JP2016103994A - Gene for imparting environment stress resistance and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene which can impart practical environment stress resistance to a plant body and use thereof.SOLUTION: The present invention provides a plant body having enhanced one or more gene selected from the group consisting of a second gene selected from a gene group consisting of a first gene sub group consisting of Os12g0150200 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a gene functionally equivalent to one of these genes and a second gene sub group consisting of Os04g0584800 gene and a gene functionally equivalent to the gene.SELECTED DRAWING: None

Description

  The present specification relates to a gene capable of imparting environmental stress tolerance and use thereof.

  Plants are protected and managed for reproduction and growth, and are used as agricultural crops in agriculture. Crop yield and quality are greatly affected by climate change. In recent years, climate change factors have increased on a global scale, and there is a need to ensure a stable food supply regardless of climate change.

  Examples of various stresses that can be caused by climate change include high temperature, low temperature, drying, flooding, salinity, and the like.

  As a plant that is resistant to such stress, for example, a transformed plant body in which salt tolerance is expressed is disclosed (Patent Document 1). Moreover, the transformed plant body which expressed drought stress tolerance is also disclosed (patent document 2).

  On the other hand, jasmonic acid is one of the plant hormones induced when a plant is subjected to some stress.

JP 2004-329210 A Special table 2014-520527 gazette

  Although climate change can be predicted to some extent, it is extremely difficult to fully predict it. In actual cultivation of crops, it is often exposed to a plurality of environmental stresses simultaneously. Then, the crop needs to have resistance to not only one stress but also a plurality of stresses. However, it is considered difficult to produce plants with tolerance to such multiple stresses, and very few examples have been realized.

  Further, according to the present inventors, the conventional evaluation results of stress tolerance at the laboratory level, which have been widely used, are not necessarily correlated with actual stress tolerance. That is, even if a positive evaluation is obtained in the laboratory level evaluation, the opposite evaluation result may be obtained in the practical level evaluation.

  Furthermore, although jasmonic acid is induced during stress, the role of jasmonic acid and its conjugates during stress is not well understood.

  The present specification provides a gene capable of imparting more practical environmental stress tolerance to a plant and use thereof.

  The present inventors newly identified a plurality of genes introduced into a plurality of FOX rice lines exhibiting salt tolerance and the like by using individual overexpression (FOX) rice lines of rice full-length cDNA. Furthermore, as a result of various stress tolerance tests using FOX rice lines against these salt tolerance candidate genes, the present inventors have contributed to salt stress tolerance at a more practical level, or resistant to other environmental stresses. We were able to identify genes that could show The present specification provides the following means based on these findings.

