support the hypothesis that such plants also serve a role in plant signaling (Brenner et al, 2003). ; iii. to identify genetic markers (SSRs) which will prove useful for analyses of phenotypic differences (Lindqvist et al, 2006). iv. to provide a well-characterized; non-redundant EST resource for advanced genomics, to generate arrays for expression studies with the view to investigate the expression of genes involved in genome interactions in naturally occurring species, and to develop codominant markers for genotyping plants (Mracek et al, 2006). v. to provide a high-quality resource, which will contribute to gaining a systemic understanding of plant diseases and - facilitate genetics-based population studies and identifying unigenes in plants (Kim et al, 2008). vi. to uncover putative sex determination genes, lipid and carotenoid metabolism enzymes, transcription factors as well as genes involved in metabolic pathways underlying secondary metabolism (Agostino et al, 2007). vii. SoyXpress is an EST database from Soybean consisting of 380, 095 ESTs derived from different cDNA libraries which was designed for the purpose of exploring potential transcriptomes differences in different plant genotypes, including genetically modified crops. Soybean EST sequences, microarray and pathway data as well as searchable and browsable gene ontology are integrated and presented in this database (Cheng and Stromvik 2008).

There are only 16 ESTs and 50 genes reported in Jatropha curcas (NCBI). Recently there have been reports about the construction and analysis of endosperm cDNA library in Jatropha curcas which gives understanding about some genes involved in fatty acid synthesis, transcription expression etc. (Zhitao et al. 2007).

In view of long-term interests in the Jatropha crop, and to provide a foundation for future research on the crop (such as gene discovery, functional genomics and marker development) the present invention has focused to provide an effort to decipher the Jatropha genome, with the initial objective of isolating expressed sequence tags (EST), and generating a searchable database of the same. The inventors of the present invention has developed a ESTs from Jatropha curcas, having utility in isolating full length genes, identifying genes involved in different biotic and abiotic stresses, and determining gene function.

OBJECTIVES OF THE INVENTION:

It is the aim of the present invention to provide nucleic acids or nucleic acid fragments and more particularly EST of Jatropha curcas.

It is the aim of the present invention to construct cDNA library of Jatropha curcas.

It is the aim of the present invention to provide cDNA library from the root of Jatropha curcas.

It is the aim of the present invention to provide EST derived from the root cDNA library

It is the aim of the present invention to provide full length genes derived from the ESTs

It is the aim of the present invention to provide functions of the full length genes. It is the aim of the present invention to collate the ESTs created from Jatropha curcas in the form of a database which will give an insight into its genome.

SUMMARY OF INVENTION

The present invention provides nucleic acids or nucleic acid fragments, such as genes and ESTs of Jatropha curcas. The present invention provides a cDNA library of Jatropha curcas, including a cDNA library from the root of Jatropha curcas. The present invention provides, for example, genes and ESTs derived from the root cDNA library. The present invention also provides full length genes derived from the ESTs. The present invention discloses functions for full length genes. The present invention provides a database of collated ESTs created from Jatropha curcas that gives insight into the genome of the plant. The present invention determines, through ESTs, contig sequences and full length genes, which genes and regulatory pathways are expressed in a given tissue, condition or disease.

The present invention provides a collection of ESTs consisting of over 700 ESTs of Jatropha curcas derived from root cDNA library. It also provides a way for obtaining full length genes using Jatropha ESTs and contig sequences, as well as a way of isolating promoters and flanking sequences using the ESTs. The ESTs and contigs provided herein are useful in identifying and mapping genes involved in developmental and metabolic processes, as well as in identifying gene functions and isolation of novel genes.

In one embodiment, the present invention provides expressed sequence tags derived from the plant Jatropha curcas. In one embodiment the present invention provides ESTs derived from the root cDNA library.

In one embodiment, the present invention provides methods of isolating promoters and other regulatory sequences using the ESTs. In other embodiment, the present invention provides methods for identifying flanking sequences using the ESTs. In one embodiment, the present invention uses ESTs to identify and map genes involved in the developmental and metabolic processes. In one embodiment, the present invention provides novel genes and their functions. In one embodiment, the present invention provides at least 72 full length novel genes from Jatropha curcas.

In other embodiments, the present invention provides an isolated nucleic acid molecule comprising a sequence that is at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a nucleotide sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 980); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 982); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 984); (d) Metallothionein (SEQ ID NO 986); (e) Profilin (SEQ ID NO 886); (f) NDP Kinase B (SEQ ID NO 936); (g) Glutathion-s- Transferase (SEQ ID NO 938); (h) S-adenosylmethionine-dependent methyltransferase (SEQ ID NO 960); and (i) Annexin (SEQ ID NO 974) hi other embodiments, the present invention provides nucleic acid molecules that are complementary to above-mentioned sequences.

In other embodiments, the present invention provides an isolated nucleic acid molecule comprising a sequence that is at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a nucleotide sequence selected from any one of SEQ ID NO. 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986. In other embodiments, the present invention provides nucleic acid molecules that are complementary to above- mentioned sequences.

In certain embodiments, the present invention is also directed to an isolated polypeptide encoded by one of the isolated nucleic acid molecules described above. In other embodiments, an isolated polypeptide may comprise an amino acid sequence at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 981); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 983); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 985); (d) Metallothionein (SEQ ID NO 987); (e) Profilin (SEQ ID NO 887); (f) NDP Kinase B (SEQ ID NO 937); (g) Glutathion-s-Transferase (SEQ ID NO 939); (h) S- adenosylmethionine-dependent methyltransferase (SEQ ID NO 961); and (i) Annexin (SEQ ID NO 915%

In other embodiments, the present invention relates to an isolated polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of SEQ ID Nos 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987.

In other embodiments, the present invention is directed to an isolated nucleic acid molecule comprising a sequence selected from any one of SEQ ID No. 737 to SEQ ID No. 843.

In other embodiments, the present invention is directed to a vector comprising at least one of any of the nucleic acid molecules described herein. The present invention also includes a host cell comprising such a vector. ^" Likewise, in certain embodiments, the present invention is directed to a transgenic plant comprising a recombinant nucleic acid molecule comprising at least one of the same nucleotide sequences described herein.

