# Patent

## Title
Polynucleotide encoding NF-YB derived from jatropha and use thereof

## Abstract

By analyzing a Jatropha genome, NF-YB-encoding genes of SEQ ID NOs: 1 to 11, fragments of NF-YB-encoding genes of SEQ ID NOs: 12 and 13, and genes relating thereto were found. By transforming Jatropha with these NF-YB-encoding genes and the like, it is possible to overexpress a NF-YB polypeptide and so on, and to significantly improve the productivity of protein synthesis involved by the NF-YB polypeptide, and to significantly improve the dry stress resistance, for example. As a result, it is possible to create dry stress resistant Jatropha capable of ensuring high growth even under water deficient conditions.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 26, 2012, is named P42720.txt and is 28,771 bytes in size.
TECHNICAL FIELD
The present invention relates to a polynucleotide encoding a NF-YB transcription factor as a novel gene of genus Jatropha, and use thereof, and in particular, use thereof for creating dry stress resistant Jatropha. 
BACKGROUND ART
Jatropha curcas attracts attention as biological resources for production of biodiesel fuel because a non-edible Jatropha oil can be produced therefrom. Further, Jatropha is known as a plant that can be cultivated even at locations unsuited for growth of other crops in terms of water and inorganic nutrients, and is believed to be very beneficial for effective utilization of semi-arid regions and for greening. On the other hand, although Jatropha plants grow in barrens, production efficiency of oils by natural cultivating is not high because fruition of the plants is once a year and the size of the fruit is significantly smaller than that of palm. For this reason, development of highly productive Jatropha is demanded.
As one measure for improving the production efficiency of a Jatropha oil, a method of transforming Jatropha so that acetyl CoA carboxylase (ACCase) can be overexpressed for increasing the oil content of the seed is known, for example, as disclosed in Japanese National Patent Publication No. 2009-536029 (PTL 1).
On the other hand, from the view point of improving the productivity of Jatropha itself, it is also conceivable to impart drought tolerance that ensures high viability even under water deficient conditions.
Generally, growth of a plant is greatly influenced by environmental factors such as dryness, salt and low temperature, and hence development of agricultural crops imparted with environmental stress resistance is expected.
As a dry stress resistant gene recombinant plant, the one wherein the stress responsive signaling intensity and mechanism are modified so as to be adaptive or responsive to dry stress, a method for improvement to achieve overproduction of a protein molecule involved in resistance (a protein responding to environmental stresses) and the like are conceivable.
Signaling pathways responsive to environmental stresses of plants are generally classified into pathways mediated by abscisic acid (ABA) which is a plant hormone, and pathways not mediated by ABA, and further sub-classified by the type of involved transcriptional regulators. Also as to proteins involved in response, regulatory proteins involved in response such as a transcription regulator, protease and protein kinase, and functional proteins responsible for resistance such as chaperone are known, and they are believed to take part in various physiological responses (Kazuo Shinozaki et al., Asakura Plant Physiology Course 5, “Environmental response”, pp. 106-1145).
Abscisic acid (ABA) is a plant hormone that is involved in seed dormancy, opening/closing of stoma and osmotic stress resistance, and ABA is known to be deeply involved in expression of a group of stress responsive genes.
For example, NPL 1 (Wen-Xue Li et al., “The Arabidopsis NFYA5 Transcription Factor Is Regulated Transcriptionally and Posttranscriptionally to Promote Drought Resistance,” The Plant Cell, Vol. 20: 2238-2251 (2008)) reports, in a mechanism of controlling dry stress resistance in Arabidopsis thaliana, that a NF-YA5 transcription factor is ABA-dependent and is strongly induced by dry stress, and that transformed Arabidopsis thaliana overexpressing NF-YA5 is superior to wild-type Arabidopsis thaliana in resistance to dry stress.
As a method of preparing environmental stress resistant Arabidopsis thaliana, Japanese Patent Laying-Open No. 2005-253395 (PTL 2) proposes a method of utilizing an activating function of a group of genes under the control of a transcription factor that activates transcription by binding with a cis element existing upstream the gene encoding a stress responsive protein expressed due to an environmental stress (a stress responsive transcription factor). Concretely, a SRK2C gene is disclosed as a novel gene encoding a signaling factor that induces expression of DREB/CBF which is a stress responsive transcription factor, and also it is disclosed that Arabidopsis thaliana transformed to overexpress the SRK2C gene shows dominantly high survival rate in comparison with a control even after stopping of water supply.
Further, NPL 2 (Donald E. Nelson et al., “Plant nuclear factor Y(NF-Y)B subunits confer drought tolerance and lead to improved corn yields on water-limited acres”, PNAS, vol. 104, No. 42, 16450-16455 (2007)) reports that a corn NF-YB factor was identified, and a corn transformed by using this showed higher productivity under the condition of water shortage in comparison with the wild type.
Furthermore, in Japanese National Patent Publication No. 2009-540830 (PTL 3), as water deficient stress resistant plants of rice, corn, soybean and cotton, plants into which a transcription unit containing a promoter operably linked with DNA encoding a NF-YB protein of Arabidopsis thaliana, corn or soybean is introduced are disclosed. It is reported that by devising a promoter or the like, yield of the transformed plant modified to be able to overexpress NF-YB is improved in comparison with that of a wild-type control even under water deficient conditions.
CITATION LIST
Patent Literature