The present invention also relates to isolated nucleic acid molecules comprising at least ten isolated expressed sequence tag (EST) nucleic acid sequences and/or contig sequences selected from any one of SEQ ID No. 737 to SEQ ID No. 843. These isolated nucleic acid molecules may comprising at least 10, 20, 50, 100, 250, 500, 700 or 1000 of the EST and/or contig nucleic acid sequences disclosed herein In one embodiment, the present invention also includes a method for identifying a gene that is over-expressed or under-expressed in a Jatropha curcas plant in response to abiotic stress, the method comprising: (a) isolating a tissue from a Jatropha curcas plant that is subjected to an abiotic stress; (b) isolating a tissue from the Jatropha curcas plant that is not subjected to an abiotic stress; (c) measuring expression of at least one isolated nucleic acid molecules disclosed herein in the plant tissue of step (a); (d) measuring expression of at least one isolated nucleic acid molecule disclosed herein in the plant tissue of step (b), wherein the sequences of the isolated nucleic acid molecules of step (c) and (d) are identical. The present invention also includes a method of detecting tolerance to abiotic stress in a plant, plant tissue or plant cell, comprising (a) isolating a tissue from a plant, and (b) measuring expression of at least one of the nucleic acid molecules described herein. A method of the present invention may also comprise identifying Jatropha curcas plants that over-express at least one gene that confers tolerance to an abiotic stress, wherein the method comprises breeding Jatropha curcas plants, and using a nucleic acid molecule disclosed herein as a probe to identify which of the Jatropha curcas plants over-express at least one gene that confers tolerance to an abiotic stress. Likewise, one may use the disclosed the nucleic acid molecules of the present invention to detect gene expression, or to identify plants that exhibit tolerance to abiotic stress.

In one embodiment, the present invention provides substantially purified protein or fragment thereof encoded by a nucleic acid that specifically hybridizes to at least one of nucleic acid molecules described above. For example, in certain embodiments, the present invention corresponds to substantially purified protein or fragment thereof encoded by at least one of nucleic acid molecules described above.

In one embodiment, the^' present invention provides a substantially purified antibody capable of specifically binding to the protein or fragment thereof encoded by a nucleic acid sequence described above. In other embodiments, the present invention provides bacterial, viral, microbial and plant cells comprising at least one of the nucleic acid molecules described herein. In another embodiment, the present invention provides methods of producing a plant containing one or more proteins encoded by amino acid sequences disclosed herein or complements thereof, expressed in a sufficient amount and/ or fashion to produce a desirable agronomic effect. Likewise, the present invention provides nucleic acids that can be used to produce transgenic plants or cells. In one embodiment, the present invention provides methods for transforming a plant cell with at least one of the nucleic acid molecule sequences described herein or complements thereof.

In one embodiment, the present invention provides nucleic acids that can be used to detect correlation between polymorphisms and plant traits. The method includes hybridizing a nucleic acid described herein specific for a polymorphism to genetic material of a plant and determining the correlation between polymorphism and plant trait. In one embodiment, the present invention provides nucleic acids that can be used for determining the genetic region and mutations in a plant. In one embodiment, the present invention provides nucleic acids that can be used as a molecular tags to isolate the genetic regions (/ e , promoters and flanking sequences), isolate genes, map genes and determine its functions.

In one embodiment, the present invention provides methods of isolating nucleic acid comprising incubating under conditions permitting a nucleic acid hybridization; a marker nucleic acid molecule, e.g. an EST, with a complementary nucleic acid molecule obtained from a plant cell or plant tissue; permitting hybridization between said marker nucleic acid molecule. In one embodiment, the present invention to collate the ESTs created from

1

Jatropha curcas in the form of a database which will give an insight into its genome.

In certain embodiments;- -the isolated" nucleic acid is from' Jatropha curcas. In related embodiments, the invention is a vector containing any of the foregoing nucleic acids, a host cell comprising such a vector. In further embodiments, the invention is a transgenic plant comprising a recombinant nucleic acid identified above. In related embodiments, the recombinant nucleic acid is over-expressed in the transgenic plant.

The nucleic acids of the invention are useful for the design of primers for amplification. Accordingly, one embodiment is a pair of primers for amplification of an isolated nucleic acid of any one of the nucleic acids herein, comprising a primer at least 15 nucleotides in length from said nucleic acid, or its reverse complement.

In further embodiments, the invention is an isolated polypeptide encoded by a nucleic acid molecule comprising a sequence at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of sequence in Table 3. In related embodiments, the invention is an isolated polypeptide encoded by a gene comprising a sequence at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of SEQ ID No 980, 982, 984, 986, 886, 936, 938, 960, 974. In certain embodiments, the polypeptide is from Jatropha curcas.

In related embodiments, the invention is an isolated polypeptide comprising a sequence at least 75%, 80%, ^"85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of Table 3.. In further embodiments, the invention is an isolated polypeptide comprising a sequence at least 75%, 80%, 85%, 90, 95%, 99% or 100% identical to a sequence selected from any one of Seq ID No 981, 983, 985, 987, 887, 937, 939, 961, nd 975.

The invention includes methods and uses'. In one embodiment, the invention is a method of identifying a plant, or plant cell or tissue, with altered gene expression that confers desirable phenotypes, such as tolerance to abiotic stress. In one embodiment, the method of identifying such -a plant, -plant cell- or plant tissue is a method of selective breeding, wherein the method comprises breeding Jatropha curcas plants, and using a isolated nucleic acid molecule of the present invention as a probe to identify, wherein the Jatropha curcas plants over-express at least one gene that confers tolerance to an abiotic stress, such as salt or drought.

In another embodiment, the invention is a method of detecting the expression of mRNA associated with abiotic stress responses, comprising identify any of the nucleic acids described herein. In related embodiments, the method includes PCR, and in another, hybridization.

The invention also encompasses the use of the nucleic acid described herein for detecting gene expression, such as detecting genes whose expression is altered by plant stress.

BRIEF DESCRIPTION OF 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 1: Flow chart describing the process of construction of the EST database in Jatropha curcas

Figure 2: Gel analysis of amplicons, obtained for Jatropha curcas L. root tissue after amplification of double-stranded (ds) cDNA libraries pools as described in the SUPER SMART cDNA construction kit. 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) have been shown. Amplicons size distributions ranged from 0.5 to >5.0 kb.

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

Lane 1: ds cDNA amplified from total RNA prepared from untreated (control) roots.

Lane 2: ds cDNA amplified from total RNA prepared from roots treated with 15OmM

NaCl. ^■

Lane 3: ds cDNA amplified from poly (A+) RNA prepared from untreated (control) roots. Lane 4: ds cDNA amplified from poly (A+) RNA prepared from roots treated with 15OmM NaCl.

Figure 3: Colony PCR using vector primers to identify colonies with inserts. M = 1 Kb ladder DNA marker. Lanes 1 to Lanes 38 are colonies showing presence of inserts in the range 0.5 Kb to 3.0 Kb.

Figure 4: Pie chart representing the functional classes into which the set of Unigenes were divided.

Figure 5: Pie chart representing the functional classes into which the full length genes were divided.

Figure 6: Functional evaluation of transformants in salt hypersensitive (shs-2) mutant. Plasmid transformation of shs-2 with five selected genes:

235, late embryogenesis abundant protein 5 (SEQ ID NO 982);

392, cytosolic ascorbate peroxidase-1 (SEQ ID NO 984);

63, metallothionein (SEQ ID NO 986);

513, mitochondrial ATP synthase 6 KD subunit (SEQ ID NO 982);

619, profilin (SEQ ID NO 886).

Single colony of each transformants and mutant strain was plated on YPD media containing range of NaCl concentrations (0 mM, 250 mM, 500 mM, 750 mM and IM).

Figure 7A: Semi quantitative RT-PCR expression levels of nine genes under oxidative stress conditions from 24hr leaf tissue:

LEA-5: Late Embryogenesis protein (SEQ ID NO 980);

JcMtATPo Mitochondria 6Kd ATP synthase (SEQ ID NO 982)

Apx-1 Cytosolic ascorbate peroxidase (SEQ ID NO 984)

MT: Metallothionein (SEQ ID NO 986)

NDPK: NDP Kinase B (SEQ ID NO 936)

GST: Glutathione-s-Transferase (SEQ ID NO 938) SAM: S-adenosylmethionine-dependent methyltransferase (SEQ ID NO960).

Figure 7B: Semi quantitative RT-PCR expression levels of same nine genes under oxidative stress conditions from 48hr leaf tissue. LEA-5: Late Embryogenesis protein, JcMtATPo: Mitochondria 6Kd ATP synthase, Apx-1: Cytosolic ascorbate peroxidase, MT: Metallothionein, NDPK: NDP Kinase B, GST: Glutathione-s-Transferase, SAM: S- adenosylmethionine-dependent methyltransferase

Figure 1C: Semi quantitative RT-PCR expression levels of same nine genes under oxidative stress conditions from 72hr leaf tissue. LEA-5: Late Embryogenesis protein, JcMtATPo: Mitochondria 6Kd ATP synthase, Apx-1: Cytosolic ascorbate peroxidase, MT: Metallothionein, NDPK: NDP Kinase B, GST: Glutathione-s-Transferase, SAM. S- adenosylmethionine-dependent methyltransferase

Figure 8: Semi quantitative RT-PCR expression levels of same nine genes under heat stress conditions. LEA-5: Late Embryogenesis protein, JcMt ATP6: Mitochondria 6Kd ATP synthase, Apx-1: Cytosolic ascorbate peroxidase, MT: Metallothionein, NDPK: NDP Kinase B, GST: Glutathione-s-Transferase, SAM: S-adenosylmethionine-dependent methyltransferase

Figure 9A: Semi quantitative RT-PCR expression levels of same nine genes under salt stress conditions in leaf. LEA-5: Late Embryogenesis protein, JcMtATPo: Mitochondria 6Kd ATP synthase, Apx-1: Cytosolic ascorbate peroxidase, MT: Metallothionein, NDPK: NDP Kinase B, GST: Glutathione-s-Transferase, SAM: S-adenosylmethionine-dependent methyltransferase i

Figure 9B: Semi quantitative RT-PCR expression levels of same nine genes under salt stress conditions in roots. LEA-5: Late Embryogenesis protein, JcMtATPo: Mitochondria 6Kd ATP synthase, Apx-1: Cytosolic ascorbate peroxidase, MT: Metallothionein, NDPK: NDP Kinase B, GST: Glutathione-s-Transferase, SAM: S-adenosylmethionine-dependent methyltransferase

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS.

An "expressed sequence tag" or "EST" is a short nucleic acid molecule of a transcribed cDNA sequence, e.g., having 100-1000, such as 400-800, nucleotides obtained from a single cycle of sequencing. "EST" herein refers to expressed sequence tags from a cDNA library of Jatropha curcas.

A "contig" as used herein refers to a nucleic acid molecule derived from combining the s sequences of two or more EST sequences.

"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.

The term "complementary" as used herein refers to nucleic acid molecules that can hybridize to one another with stability to allow to remain annealed to one another. The term "complementarity" or "complementary" also means that a nucleic acid can form hydrogen bonds, i.e., hybridize, with another nucleic acid molecule. A nucleic acid molecule comprising two or more nucleic acids may be partially or completely (100%) complementary to another nucleic acid molecule, for example, with regard to corresponding nucleic acids that are capable of forming a double stranded molecule. A percent "complementarily" or "complementary" indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence. For instance, a first sequence is 95% complementary to a second sequence if 19 out of 20 contiguous nucleotides in the first sequence form hydrogen bonds with 19 out of 20 contiguous nucleotides in the second sequence. "Completely complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous Tesidues in a second nucleic acid sequence.

The term "percent homology" as used herein refers to percent identity over the region of similarity. Likewise, a sequence that is a certain "% identical" to another sequence means that the first sequence has nucleotides or amino acids that have that certain % identity as compared to the second sequence. For instance, a first sequence having 20 nucleotides is "at least 95%^' identical" to a second sequence having 20 nucleotides if 19 out of 20 nucleotides in the first sequence are exactly the same in identity and order as 19 out of 20 of the nucleotides in the second sequence.

The term "isolated" with regard to nucleic acid molecule(s) refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. However, a nucleic acid molecule contained in a clone that is a member of a mixed clone library {e.g , a genomic or cDNA library) and that has not been isolated from any other clones of the library, or a chromosome isolated or removed from a cell or a cell lysate, is not

The term "stress" as used herein refers to a condition pertaining in plants 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 the plant.

The term "tolerance" or "tolerant" refers to the ability of a plant to resist stress, such as the ability to grow or survive despite a condition that would decrease growth, prevent growth, or kill the plant 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, high salinity includes water with EC 10-28 dS/m, and over 28 dS/m.