    PTL 1: Japanese National Patent Publication No. 2009-536029
    PTL 2: Japanese Patent Laying-Open No. 2005-253395
    PTL 3: Japanese National Patent Publication No. 2009-540830


Non Patent Literature


    NPL 1: Wen-Xue Li et al., “The Arabidopsis NFYA5 Transcription Factor Is Regulated Transcriptionally and Posttranscriptionally to Promote Drought Resistance”, The Plant Cell, Vol. 20: 2238-2251 (2008)
    NPL 2: Donald E. Nelson et al., “Plant nuclear factor Y(NF-Y)B subunits confer drought tolerance and lead to improved corn yields on water-limited acres”, PNAS, vol. 104, No. 42, 16450-16455 (2007)


SUMMARY OF INVENTION
Technical Problem
A mechanism of signaling pathway for environmental stresses is complicated, and various transformation methods have been proposed for creating a dry stress resistant plant as described above. However, as to Jatropha, a regulatory protein related with dry stress, a functional protein related with resistance and the like have not been clarified.
It is an object of the present invention to create dry stress resistant Jatropha capable of ensuring high growth even under water deficient conditions, and to provide a gene or the like capable of transforming wild-type Jatropha to be dry stress resistant.
Solution to Problem
For achieving the object, as a result of examination of a gene for transforming Jatropha to be dry stress resistant, inventors of the present invention clarified a genomic sequence of Jatropha, and succeeded in isolating and identifying 13 genes encoding NF-YB, and accomplished the present invention. Specifically, the present invention is as follows.
[1] An isolated polynucleotide selected from the following polynucleotides:

    
    
        (a) a polynucleotide represented by any one of SEQ ID NOs: 1 to 11;
        (b) a polynucleotide encoding a NF-YB polypeptide derived from Jatropha, comprising a polynucleotide fragment represented by SEQ ID NO: 12 or 13; and
        (c) a polynucleotide represented by a nucleotide sequence having a homology of 90% or higher with the nucleotide sequence of the polynucleotide of either one of (a) and (b), wherein a polypeptide encoded by the polynucleotide maintains dry stress resistance of the NF-YB polypeptide encoded by either one of the polynucleotides of (a) and (b).
    
    


[2] The isolated polynucleotide as described in [1] selected from the polynucleotides of (a) and (b).
[3] An isolated NF-YB polypeptide selected from the following polypeptides:

    
    
        (a) a NF-YB polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 14 to 24;
        (b) a NF-YB polypeptide derived from Jatropha comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 25 or 26; and
        (c) a polypeptide represented by an amino acid sequence having a homology of 90% or higher with the amino acid sequence of the polypeptide of either one of (a) and (b), wherein the polypeptide maintains dry stress resistance of either one of the NF-YB polypeptides of (a) and (b).
    
    


[4] The isolated NF-YB polypeptide as described in [3] selected from the polypeptides of (a) and (b).
[5] A polynucleotide encoding the polypeptide as described in [3] or [4].
[6] A Jatropha plant transformation vector, wherein the polynucleotide as described in [1], [2] or [5] is incorporated.
[7] A transformant containing the vector as described in [6].
[8] A Jatropha plant transformed by using the vector as described in [6], wherein the plantlet is a dry stress resistant transformed Jatropha capable of overexpressing a NF-YB polypeptide compared with a wild type.
[9] A seed harvested from the dry stress resistant transformed Jatropha as described in [8].
[10] A method of producing a Jatropha oil by squeezing the seed as described in [9] and purifying it.
[11] A Jatropha oil produced by the production method as described in [10].
Advantageous Effects of Invention
When Jatropha is transformed by using the polynucleotide according to the present invention, the transformed Jatropha allows expression of a NF-YB polypeptide derived from Jatropha of the present invention, or a polypeptide equivalent thereto. With these polypeptides, it is possible to significantly improve the productivity of protein synthesis in which a NF-YB polypeptide is involved, and to significantly improve, for example, dry stress resistance.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a chart showing a molecular phylogenetic tree of NF-YB of Arabidopsis thaliana and Jatropha. 
FIG. 2 is a photograph showing a result of agarose electrophoresis of NF-YB1 to NF-YB5.
FIG. 3 is a gene map of a pGWB11 plasmid (see Nakagawa et al., “Development of Series of Gateway Binary Vectors, pGWBs, for Realizing Efficient Construction of Fusion Genes for Plant Transformation”, Journal of Bioscience and Bioengineering Vol. 104 (2007), No. 1 p. 38).