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 500 mm of rainfall/year, but can grow in Cape Verde Islands on 250 mm/year because of the high ambient humidity. [How does this last sentence relate to our definition of drought stress? For example, does drought stress mean less than 500 mm rainfall/yr? Or less than 250 mm/year?

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+, SO₄ ^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, as well as less than 6, such as less than 5.3, would constitute "low pH." A pH about 9.0, as well as greater than 9.0, such as 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.

"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.

"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 plant and of any variety not limited to Jatropha curcas.

Genetics of Jatropha

Only limited genetic information is available for Jatropha curcas despites its popularity as a

'¹M P source of biodiesel. Current efforts to improve Jatropha curcas include transgenic Jatropha deficient in the curcin toxin, improved oil biosynthesis, and tolerance environmental conditions. Identification of genes in Jatropha responsible for oil biosynthesis is a major field of research that has gained importance world-wide. With regard to the present scenario where lack of genome information is a key limitation to research, the present invention herein provides an EST database of Jatropha curcas obtained from the root tissue.

Expressed sequence tags (ESTs) are short (100-1000 nucleotide bases in length), randomly selected single-pass sequence reads derived from cDNA libraries. EST sequencing projects are underway for numerous organisms, generating a vast amount of publicly available sequence data from plant species. Such data are useful for understanding various aspects of the transcriptomes of plant species. EST databases have multiple utilities. For example:

(i) provide accurate gene annotation and a dedicated platform for storage, processing and retrieval of sequence information, such as in genome construction (ii) uncover conserved and novel genes (iii) identify genetic markers (SSRs) which will prove useful for analyses of phenotypic differences;

(iv) identify codiminant markers for genotyping plants; (v) create nucleic acid arrays; (vi) track expression of genes and regulatory pathways in response to external stimuli, including pathogens, stressors, nutrients, etc; (vii) facilitate genetics-based population studies and identifying unigenes in plants (Kim et al, 2008); (viii) uncover putative sex determination genes, lipid and carotenoid metabolism enzymes, transcription factors as well as genes involved in metabolic pathways underlying secondary metabolism.

In view of long-term interests in the Jatropha crop, and to provide a foundation for future research on the crop (such as gene discovery, functional genomics and marker development) the present invention has focused to provide an effort to decipher the Jatropha genome, with the initial objective of isolating expressed sequence tags (ESTs), and generating a searchable database of the same. The inventors of the present invention have developed an EST library from Jatropha curcas, having utility in isolating full length genes, identifying genes involved in different biotic and abiotic stresses, and determining gene function.

The present invention provides an EST database consisting of at least 1240 ESTs from a Jatropha curcas root cDNA library. The database provides a way for obtaining full length genes using Jatropha ESTs and a way of isolating promoters and flanking sequences using the ESTs. The ESTs provided here will be useful in identifying and mapping genes involved in developmental and metabolic "processes." It will^' be useful ϊn identifying gene functions and isolation of novel genes.

The present invention identified ESTs from the cDNA library of Jatropha curcas roots. Roots are involved in the first uptake of resources from the soil and are affected by drought, salinity, pH, ion and other abiotic stressors, making roots a good source for isolating immediate and early responsive genes mediating tolerance to abiotic stresses. The present invention focused on isolating responsive genes mediating tolerance to abiotic stress.

In view of expanding the EST database, cDNA libraries are created from stressed leaf tissue with the aim to isolate stress induced and leaf specific genes. In addition, ESTs are obtained from cDNA libraries created from early flower and immature embryo with aim to isolate tissue specific genes.

The present invention also provides nucleic acid molecules and more specifically EST nucleic acid molecules or fragments thereof that can be used to isolate agronimically significant genes of abiotic stress not limited to genes involved in absorption, storage, anchorage, transport, propagation.

In another embodiment, the present invention has identified at least 72 full length genes that are involved in metabolic process, protein metabolism, growth and development, stress responses, signaling and defense, structural and ribosomal proteins, membrane and secretory proteins, transport and metal binding proteins, storage proteins, hypothetical proteins and unknown proteins.

The present invention has also provided ESTs that were used to identify genes for novel proteins or whose functions-are yet to be identified.

The cDNA library was obtained using the Super SMART cDNA synthesis kit. The vector used was pYES2.1 TOPO TA cloning vector.

The present invention provides nucleic acid molecules that can be used as molecular tags or marker molecules for determination of an attribute or feature {e.g., presence or absence, location, correlation, etc.) of a gene. Such methods include microarray techniques wherein sequences to be compared are analyzed by hybridization to a set of oligonucleotides. The present invention provides nucleic acids or proteins or fragments thereof to be utilized in microarray methods that are homologs of known or unknown genes.

The present invention provides nucleic- acids that- can be used for -producing genetically transformed plants comprising: inserting into the genome of a plant cell a recombinant double stranded DNA molecule and obtaining a transformed plant cell with said nucleic acid molecule is transcribed and results in expression of said protein(s) and regeneration from the transformed plant cells a genetically transformed plant.

The present invention also provides an essential application of the ESTs by creating an database of Jatropha curcas. for data management and analysis. The Jatropha EST database server is composed of a web interface and a management system. The web interface is implemented for querying the database to allow retrieval of unigenes based on BLASTX hits and other functional annotation results. The management system is used to store the collected sequence information and the analyzed data. Jatropha ESTs can be viewed individually by the "EST type" option! Under this option are two main categories i.e., "RAW ESTs" and "Unigenes set." Under the "Unigenes set" are two sub categories i.e., "Singleton ESTs" and "Contigs", with each having its own ID numbering. RAW ESTs are the total number of ESTs derived from the root cDNA library, numbering JcrMe RLOOOl to JcrME_RL1240. Under the "Unigenes set," the singleton ESTs are the unique ESTs which did not form a part of any- clusters, numbering JcrME RLSOOOl to JcrME_RLS0736. Contigs have been derived from clustering of 504 RAW ESTs that is also a part of the unigenes set, with numbering JcrME RLCOOOl to JcrME_RLC0111. There is a separate option for full length genes where all the full length genes can be viewed, for example, with numbering JcrME_RLF0001 to JcrME_RLF0073 (except JcrME_RLF0014). All ID numbers are linked to the nucleotide sequences and their related information. In certain embodiments ; the present invention is also directed to a computer program product for use in conjunction with a computer system, wherein the computer program product comprises a computer readable storage medium and a computer program mechanism embedded therein, wherein the computer readable storage medium comprising an electronic database comprising, for example, at least 10, 50, 100, 500 or 700, or over 900 nucleotide and/or amino acid sequences selected from SEQ ID NO: 1 to SEQ ID NO: 987 or complements thereof. ^{; ;}

Regarding a database server, a "Search" menu holds four different tab-style submenus for querying; these are "Keywords", "ID numbers", "Tissue" and "BLAST" search. The "Keywords" option brings the user to a page where it is possible to search against keyword and other parameters. The "ID numbers" allow the user to search and download each EST sequence. The "Tissue" search allows the user to search for ESTs based on different tissue- specific cDNA libraries.

An electronic database such as that describe herein can also include more ESTs derived from cDNA libraries of different tissues from Jatropha curcas. Users can use the BLAST search to compare their own sequences with in-house sequences in the Jatropha EST database. The BLAST option will have searches based on BLASTN, BLASTP and BLASTX. The display of search results will contains links to singleton and contig sequences. This provides a way to examine the relationship of the putative homolog to the gene being queried. In addition, all of the EST sequences will be deposited in dbEST at the NCBI.

The EST data from Jatropha curcas cDNA library described here can be used to generate probes to isolate genomic DNA containing the corresponding genes and to provide markers for physical maps. The ESTs can be converted into molecular markers. Finally, the EST database may be of use to other scientists who have obtained purified proteins of interest from Jatropha. The partial peptide sequence of a purified protein could be compared against translated EST sequences. The Jatropha EST database will provide a high-quality resource for Jatropha curcas EST analysis and also for comparative genomics among other species of Jatropha.

EXAMPLES

The following examples are included to demonstrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute exemplary 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 which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: Plant growth conditions

In marginal and poor soils "drought and salt stress affect the roots; and so cDNA libraries were constructed from root tissue exposed to salt stress, which also causes stress associated with drought. cDNA libraries were also prepared from both salt treated and untreated separately and later mixed before constructing library. The inventors indetended to identify genes that are expressed when exposed to salt- stress, but not under normal conditions. Therefore both cDNAs wer mixed as it will reduce the amount of work while screening library

Jatropha curcas seeds obtained from Jagdalpur district in Chhattisgarh 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, and. maintained at 23-25 C at 50- 60% relative humidity, under long day conditions as described previously (Deore and Johnson, 2008). 3-4 week old in vitro germinated Jatropha curcas plantlets were removed from the media, and separated into groups of 15-20 and placed either in sterile water or into 15OmM NaCl solution for 1.5-2.0 hours. Jatropha root tissue was dissected, frozen in liquid

O nitrogen, and stored at -80 C.

EXAMPLE 2: Construction of Jatropha curcas cDNA expression libraries from untreated and salt treated root tissue

Root tissue samples were homogenized to a fine powder in liquid nitrogen, and total RNA was extracted as described in the Plant mini RNA prep kit (Qiagen, Germany). Quantity and yield of total RNA was estimated spectrophotometrically at 230, 260 and 280nm (Nanodrop spectrophotomer). 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 C, and the mRNA fraction was enriched using oligo-d(T) beads (Oligotex, Qiagen, Germany).

According to the protocol described in the Super SMART cDNA synthesis Kit (Clontech), 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). Following! first strand synthesis, double stranded DNA was generated though PCR amplification using the conditions described in Table 1. cDNA pools were size separated using NucleoSpin columns (BD Clontech, USA) and the yield and quality of the amplicons were monitored on agarose gel. cDNA was cloned into pYES 2.1 TOPO, a yeast expression plasmid, permitting not only analysis of the sequence, but also screening for phenotypic properties such as salt tolerance in a yeast background. Clones were transformed into E coli TOP 1OF' chemically competent cells (Invitrogen, Carlsbad, USA), revived in SOC, plated on LB plates supplemented with 100ug/ml ampicillin, and

O grown overnight at 37 C (as recommended by the manufacturer).

Table 1: 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). as in Example 3

A schematic diagram elaborating the construction of Jatropha root cDNA libraries is outlined in Figure 1. 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 2. ^■ • The number of colonies to examine in screen was determined by comparison with other plant species. While plant genomes display large size variations, the number of genes involved in cellular processes are more conserved. Based on the information available for model plants, Arabidopsis and rice, ~3000-5000 genes out of a transcriptome of -27000 genes are 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 Jatropha curca has an estimated genome size of -450MB (Carvalho et al., 2008; Gregory, 2002). Transformation of Jatropha cDNA library pools cloned in pYES2.1 TOPO, yield -48,000 c.f.u's, in each pool, representing un-amplified libraries. Unlike previously reported cDNA library synthesis methodologies, that yield 3' end biased partial cDNA, the SMART cDNA synthesis system is expected to yield a large fraction of full-length cDNAs (Chenchik et al., 1994; Chenchik et al., 1998).

EXAMPLE 3: Colony PCR and Plasmid isolation and cDNA sequencing

E coli colonies 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 provide in (Table 1). A profile showing colony PCR using vector specific primers in 38 colonies is shown in Figure 3. Subsequently, these E. coli colonies were grown and plasmid ¹DNA extracted as described in Qiagen plasmid miniprep kit (Qiagen, Germany). Sequencing of the inserts {Jatropha cDNA) was performed with vector specific GALl and V5/6XHIS primers with the BigDye Sequencing Kit (ABI, USA), and analyzed on Genetic Analyzer (ABI, USA), subsequent to clean up with Montage Kit (Millipore, France).

EXAMPLE 4: Computer Analyses

Prior to annotation, the sequences were subjected to quality checking and vector masking using NCBI's UniVec (www.ncbi.nlm.nih.gov/blast/). Computational searches were performed against sequence databases at NCBI (www.ncbi.nlm.nih.gov/blast/) and TIGR plant transcript assemblies (tigrblast.tigr.org/euk-blast/plantta_blast.cgi) using the BLAST algorithm (Altschul et al., 1990; Altschul et al., 1997; Gish and States, 1993). Hits (i e., sequences identified in a search) to plant specific sequences from other related plant species that that have been implicated in tolerance to salinity, drought and related stresses, suggest conservation in some of the genetic pathways of stress response (Seki et al., 2003; Sreenivasulu et al., 2007; Tuteja, 2007; Vashisht and Tuteja, 2006). Several of genes isolated in this screening process corresponded to yet uncharacterized but conserved hypothetical protein, or unknown or novel genes. AU those sequences that have shown match with unknown sequences or protein, have not been functionally identified. Means, their biological role is yet unidentified. We have named all those sequences as unknown/hypothetical) .