DESCRIPTION OF EMBODIMENTS
JcNF-YB Gene
The isolated novel Jatropha gene according to the present invention is a polynucleotide encoding a wild-type transcription factor NF-YB of Jatropha, and is a family of 13 genes individually existing in a Jatropha genome. Concretely, the present invention includes (a) polynucleotides represented by SEQ ID NOs: 1 to 11 (named “JcNF-YB1 gene” to “JcNF-YB11 gene” sequentially); (b) polynucleotides encoding a NF-YB polypeptide derived from Jatropha, comprising polynucleotide fragments represented by SEQ ID NOs: 12 and 13 (respectively, named “JcNF-YB12 gene” and “JcNF-YB13 gene”); and (c) a polynucleotide represented by a nucleotide sequence having a homology of 90% or higher with the nucleotide sequence of the polynucleotide of either one of (a) and (b), wherein the polypeptide encoded thereby maintains dry stress resistance of the NF-YB polypeptide encoded by the polynucleotide of either one of (a) and (b). The nucleotide sequence of the polynucleotide of (c) has a homology of preferably 95% or higher, more preferably 98% or higher, and particularly preferably 99% or higher, with the nucleotide sequence of the polynucleotide of either one of (a) and (b).
Polypeptides that can be obtained by expression of each gene of the present invention include, for example, (a) NF-YB polypeptides of Jatropha wild-type transcription factors JcNF-YB1 to JcNF-YB11 (having amino acid sequences of SEQ ID NOs: 14 to 24); (b) NF-YB polypeptides derived from Jatropha comprising polypeptides having amino acid sequences represented by SEQ ID NOs: 25 and 26; and (c) a polypeptide represented by an amino acid sequence having a homology of 90% or higher with the amino acid sequence of the polypeptide of either one of (a) and (b), wherein the polypeptide maintains dry stress resistance of the NF-YB polypeptide of either one of (a) and (b). The polypeptide of (c) has a homology of preferably 95% or higher, more preferably 98% or higher, and particularly preferably 99% or higher, with the amino acid sequence of the polypeptide of either one of (a) and (b).
The nucleotide sequence of the gene of the present invention also includes polynucleotides encoding polypeptides of the above (a) to (c). For example, part of bases may be substituted as far as polypeptides of (a) and (b) are encoded, and in the JcNF-YB1 DNA represented by SEQ ID NO: 1, by substituting the sixth base G with the base T (SEQ ID NO: 39), it is possible to make the translation efficiency higher than that of the wild type.
Hereinafter, by the term “JcNF-YB gene”, polynucleotides of the present invention are collectively referred to.
A method of preparing the JcNF-YB gene of the present invention is not particularly limited. For example, a PCR product of a target gene may be directly obtained by conducting a PCR reaction using a Jatropha genome as a template and primers designed for each JcNF-YB gene, or a PCR product of a target polynucleotide may be obtained by a RT-PCR method using the following primer set, from mRNA that is obtained by crushing part of a Jatropha plant, preferably leaves exposed to dry stress. Also, predetermined bases may be substituted, deleted or added according to an ordinary technique.
A method of directly obtaining a PCR product of a target gene using a Jatropha genome extracted according to a method of Sudheer et al., (Indian Journal of Biotechnology, Vol. 8 (2009) p. 187-192) is preferred. The method of Sudheer et al. has a feature in that NaCl concentration is regulated from in an extraction buffer to be used, to in a solution used for DNA precipitation, and that treatment in the purification step is conducted with Tris-saturated phenol, followed by a mixture of chloroform and isoamyl alcohol, and that 80% ethanol is used in the precipitation step.
mRNA may be prepared by a generally conducted method. For example, after grinding a frozen plant in a mortar or the like, a crude RNA fraction may be extracted and prepared from the obtained ground matter by a glyoxal method, a guanidine thiocyanate-cesium chloride method, a lithium chloride-urea method, a proteinase K-deoxyribonuclease method or the like. Also, a commercially available kit may be used.
Determination and confirmation of a nucleotide sequence of an obtained PCR product may be conducted by a conventionally known method, for example, a Maxim-Gilbert chemical modification method or a dideoxynucleotide chain termination method using M13 phage.
[Creation of Dry Stress Resistant Transformed Jatropha]
The dry stress resistant transformed Jatropha of the present invention is created by gene introduction of an expression cassette having the JcNF-YB gene operably linked with a promoter for expression or expression regulation, into a wild-type Jatropha. 
The species of Jatropha intended by the present invention are not particularly limited, and Jatropha curcas, Jatropha potagurica, Jatropha multifida, Jatropha berlandieri, Jatropha integerrima and the like may be used. Among these, from the view point of large oil content, Jatropha curcas is preferably used.
The gene introduction may be achieved by any method including methods of directly introducing DNA into a cell such as a method of fusing protoplasts, an electroporation method and a gene shotgun method; and methods of indirectly introducing DNA by using Agrobacterium tumefaciens or R. rhizogenes, and a method of using an agrobacterium is preferred. In the following, a transformation method using an agrobacterium is described.
An agrobacterium is a plant pathogen, and has a Ti plasmid having a region sandwiched between LB (left border) and RB (right border) (a T-DNA (Transferred DNA) region) that can be cut out and inserted into a host genome. When a host plant is infected with an agrobacterium having a plasmid incorporating a gene to be introduced, namely a JcNF-YB gene in this T-DNA region, the T-DNA region is cut out, and forms a complex with a protein group encoded by a vir region, and enters a plant cell, and further insertion into a host genome is achieved.
As a transformation method using an agrobacterium, a binary vector method is preferred. The binary vector method is a method of inserting a target gene into a plant genome by introducing into an agrobacterium, a plasmid having a target exogenous gene incorporated into a T-DNA region of a plasmid having borders (LB and RB) of the T-DNA region, in addition to a T-DNA-deficient plasmid of a Ti plasmid (such as pAL4404), and infecting a plant with the agrobacterium. 
An expression cassette used for creation of transformed Jatropha using the binary vector method includes the JcNF-YB gene according to the present invention, a promoter for expression of the nucleotide, a marker gene and a reporter gene in the T-DNA region.
As a promoter, a 35S cauliflower mosaic virus promoter, a nopaline synthase (NOS) promoter, and other endosperm-specific promoters such as β phaseolin, napin and ubiquitin can be recited.
As a selection marker gene, a gene that imparts resistance to a selection agent such as an antibiotic or a herbicide is used. Concrete examples thereof include a kanamycin resistant gene, a paromomycin B resistant gene, or a resistant gene against herbicides such as glufosinate and glyphosate. Also usable is a gene that expresses a selection marker enabling visual identification of a transformant, for example, a chromogenic or fluorescent protein such as luciferase or green fluorescent protein (GFP), or a gene that expresses β glucuronidase or GUS for which various chromogenic substrates are known. Such a selection marker may be used also as a reporter gene.
If necessary, an enhancer, a terminator, a tag and the like may further be included. An enhancer is used for improving expression efficiency of a target gene, and for example, an enhancer region including an upstream sequence in a CaMV 35S promoter can be recited. A terminator may be any sequence capable of terminating transcription of a gene transcribed by a promoter, and for example, a terminator of a nopaline synthase (NOS) gene, and a terminator of an octopine synthase (NOS) and a CaMV 35S RNA gene are recited.
As a binary vector for use in transformation of Jatropha by the binary vector method, those including the aforementioned expression cassette in a T-DNA region, and concretely, those prepared by incorporating the aforementioned expression cassette into commercially available vectors such as pBI series, pPZP series, pSMA series, and pGWB series may be used. In particular, a binary vector for plant transformation to which a cloning system of Gateway (registered trade name) is applicable is preferred, and as such a vector, pGWB series vectors can be recited. In these pGWB series vectors, a target gene and a reporter are operably linked using a cauliflower mosaic virus (CaMV) 35S promoter as a promoter; a hygromycin resistant gene or a kanamycin resistant gene as a selection marker gene; β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP) as a reporter; and 6×His (SEQ ID NO: 40), FLAG, 3×HA, 4×Myc, GST, or T7-epitope as a tag. Further, there are sequences that encode a reporter and a tag for allowing fusion at both the N terminal and the C terminal.
The Gateway (registered trade name) cloning system facilitates construction of an expression vector by using the Gateway (registered trade name) signal (att). In this method, by a reaction (BP reaction) between a donor vector having attP1 and attP2 sequences, and a target gene having attB1 and attB2 sequences added on each terminal, an entry vector having the target gene incorporated therein (having attL1 and attL2 sequences on each terminal) is created, and then by a recombination reaction (LR reaction) between this entry vector and a destination vector having a promoter required for expression incorporated therein (added with attR1 and attR2 sequences), a vector having the target gene inserted therein (expression vector) is created.
Therefore, first, a cloned JcNF-YB gene is allowed to undergo a BP reaction with a donor vector to prepare an entry vector having cloned JcPPAT cDNA incorporated in the donor vector, and then by a LR reaction between the entry vector and a destination vector (pGWB), an expression vector having the target DNA (JcNF-YB) incorporated therein can be created.
A detailed description for construction of an expression cassette for plant transformation using the Gateway binary vector (pGWB) is found in Nakagawa et al., “Development of Series of Gateway Binary Vectors, pGWBs, for Realizing Efficient Construction of Fusion Genes for Plant Transformation”, Journal of Bioscience and Bioengineering, Vol. 104, No. 1, 34-41 (2007).
The expression vector created as described above (plant transformation vector) can be amplified in Escherichia coli. The amplified transformation vector may be introduced into an agrobacterium by an electroporation method or the like. The agrobacterium into which the expression vector is introduced in this manner is used for transformation of Jatropha. 
Introduction of a target gene (JcNF-YB gene) into Jatropha by infection of an agrobacterium having the plant transformation vector can be achieved by using a known method such as a leaf disc method.
Concretely, a bacterial liquid for infection in which an agrobacterium is suspended in a MS medium is prepared, and the bacterial liquid and part of Jatropha which is a host (preferably, cut pieces of cotyledons, hereinafter referred to as “Jatropha leaf pieces”) are co-cultivated for about 3 days. The leaf pieces of Jatropha are dipped in a MS medium for about 2 days prior to the co-cultivation, and are preferably sonicated. In this way, it is possible to improve the efficiency of introduction. Also preferred is a Sandvortex method that applies vibration to a suspension of an agrobacterium into which sand has been added because infectability of the agrobacterium is improved.
As a co-cultivation medium, a MS medium or the like incorporating a plant hormone such as 3-indolebutyric acid (IBA) or 6-benzylaminopurine (BA) is used.
Following the co-cultivation, the Jatropha leaf pieces are washed, and transferred into a selection medium (containing an antibiotic corresponding to the selection marker gene used in the expression cassette in the transformation vector), and incubated, and then calluses formed in the leaf pieces are cut out, and transferred to a selection medium, and further screening of the transformed Jatropha (recombinant cell) is conducted.
As the selection medium, the one prepared by adding an antibiotic (kanamycin, hygromycin) which is a substance for selection to the medium (MS medium or the like) used for pre-culture, which contains IBA, BA, thidiazuron (TDZ) or the like as a plant hormone is preferably used.
Next, the selected calluses are transferred into a medium such as a RI medium or a MS medium, and allowed to root and redifferentiate into a plantlet. Induction of redifferentiation can be achieved by appropriately setting kinds and quantities of various ingredients including plant growth regulation substances such as auxin and cytokinin, and carbon sources in the medium, and light, temperature and so on.
[Transformed Jatropha]
The transformed Jatropha of the present invention is able to overexpress a transcription product of a JcNF-YB gene from a gene encoding a transcription factor JcNF-YB involved in transcription of a dry stress resistant gene, in comparison with the wild type. Therefore, it is possible to activate transcription and expression of the dry stress resistant gene. As a result, even in a dry condition, higher plant growth is achieved in comparison with the wild type.
The transformed plant of the present invention embraces not only “T1 generation” subjected to the transformation treatment, but also progeny plants including “T2 generation” which are succeeding generations obtained from a seed of this plant, and a next generation (T3 generation) obtained by self-fertilization of a flower of the plant of “T2 generation” which is proved to be a transformant by drug selection or analysis by a Southern method or the like.
[Production of Jatropha Oil]
A Jatropha oil can be produced from a seed harvested from the transformed Jatropha of the present invention according to a routine method. For example, a Jatropha oil that can be used as biodiesel can be produced by obtaining a material oil by squeezing a seed, and filtering the material oil through a filter. When the Jatropha oil is intended to be further purified, for example, it can be purified by distillation, and phorbol ester can be removed by the method described in Japanese Patent Laying-Open No. 2010-209177.
EXAMPLES
Embodiments for practicing the present invention will be described by way of examples. The following examples are not given to limit the scope of the present invention.
[Isolation of JcNF-YB-Encoding DNA in Jatropha and Construction of Transformation Plasmid]
(1) Preparation of Jatropha Genomic DNA
Thailand line Jatropha (Jatropha curcas) distributed from the agricultural department of Tottori University was used. From mature leaves of this Jatropha, genomic DNA was prepared according to the method of Sudheer et al. (Indian Journal of Biotechnology, Vol. 8 (2009) p 187-192).
The leaves of Jatropha were washed with distilled water, and the moisture was blotted with tissue paper, and 1 g was ground into powder in a mortar. The resultant powder was sufficiently mixed with 10 mL of an extraction buffer (2% CTAB, 100 mM Tris-HCl, 3.5 M NaCl, 20 mM EDTA, 1% β-mercaptoethanol) at 65° C. The mixture was incubated in a water bath at 65° C. for 90 minutes, and then cooled for 5 minutes. An equivalent amount of a mixture of chloroform and isoamyl alcohol (24:1) was added, and slowly mingled to give a uniform emulsion. This emulsion was centrifuged at 10,000×g for 15 minutes, and then the water phase was separated. The separated water phase was again added with an equivalent amount of a mixture of chloroform and isoamyl alcohol (24:1) and slowly mingled to give a uniform emulsion. After centrifuging this emulsion at 10,000×g for 15 minutes at 4° C., the water phase was collected. The collected water phase was added with an equivalent amount of isopropyl alcohol, and cooled at −20° C. for 30 minutes, and then centrifuged at 10,000×g for 30 minutes at 4° C. to obtain a DNA pellet. This DNA pellet was washed with 70% ethanol, and then suspended again in a TE buffer. The obtained DNA pellet was dissolved in a TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 20 mg/mL of RNase to give a genomic DNA sample.
The obtained extracted genomic DNA was fragmented by culturing together with EcoRI, HindIII and SauIII, and sequenced by a sequencer.
(2) Cloning and Amplification of JcNF-YB-Encoding Gene
Based on the genome information (Contig Map) of Jatropha obtained from (1), a gene showing a homology with Arabidopsis thaliana NF-YB was searched by TBLASTN. For gene information of NF-YB of Arabidopsis thaliana, gene registration information of NCBI (www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=full_report&list_uids=8184 72&itool=HomoloGeneMainReport) was referred to.
As a result of search, those annotated as encoding JcNF-YB are as follows.