ESTs that were identified as likely derived from a single mRNA transcript were assembled into contigs (set of overlapping nucleotide segments derived from a single genetic source) using CAP3 (Hunag and Madan, 1999) with default values. ClustalW sequence alignment (Thompson et al, 1994) was performed using the EBI web interface (www.ebi.ac.uk/clustalw).

EST library

The average insert size^" in the cDNA library was 1000 bp. 1282 ESTs were subject to random 5' sequencing, resulting in 1240 sequences (97%) passing quality check (see Table 2). After confirming EST quality and trimming the vector sequence to obtain high-quality sequence, the EST collection was computationally clustered using CAP3 and assembled to produce a non-redundant (unigene) sequence set. The resulting unigene set contains a total of 843 unigenes (non-redundant sequence assemblies). This" includes 736 singletons and 107 multimember EST clusters (or "contigs"). A singleton is defined as an EST clone that does not coherently overlap with any other EST and that contains a minimum of 100 consecutive base pairs of non-repetitive sequence. A contig is defined as a candidate gene cluster containing sequences from more than one EST. Most of the 107 contig unigenes contain fewer than 5 ESTs. Out of 107, 58 contigs contains only 2 ESTs. Only 4.5% of the contigs contain more than 10 ESTs. Table 2: EST and full length genes collection statistics

Non redundant set of contigs

Table 3 lists all contigs obtained and the number of ESTs involved in the formation of each contig.

Annotation and Functional classification

Each EST and contig was compared against all sequences in the non-redundant database at the U.S. National Center for Biotechnology Information (NCBI) using the program BLASTX, which compares predicted translated nucleotide sequences with protein sequences; and against the plant transcript assemblies database at The Institute for Genomic Research (TIGR) using the program BLASTN, which compares nucleotide sequences with nucleotide sequences. The results of each comparison were screened manually. Sequences deemed to be of bacterial origin were removed from the collection.

Of the 843 ESTs and and contigs, 586 had significant homology to previously identified genes. Although the BLAST scores and P values were considered, the assessment of whether a given homology was significant was not only judged by absolute numerical cutoffs but was also determined by investigator judgment. The annotations of genes with similarities to an EST or contig were used to assign a putative identification to the cDNA represented by the EST. 229 ESTs did not show similarities with known genes but did show similarities with hypothetical proteins or unknown proteins. The remaining 28 ESTs or contig did not give any hits in the databases and are, therefore, completely novel. All 843 ESTs and contigs have been divided into 14 functional classes as represented in Figure 4. The distribution of the functional classes in terms of percentage is listed in Table 4. Table 4: List of functional classes and percentage of each for set of Unigenes

Abundantly expressed transcripts

The relative abundance of the mRNA in a tissue is approximately reflected in the abundance of its corresponding cDNA in non-normalized libraries. Random sequencing of cDNAs therefore yields information about the relative expression levels of the genes represented by the ESTs (Covitz et al, 1998). Table 5 lists the most abundantly expressed transcripts in our root cDNA library. Two of these, expressed plant transcript and hypothetical proteins, are the ESTs which show similarities with other plant transcripts in databases but are still unknown. The most abundantly expressed gene was metallothionein. The metallothioneins are small, high-Cys-containing proteins that play a role in heavy metal detoxification, especially in respect to cadmium, copper and zinc, and enhance tolerance to oxidative and salinity stress. Aquaporins in plants are shown to be involved in root water uptake, reproduction or photosynthesis which is another abundantly expressed gene obtained from our root library (Kaldenhoff and Fischer 2006). Plant annexins are known to bind Ca2+ and phospholipids and are abundant proteins (Delmer and Potikha 1997). The other abundantly expressed gene Thioredoxin is known to be involved in stress responses by acting as antioxidants (Gelhaye et al, 2004).

Table 5: List of Abundantly^* expressed transcripts in Jatropha cDNA library

Full length genes

The full cDNA sequences of 72 genes were obtained' from the library The list of full length genes with their length and their % identity with the exiting databases (NCBI) is listed in Table 6A. Out of these 72 genes, the first 37 (see Table 6A) were obtained from the EST itself by single-pass sequencing. The next 29 (38-62 and 70-73 of Table 6A) were obtained by sequencing the insert with both the forward and reverse primer. The last six genes (63-68 of Table 6A) were obtained from the contigs that were formed from the ESTs after clustering the ESTs using CAP3 program. Each gene was compared against all the sequences in the database of NCBI using the program BLASTX. Putative functions were assigned to 69 of the genes, and three showed homology to unknown proteins. The different functional classes into which all the full length genes fall is represented by Figure 5.

Table 6A: List of 72 full length genes obtained from root cDNA library

Table 6B: Putative functions of 72 full length genes

EXAMPLE 5. Gene-expression analysis of selected Jatropha sequences involved in salinity stress tolerance using semi-quantitative RT-PCR

The change in gene expression in response to salt and drought stress was examined for nine novel genes identified herein: Late embryogenesis protein-5 (LEA-5) (SEQ ID NO 980), mitochondria^*ATP 6Kd synthase protein (JcMt ATP) (SEQ ID NO 982), cytosolic ascorbate peroxidase (Apx-1) (SEQ ID NO 984), metallothionein (MT) (SEQ ID NO 986), profiling (SEQ ID NO 886, NDP kinase B (NDPK) (SEQ ID NO 936), glutathione-s- transferase (GST) (SEQ ID NO 938), S-adenosylmethionine-dependent methyltransferase (SAM) (SEQ ID NO 960)and annexin (SEQ ID NO 974) These genes were identified in the EST screen expressed under salt stress^" and,^" based on other plant ^'rnόdelsTare considered to represent diverse functional classes and varied mechanisms implicated for stress avoidance/tolerance pathways in model plants.

Semi-quantitative RT-PCR expression of selected genes was performed with specific oligonucleotide primers (Table 7) on first strand cDNA synthesised from RNA isolated from root and leaf tissues samples of J curcas seedlings exposed to different stressors for periods of time, described below in greater detail. The total RNA was extracted with Plant mini RNA prep kit (Qiagen, Germany). Subsequently the quantity and yield of total RNA was estimated spectrophotometrically at 230, 260 and 280nm (Nanodrop). Eight hundred ng of total RNA was taken to synthesize the first strand cDNA with oligodT primers using Superscript reverse transcriptase (Invitrogen) following the manufacturer's protocol. For PCR, 1 μL of cDNA was used as a DNA template in a reaction volume of 50 μL using PCR master mix (Lucigene) with cycling conditions of 95⁰C for 5 min, 95⁰C for 1 min (denaturation), 60⁰C for 1 min (annealing), 72⁰C for 1 min (elongation). The amplification reaction was carried out for 32 cycles for all genes with a final elongation of 72⁰C for 10 min. J curcas actin gene was used as an internal control for analysis of gene expression. RT-PCR analyses were carried out with three independent total RNA samples.

Quantitation of RT-PCR product was determined by densitometer. Two μl of RT-PCR products derived from the target gene and actin gene were resolved on 2 % agarose gels stained with ethidium bromide. Densitometeric scan analysis was carried out using Kodak MI Imaging software program as per the supplier's instructions. Percent gene expression was determined by normalizing values against the actin internal control. Values were represented as per cent of gene expression with respect to corresponding controls, which were plotted using Microsoft Excel.

Table 7: List of forward and reverse primers used for semi-quantitative RT-PCR

I \ I

Salt stress

28-days old Jatropha curcas seedlings were subjected to salt stress by placing the plant in series of sodium chloride concentration (OmM, 15OmM, 25OmM, 75OmM and IM) and monitored at different time points of 0 hrs, 2 hrs, 4 hrs and 8 his. Photographs were taken of all plants at all concentrations and different time points. The plant material (0.5 g) from the 15OmM salt treated leaves and roots at different time points such as Ohrs, 2hrs and 8hrs were collected and frozen. To salt stress roots, the roots were placed in NaCl solutions. After exposing seedlings to 15OmM NaCl, expression of nine selected genes (described in Table 7) were analyzed by semi qRT-PCR both in leaf and root tissues. LAE-5 and SAM expression was increased in leaf tissue (FIG 9A), whereas other genes were repressed in contrast. LAE-5 was repressed in root tissue, while increase in Profilin, NDPK expression was observed. Interestingly SAM expression in root tissue was close to 5-fold (FIG 9B). Analysis of the gene-expression data suggests dynamic changes in the transcript abundance of these genes, with changes in transcript level being apparent from early 2h time-point, indicting an early regulation of these genes in response to salt and desiccation stresses. Gene-expression of normalized transcripts suggests up-regulation of these transcripts upon prolonged stress. We note an increase in expression of all genes the highest being SAM in root tissue within the 2-hour to 8-hour time-points, of exposure to 150 mM salt stress, that is consistent with its previously reported function during salt stress (Ramachandran et al, 2000).

Heat stress

Jatropha seedlings (28-days old) were subjected to heat stress by placing them at 42⁰C incubator for 20 hrs. Photographs were taken at 0 hrs and 20hrs after heat stress. The plant material (0.5 g) of leaves from control plants and heat stressed plants were collected and frozen. After 20hr of heat stress, only JcMTATPό transcript accumulation was high indicating its role in heat stress. Increase in JcMTATPό transcript levels both under heat stress and oxidative stress conditions directly suggest its role in stress tolerance. MTATP6 was first isolated from mitochondrial F₁Fo ATPase. Therefore increase in JcMTATPό must have a role in maintaining or intensifying the activity of F₁F₀ ATPase under stress conditions, indicating its role in stress tolerance (FIG 8).

Oxidative stress

Oxidative stress was induced by treating 28-day old seedlings with 0, 100 and 200μM methyl viologen in 0.1% Tween 20. Observations were taken at different time points such as 0 hrs, 24 hrs, 48 hrs and 72 hrs. The plant material (0.5g) of leaves was collected from plants sprayed with different concentrations of methyl viologen and at different time points and frozen, under light conditions 100 μmol m^"2 s^"1 After 24hr increase in transcript levels of LEA-5, Apx-1, MT_vProfilin, SAM and Annexin was noticed in leaf tissues (FIG 7A). After 48hr post treatment, in addition to above genes increase in transcript abundance of JcMTATPό was observed, while Annexin levels have been repressed (FIG 7B). After 72hr post treatment high-levels of MT, SAM and Annexin was notices indicating their strong role in oxidative stress (FIG 7C).

EXAMPLE 6: Functional analysis of genes in yeast

To demonstrate if cDNA clones isolated from salt-stressed roots confer salinity tolerance, plasmids were transformed into the salt hypersensitive mutant (shs-2) yeast and evaluated for growth on high-salt, at and beyond 75OmM. upto 2M. Screening for saline tolerant yeast strain and isolation of shs mutants

To identify and define the condition that lead to salt stress in wild-type yeast BY4741 (yeast strain obtained from EUROSCARF, Germany), we measured relative growth of yeast (YPD media 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. Salt-hyper-sensitive mutants were isolated though UV induced random mutagenesis, followed by selection on salt containing plates by replica-plating.

Yeast transformation

To increase the probability of representation of each individual clone within these yeast expression libraries and to efficiently transform yeast, we amplified the copy numbers of clones contained in E. coli. Each library pool was plated completely into multiple amplified LB antibiotic plates (supplemented with 100 μg/ml ampicillin) and grown overnight at 37⁰C. Plasmid DNA was extracted using Plasmid Midi preparation Kit (Qiagen, Germany).

Plasmid transformation of yeast

Plasmid transformation of yeast (Saccharomyces cerevisiae) mutant shs-2 was accomplished using PEG-lithium acetate based transformation protocols (Becker and Lundblad 2001), 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 containing cloned inserts derived from J curcas, regulated by the galactose-inducible GALl promoter, were transformed into yeast mutant shs-2; following heat-shock at 15 min at 42⁰C, the yeast cells were revived in YPD media and plated on synthetic minimal medium plates lacking uracil and placed at 23-25⁰C for 48-96 hours.