    Contig1977.1.1
    Contig21632.1.1
    Contig30054.1.1
    Contig31310.1.2
    Contig31788.1.2
    Contig3182.1.1
    Contig8131.1.1
    F4IDXKH14IHOZQ.1
    HYB_Contig17630.1.2
    HYB_Contig31673.1.1
    HYB_Contig46618.1.1
    HYB_Contig46864.1.1
    HYB_Contig61720.1.1.1
    Jatropha454_3Run_c74008.1


Among these sequences, since DNA nucleotide sequences of HYB_Contig46618.1.1 and HYB_Contig61720.1.1.1 are proved to perfectly coincide with each other except for the terminal parts, we decided to use HYB_Contig46618.1.1 for prediction of a JcNF-YB gene. Therefore, it is supposed that 13 kinds of NF-YB genes are present in Jatropha (these genes are named NF-YB1 to NF-YB13). As a result of homology search, relations between Jatropha DNA contained in each of the above genome fragments and an Arabidopsis thaliana NF-YB gene are as shown in Table 1. Nucleotide sequences of JcNF-YB genes are shown in SEQ ID NOs: 1 to 13 in the sequence list. Amino acid sequences of polypeptides obtained by translating these polynucleotides are sequentially shown in SEQ ID NOs: 14 to 26. Further, CLASTALW analysis was conducted for JcNF-YB1 to JcNF-YB13 and NF-YB family of Arabidopsis thaliana (AtNF-YB1 to AtNF-YB13), and a molecular phylogenic tree was prepared. The result of preparation is shown in FIG. 1.









TABLE 1







NF-YB gene of



Novel isolated
Arabidopsis thaliana



NF-YB gene
showing highest


Jatropha genomic DNA
name of Jatropha
homology







Contig8131.1.1
JcNF-YB1
AtNF-YB3


Contig3182.1.1
JcNF-YB2
AtNF-YB5


Contig31310.1.2
JcNF-YB3
AtNF-YB7


Contig1977.1
JcNF-YB4
AtNF-YB5


HYB_Contig46864.1.1
JcNF-YB5
AtNF-YB3


Contig21632.1.1
JcNF-YB6
AtNF-YB1


HYB_Contig17630.1.2
JcNF-YB7
AtNF-YB1


Contig31788.1.2
JcNF-YB8
AtNF-YB13


HYB_Contig46618.1.1
JcNF-YB9
AtNF-YB11


HYB_Contig31673.1
JcNF-YB10
AtNF-YB2


Contig30054.1
JcNF-YB11
AtNF-YB6


F4IDXKH14IHOZQ
JcNF-YB12
AtNF-YB11


jatropha454_3Run_c74008
JcNF-YB13
AtNF-YB4









Next, JcNF-YB1 to JcNF-YB5 genes were amplified by conducting a PCR reaction using Jatropha (Thailand line breed) genomic DNA as a template, and respective primer sets (SEQ ID NOs: 27 to 36) shown in Table 2.









TABLE 2





Target




gene
Forward primer
Reverse primer



















NF-YB1
SeqID: 27
5′-AAAAAGCAGGCTAAACAATGGCT
SeqID: 28
5′-AGAAAGCTGGGTCCCTTGAATT




GATTCCGACAATGAATCTGGA-3′

GCCGGAGCCACC-3′


 


NF-YB2
SeqID: 29
5'-AAAAAGCAGGCTCAACAATGGTT
SeqID: 30
5′-AGAAAGCTGGGTAAAATCGCC




GACAATGCAAGCAATAATTCAGAC-3′

TGGAAACAGAACTGTTATTGC-3′


 


NF-YB3
SeqID: 31
5′-AAAAAGCAGGCTCAACAATGGAA
SeqID: 32
5′-AGAAAGCTGGGTATAACTTCAT




GAAGAGAGCCATGCCAGTG-3′

ATCTTGCCAATGCCC-3′


 


NF-YB4
SeqID: 33
5′-AAAAAGCAGGCTCAACAATGAAG
SeqID: 34
5′-AGAAAGCTGGGTAAGATGGCC




CAAATTTTGCCTCCTAATGCAAAAAT

TTGAACTTCTAGAGCTATTC-3′




C-3′




 


NF-YB5
SeqID: 35
5'-AAAAAGCAGGCTCAACAATGGCT
SeqID: 36
5′-AGAAAGCTGGGTAGTTATATCC




GGAAAAAGAAACCAAATAACCAGC-3′

ATAGCCGCTTTTAGGAGTAATTA-3′









The reaction liquid used for PCR is as follows.