Evaluation of transformants in hyper-salt sensitive mutant

In addition to screening the library in yeast, individual clones were transformed into yeast hyper-salt sensitive mutant (shs-2) according to the above protocol. Five genes were LEA 5, mitochondrial ATP synthase 6 KD subunit, cytosolic ascorbate peroxidase, metallothionein and profiling, described above. (See Figure 6). Single colony of each transformants and mutant strain was dissolved in lOOμl of sterile water. YPD media containing range of NaCl concentrations (OmM, 250 mM, 500 mM, 750 mM and IM) were prepared in Petri dishes. Each Petri plate was divided into six sectors. lOμl of each yeast transformants and the mutant strain were patched on the salt series plates uniformly and incubated overnight at 30⁰C incubator. Survival of transformants was scored against hyper- salt sensitive mutant strain.

Figure 6 and Table 8 A demonstrates the ability of these plasmids encoded genes derived from J curcas confer tolerance to ^"salt in salt-hypersensitive yeast. These data provides functional support for the consistent ability of these recovered genes to confer salt tolerance, and support cell growth at conditions that would otherwise be detrimental to cell survival.

Table 8A

τ,4.7.f.,

In order to validate function, Yeast hypersensitive mutants were used. Survival of certain clones at and beyond 75OmM NaCl has-been noticed in shs-2 mutants indicating that tested genes were salt tolerant. Again, semi-quantitative RT-PCR was performed on salt stressed tissues. Genes that conferred tolerance in shs-2 need not show expression in plant tissue. In fact, in our studies we found that same gene expressed differentially in roots and leaves indicating complex framework of gene regulation during stress adaptation

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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.

Claims

WE CLAIM:

1. An isolated nucleic acid molecule comprising a sequence that is at least 95% identical to a nucleotide sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 980); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 982); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 984); (d) Metallothionein (SEQ ID NO 986); (e) Profilin (SEQ ID NO 886); (f) NDP Kinase B (SEQ ID NO 936); (g) Glutathion-s-Transferase (SEQ ID NO 938); (h) S-adenosylmethionine-dependent methyltransferase (SEQ ID NO 960); and (i) Annexin (SEQ ID NO 974).

2. An isolated nucleic acid molecule comprising a sequence at least 95% identical to a sequence selected from any one of SEQ ID NO 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 984, 986.

3. The isolated nucleic acid molecule of claim 1, comprising a sequence at least 99% identical to a sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 980); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 982); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 984); (d) Metallothionein (SEQ ID NO 986); (e) Profilin (SEQ ID NO 886); (f) NDP Kinase B (SEQ ID NO 936); (g) Glutathion-s-Transferase (SEQ ID NO 938); (h) S-adenosylmethionine-dependent methyltransferase (SEQ ID NO 960); and (i) Annexin (SEQ ID NO 974).

4. The isolated nucleic acid molecule of claim 2, comprising a sequence at least 99% identical to a sequence selected from any one of SEQ ID NO 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 984, 986

5. The isolated nucleic acid molecule of claim 3, comprising a sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 980); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 982); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 984); (d) Metallothionein (SEQ ID NO 986); (e) Profilin (SEQ ID NO 886); (f) NDP Kinase B (SEQ ID NO 936); (g) Glutathion-s-Transferase (SEQ ID NO 938); (h) S-adenosylmethionine-dependent methyltransferase (SEQ ID NO 960); and (i) Annexin (SEQ ID NO 974).

6. The isolated nucleic acid molecule of claim 4, comprising a sequence selected from any one of SEQ ID NO 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 984, 986

7. An isolated polypeptide ejicoded by a nucleic acid molecule comprising at least one of the isolated nucleic acid molecules of any one of claims 1-6.

8. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from any one of: (a) Late Embryogenesis Protein-5 (SEQ ID NO 981); (b) Mitochondria ATP 6Kd Synthase protein (SEQ ID NO 983); (c) Cytosolic ascorbate peroxidase (SEQ ID NO 985); (d) Metallothionein (SEQ ID NO 987); (e) Profilin (SEQ ID NO 887); (f) NDP Kinase B (SEQ ID NO 937); (g) Glutathion-s- Transferase (SEQ ID NO 939); (h) S-adenosylmethionine-dependent methyltransferase (SEQ ID NO 961); and (i) Annexin (SEQ ID NO 975).

9. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from anyone of SEQ ID NO 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987

10. A vector comprising the nucleic acid molecule of claim 1 or 2.

11. A host cell comprising a vector comprising the nucleic acid molecule of claim 1 or 2.

12. A transgenic plant comprising a recombinant nucleic acid molecule comprising the same nucleotide sequence as the nucleic acid molecule of claim 1 or 2.

13. An isolated nucleic acid molecule comprising a sequence selected from any one of SEQ ID NO 737 to SEQ ID NO 843

14. Isolated nucleic acid molecules comprising at least ten isolated expressed sequence tag (EST) nucleic acid sequences and/or contig sequences selected from any one of SEQ ID NO 737 to SEQ ID NO 843

15. Isolated nucleic acid molecules of claim 14, comprising at least 50, 100, 500 or 1000 EST and/or contig nucleic acid sequences of claim 14.

16. A method for identifying a gene that is over-expressed or under-expressed in a Jatropha curcas plant in response to abiotic stress, the method comprising: (a) isolating a tissue from a Jatropha curcas plant that is subjected to an abiotic stress; (b) isolating a tissue from the Jatropha curcas plant that is not subjected to an abiotic stress; (c) measuring expression of the isolated nucleic acid molecules of claim 14 in the plant tissue of step (a); (d) measuring expression of the isolated nucleic acid molecules of claim 14 in the plant tissue of step (b), wherein the sequences of the isolated nucleic acid molecules of step (c) and (d) are identical.

17. A method of detecting tolerance to abiotic stress in a plant, plant tissue or plant cell, comprising (a) isolating a tissue from a plant, and (b) measuring expression of the nucleic acid molecule of claim 1 or 2 or the nucleic acid molecules of claim 14 in the tissue.

18. The method of claim 17, further comprising identifying Jatropha curcas plants that over-express' at least one gene that confers tolerance to an abiotic stress, wherein the method comprises breeding Jatropha curcas plants, and using the nucleic acid molecule of claim 1 or 2 as a probe to identify which of the Jatropha curcas plants over-express at least one gene that confers tolerance to an abiotic stress.

19. The use of the nucleic acid molecule of any one of claims 1, 2 and 13 for detecting gene expression.

20. The use of the nucleic acid molecule of any one of claims 1, 2 and 13 for identifying a plant that exhibits tolerance to abiotic stress.

21. The isolated nucleic acid of Jatropha curcas, its method and its use as claimed above exemplified herein substantially in the examples and figures.