    
    
        1.25 Unit Ex taq (TAKARA BIO)
        1×Ex taq buffer (TAKARA BIO)
        0.2 mM dNTPs (TAKARA BIO)
        1 μM Forward primer
        1 μM Reverse primer
    
    


The reaction liquid prepared in the above was added with 1 μL of a Jatropha genomic DNA solution diluted 100 folds to make the total amount 50 μL, and a PCR reaction was conducted under the following conditions.
After retaining at 96° C. for 5 minutes, a cycle of [96° C., 30 seconds→60° C., 30 seconds→72° C., 1 minute] was repeated 30 times, and then the reaction was retained at 72° C. for 5 minutes, and cooled to 4° C.
After end of the reaction, DNA obtained by amplification was identified by agarose gel electrophoresis. Results of electrophoresis of JcNF-YB1 to JcNF-YB5 are shown in FIG. 2. Further, sequences of the obtained PCR products were determined by a DNA sequencer. Respective nucleotide sequences of JcNF-YB1 to JcNF-YB5 polynucleotides are as shown in SEQ ID NOs: 1 to 5 in the sequence list.
For applying a Gateway (registered trade name) cloning system for DNA represented by SEQ ID NO: 1 (JcNF-YB1), a PCR reaction for adding the following adaptor sequences attB1 (SEQ ID NO: 37) and attB2 (SEQ ID NO: 38) was conducted.









[Chemical formula 1]



attB1: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′



 



attB2: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′






The PCR reaction was conducted with the one prepared by adding each 1 μL of a DNA solution to the following reaction liquids to make the total amount 50 μL.














1.25
Unit
Ex taq (TAKARA BIO)








1x
Ex taq buffer (TAKARA BIO)









0.2
mM
dNTPs (TAKARA BIO)


1
μM
attB1_adapter


1
μM
attB2_adapter









The PCR reaction was conducted with the following temperature cycle. After retaining at 94° C. for 1 minute, a cycle of [94° C., 15 seconds→45° C., 30 seconds→68° C., 1 minute] was repeated 5 times, and then a cycle of [94° C., 15 seconds→+55° C., 30 seconds→68° C., 1 minute] was repeated 20 times, and then the reaction was cooled to 4° C. After end of the reaction, amplified DNA was checked by agarose electrophoresis.
(3) Construction of Transformation Plasmid
A JcNF-YB1 gene was cloned by using a donor vector (pDONR221) of the Gateway (registered trade name) system of Invitrogen. Concretely, by conducting a recombination reaction (BP reaction) using BP Clonase (Invitrogen) after mixing the JcNF-YB1 gene (having attB1 and attB2 on each terminal) amplified in the above by PCR and the donor vector pDONR221, pENTRJcNF-YB1 which is to be an entry vector was obtained, and introduced into an Escherichia coli DH5α strain. pDONR221 has a kanamycin resistant gene introduced as a marker gene.
For construction of a plasmid for plant transformation, a pENTRJcNF-YB1 plasmid was extracted from the Escherichia coli, and mixed with a plasmid vector (destination vector) pGWB11 that was straight-chained by a restriction enzyme XhoI (TAKARA BIO), and then a recombination reaction was conducted using LR Clonase (Invitrogen).
As shown in FIG. 3, pGWB11 has a 35S promoter as a promoter, and has a FLAG tag added to its C terminal. Also, a 35S promoter —R1-Cmr-ccdB-R2-FLAG is inserted between HindIII and SacI. The part of R1-Cmr-ccdB-R2 can be substituted by attB1-(PPAT)-attB2 by the LR reaction with the entry vector. In this manner, pGWB11JcNF-YB1 which is to be a vector for plant recombination was obtained.
[Creation of Transformant]
(1) Preparation of agrobacterium for Transformation
The aforementioned vector for recombination was introduced into an agrobacterium by an electroporation method to achieve transformation. This transformed agrobacterium was shake-cultured in a YEB liquid medium (added with 50 mg/L kanamycin, 50 mg/L hygromycin) at 30° C. for 2 days, and then harvested by centrifugation. The harvested bacterial cells were resuspended in the YEB medium, to prepare a bacterial liquid for infection.
(2) Transformation of Jatropha 
As a Jatropha cell which is to be a host, Thailand line of Jatropha (Jatropha curcas) which is the same species of Jatropha as that used for genome extraction was used. Using mature leaves of the Jatropha, transformation was conducted by a leaf disc method. Concretely, first, cut pieces of mature leaves of Jatropha which are to be a host (about 25 mm2, hereinafter, referred to as a “Jatropha leaf pieces”) is sterilized with diluted kitchen bleach, and kept still at 25° C. for 2 days on a Pre-conditioning agar medium prepared by adding plant hormones (TDZ, IBA, BA) to a MS basal medium. A bacterial liquid for infection is prepared by suspending an agrobacterium in a MS medium, and the aforementioned Jatropha leaf pieces are dipped in the bacterial liquid, and shaken for 10 minutes. Then, co-cultivation is conducted on an agar medium at 25° C. for 3 days in a light-shielded environment. As a co-cultivation medium, a Co-cultivation medium prepared by adding acetosyringone to a Pre-conditioning medium is used.
(3) Screening of Transformed Jatropha 
A transformant having the expression cassette prepared in the above stably inserted into a chromosomal genome of Jatropha is screened.
Concretely, Jatropha leaf pieces after co-cultivation are washed with an aqueous solution of cefotaxime sodium (200 mg/L), and transformed Jatropha (a recombinant cell) is screened. As an antibiotic for screening, kanamycin (20 mg/L) is used. Following transfer to a Shoot regeneration I agar medium (SR-I), the leaf pieces in which formation of calluses are observed after culturing at 25° C. are transferred to a Shoot regeneration II (SR-II) agar medium.
Next, the selected calluses are transferred to a Shoot elongation I agar medium (SE-I) and a Shoot elongation II agar medium (SE-II), and an embryoid is allowed to differentiate, and rooting is induced in the Root induction agar medium (RI), to obtain a redifferentiated Jatropha plant (T1).
A culture medium composition used herein is shown below.
<MS Basal Medium>
















MS
1x, (pH 5.8)



Sucrose
  3%



Myo-inositol
100 mg/L



Thiamine hydrochloride (pH 5.8)
 10 mg/L



Agar
0.8%










<Pre-Conditioning Medium>











MS basal medium


















Thidiazuron (TDZ)
 0.5 mg/L



6-benzylaminopurine (BA)
   1 mg/L



3-indole butyric acid (IBA)
0.075 mg/L










<Co-Cultivation Medium>











MS basal medium


















Thidiazuron (TDZ)
 0.5 mg/L



6-benzylaminopurine (BA)
   1 mg/L



3-indole butyric acid (IBA)
0.075 mg/L



Acetosyringone (AS)
  20 mg/L










<SR-I Medium>











MS basal medium


















Thidiazuron (TDZ)
 0.5 mg/L



6-benzylaminopurine (BA)
   1 mg/L



3-indole butyric acid (IBA)
0.075 mg/L



Cefotaxime sodium
  200 mg/L



Kanamycin
  20 mg/L










<SR-II Medium>











MS basal medium


















6-benzylaminopurine (BA)
 3 mg/L



3-indole butyric acid (IBA)
 0.5 mg/L



Cefotaxime sodium
200 mg/L



Kanamycin
 20 mg/L










<SE-I Medium>











MS basal medium


















6-benzylaminopurine (BA)
 2 mg/L



Cefotaxime sodium
200 mg/L



Kanamycin
 20 mg/L










<SE-II Medium>











MS basal medium


















6-benzylaminopurine (BA)
 2 mg/L



Kanamycin
20 mg/L










<RI Medium>











MS basal medium (MS of 1⁄2 concentration)


















3-indole butyric acid (IBA)
0.2 mg/L










(4) Confirmation of JcNF-YB Gene Expression

It is checked that JcNF-YB1 transcription factor is overexpressed in a transformant selected by the screening.
A transformed cell (transformed dicotyledonous cell that expresses a NF-YB polypeptide by a promoter) and a control (dicotyledonous cell of wild-type Jatropha) are respectively cultured, and mRNA is extracted. The amount of mRNA of JcNF-YB1 transcription factor of the transformed cell is compared with that in the control.
(5) Confirmation of Dry Stress Resistance of Transformed Jatropha 
A transformed plantlet obtained by redifferentiation is sand cultured, and cultured under a water deficient condition after irrigation is stopped at an arbitrary point of time, and the photosynthetic rate and chlorophyll fluorescence, transpiration rate, and yellowing, curling and falling of mature leaves of the transformed plantlet are compared with those of the wild type, and dry stress resistance is evaluated.
INDUSTRIAL APPLICABILITY
The novel isolated gene of the present invention can be used for creation of dry stress resistant Jatropha, and hence Jatropha capable of growing in a dry area can be provided.

## Claims

1. A vector comprising a heterologous nucleotide sequence and a polynucleotide
with at least 99% identity to SEQ ID NO: 1, wherein expression of the polynucleotide enhances stress resistance in Jatropha compared to wild-type Jatropha.

2. The vector according to claim 1, comprising SEQ ID NO:1.

3. The vector according to claim 1, which is a Jatropha plant transformation vector.

4. A transformant containing the vector according to claim 3.

5. A transgenic Jatropha plant transformed with the vector according to claim 3, wherein the transgenic Jatropha plant has increased stress resistance compared to a corresponding control Jatropha plant.

6. A method of preparing a stress resistant Jatropha plant, said method comprising: preparing an isolated polynucleotide
with at least 99% identity to SEQ ID NO: 1,
wherein expression of the polynucleotide enhances stress resistance in Jatropha compared to wild-type Jatropha; 
preparing a Jatropha plant transformation vector comprising said isolated polynucleotide;
and transforming a Jatropha plant with said vector.