(1) Os12g0150200 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and the first group of genes functionally equivalent to any of these genes, the Os04g0584800 gene and this gene A plant having enhanced expression of one or more genes selected from a gene group consisting of a second gene subgroup consisting of functionally equivalent genes.
(2) The plant according to (1), wherein environmental stress tolerance is enhanced.
(3) The plant according to (1) or (2), wherein tolerance to salt stress is enhanced.
(4) The plant according to (3), wherein other environmental stress tolerance is further enhanced.
(5) The plant according to any one of (1) to (3), wherein expression of the first gene is enhanced.
(6) The plant according to (5), wherein the first gene is selected from the group consisting of at least the Os12g0150200 gene and a gene functionally equivalent to this gene.
(7) The plant according to any one of (1) to (6), wherein the first gene encodes any one of the following proteins (a) to (f):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of converting activated jasmonic acid into inactive jasmonic acid (c) comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and inactivating activated jasmonic acid Protein having activity of converting to type jasmonic acid (d) Protein coated with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 (e) 90% or more identity with the base sequence represented by SEQ ID NO: 1 Encoded by a polynucleotide having a nucleotide sequence having an activity and having an activity of converting active jasmonic acid to inactive jasmonic acid Protein (f) encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or a complementary nucleotide sequence, and converts activated jasmonic acid into inactive jasmon Protein having activity to convert into acid (8) The plant according to any one of (1) to (7), wherein expression of the second gene is enhanced.
(9) The plant according to (8), wherein expression of at least the Os04g0584800 gene is enhanced.
(10) The plant according to any one of (1) to (9), wherein the second gene encodes any one of the following proteins (a) to (f).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 26 (b) comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 26; A protein having an activity of enhancing the resistance to salt stress (c) comprising the amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 26, and being enhanced in the plant A protein having an activity of enhancing resistance to salt stress (d) and a protein coated with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 25 (e) 90% of the base sequence represented by SEQ ID NO: 25 It is encoded by a polynucleotide comprising a base sequence having the above identity and is enhanced in a plant body. A protein having an activity of enhancing resistance to salt stress (f) encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the base sequence represented by SEQ ID NO: 25 or a complementary base sequence The plant according to any one of (1) to (10), which is a protein (11) dicotyledonous having an activity of enhancing resistance to salt stress when enhanced in the plant .
(12) The plant according to (11), which is soybean.
(13) The plant according to any one of (1) to (10), which is a monocotyledonous plant.
(14) The plant according to (13), which is a gramineous plant.
(15) The plant according to (14), which is rice.
(16) The plant according to (14), which is a sugarcane.
(17) The plant according to (14), which is corn.
(18) An expression vector for imparting environmental stress resistance to a plant body,
Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes and Os04g0584800 gene and this gene functionally A vector comprising one or more genes selected from a gene group consisting of a second gene subgroup consisting of equivalent genes.
(19) A transformant comprising the expression vector according to (18).
(20) A transformed plant comprising the expression vector according to (18).
(21) a first group of genes consisting of the Os12g0150200 gene, the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, the AT3G48520 gene and the AT2G27690 gene, and a gene functionally equivalent to any of these genes, the Os04g0584800 gene and the gene A step of enhancing an endogenous or exogenous gene that is one or more genes selected from a gene group consisting of a subgroup of second genes consisting of functionally equivalent genes,
A method for imparting environmental stress tolerance to a plant body.
(22) a first group of genes consisting of the Os12g0150200 gene, the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, the AT3G48520 gene, the AT2G27690 gene, and a gene functionally equivalent to any of these genes, the Os04g0584800 gene and this gene A step of enhancing an endogenous or exogenous gene that is one or more genes selected from a gene group consisting of a subgroup of second genes consisting of functionally equivalent genes,
A method for producing a plant body.
(23) The first gene subgroup comprising the Os12g0150200 gene, the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, the AT3G48520 gene and the AT2G27690 gene, and a gene functionally equivalent to any of these genes, the Os04g0584800 gene, and this gene and function Selecting plants by mating using as an index the expression of one or more genes selected from a gene group consisting of a second gene subgroup consisting of genetically equivalent genes,
A method for producing a plant body.
(24) A method for producing crops,
A method comprising: cultivating a crop which is a plant according to any one of (1) to (17).
(25) Os12g0150200 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene, and a first group of genes functionally equivalent to any of these genes, Os04g0584800 gene and this gene Screening one or more plants using as an index the expression of one or more genes selected from a gene group consisting of a second gene subgroup consisting of functionally equivalent genes; ,
Evaluating the salt tolerance of the plant body having a high expression level of the one or more genes,
A plant body screening method comprising:

It is a figure which shows the molecular phylogenetic tree (top) and amino acid sequence alignment (bottom) of CYP94C1 (AT2g27690) of Arabidopsis thaliana and a rice homolog. It is a figure which shows alignment of the amino acid sequence of CYP94C1 (AT2g27690) of Arabidopsis thaliana and Os12g0150200 which is a homologue of rice. It is a figure which shows the salt tolerance evaluation result of various rice full length cDNA overexpression (FOX) rice type | system | groups. It is a figure which shows the salt tolerance evaluation result of a wild type, a CYP94C2b FOX rice strain | stump | stock (system | strain name: FE047), and three CYP94C2b overexpression rice lines produced separately. It is a figure which shows the salt tolerance evaluation result (damage degree) of a wild type and CYP94C2b overexpression type | system | group rice (FE047). It is a figure which shows the salt tolerance evaluation result (survival rate) of a wild type and CYP94C2b overexpression type | system | group rice (FE047). It is a figure which shows the salt tolerance evaluation result (fruit number) of a wild type and CYP94C2b overexpression type | system | group rice (FE047). It is a figure which shows the salt tolerance evaluation result (appearance) of CYP94C2b overexpression type | system | group rice (FE047). It is a figure which shows the change of the accumulation amount of the jasmonic acid induced | guided | derived after a wound process, an active jasmonic acid, and an inactive jasmonic acid. It is a figure which shows the response (shoot length) with respect to jasmonic acid of a wild type and FE047 system | strain. It is a figure which shows the response (root elongation) with respect to jasmonic acid of a wild type and FE047 strain | stump | stock. It is a figure which shows the response (shoot length) with respect to coronatine (COR) of a wild type and FE047 strain | stump | stock. It is a figure which shows the response (root elongation) with respect to coronatine (COR) of a wild type and FE047 strain | stump | stock. It is a figure which shows the expression level of the JA responsive gene (JAmyb) after the wound process to a wild type and FE047 strain | stump | stock. It is a figure which shows the expression level of JA responsive gene (JAZ11) after the wound process to a wild type and FE047 strain | stump | stock. It is a figure which shows the evaluation result of the senescence (senescence, external appearance) of the leaf induced | guided | derived to a wild type and the FE047 strain | stump | stock by salt stress. The degree of leaf yellowing (senescence) induced in wild-type and FE047 lines by salt stress was evaluated by leaf color (green: dark gray, yellow-green: gray, yellow to brown: light gray). It is a figure which shows a result. It is a figure which shows the evaluation result of the yellowing of the leaf induced | guided | derived to a wild type and FE047 strain | stump | stock by salt stress (Senescence, upper: aging stress marker gene (SGR gene), lower: cytokinin responsive marker gene (OsRR10 gene)). . It is a figure which shows the expression level of CYP94C2b and salt tolerance evaluation in a wild type, FE047 line | wire (T2 generation), and the CYP94C2b overexpression rice line | wire produced separately. As for individual viability, + indicates survival and-indicates death. It is a figure which shows the expression level (relationship between ranking of an expression level, and survival rate) of the expression level of CYP94C2b in a wild type, FE047 line | wire (T2), and an independent overexpression line | wire. It is a figure which shows the identification result of the rice gene which provides high salt tolerance by overexpression of cDNA. It is a figure which shows provision of salt tolerance by overexpression of Os12g0150200 gene and Os04g0584800 gene. It is a figure which shows the provision of high temperature stress tolerance by overexpression of Os12g0150200 gene and Os04g0584800 gene. It is a figure which shows the provision of hyperosmotic stress tolerance and ion stress tolerance by overexpression of the Os04g0584800 gene. It is a figure which shows the expression level (C) of the Os12g0150200 gene and the result of the salt tolerance test about a standard rice variety (Nipponbare) and a salt tolerance rice variety (Heitai, Pokkali).

  The disclosure of the present specification relates to a gene that imparts environmental stress tolerance by enhancing its expression and use thereof. The present disclosure is based on the successful identification of genes that contribute to new salt stress tolerance by enhancing their expression.

  The present inventors paid attention to problems in the evaluation method in evaluating environmental stress tolerance. That is, based on two parallel evaluation methods, a gene that can impart excellent environmental stress tolerance with higher accuracy at a practical level was searched. As a result, we found a gene that does not show clear tolerance at the experimental level, but exhibits higher or diverse environmental stress tolerance at the practical level.

  In addition, any of the genes identified in the present disclosure can exhibit resistance to two or more kinds of environmental stresses, and can easily cope with complex environmental stresses that can occur in the natural environment. Was also found. With these genes, two or more types of environmental stress tolerance can be conferred by high expression of one kind of gene, so that appropriate stress tolerance genes can be selected and introduced and overexpressed in plants sensitive to multiple stresses. Thus, it is possible to create a plant body that is excellent in environmental stress tolerance.

  In addition, the present inventors have confirmed that the expression level of the gene identified in the present disclosure, for example, the gene identified by Os12g0150200 is enhanced even under non-stress conditions of existing salt-tolerant rice varieties. did. That is, the gene specified by the present inventors can avoid or suppress the adverse effect on the plant body due to the enhanced expression thereof, and can create a plant body excellent in environmental stress resistance such as salt tolerance.

  In this specification, the expression of a gene is enhanced that the expression level of the gene is increased and the protein encoded by the gene is enhanced (for example, the protein amount is increased or the protein is increased). Improved activity). Therefore, in addition to introducing and enhancing a specific gene as an exogenous gene, the expression regulatory region such as the promoter of the endogenous gene may be modified to enhance the expression of the endogenous gene by the expression regulatory gene. .

  Hereinafter, with respect to the disclosure of the present specification, genes, expression vectors, plants, plant production methods, crop production methods, and the like that impart environmental stress tolerance will be described in order.

(Gene that can confer environmental stress tolerance)
The gene capable of imparting environmental stress tolerance is a gene identified by Os12g0150200 (herein simply referred to as Os12g0150200 gene), Os05g0445100 gene, Os11g0151400 gene, Os01g0858350 gene, AT3G48520 gene and AT2G27690 gene and any one of these genes One or two selected from the gene group consisting of the first gene subgroup consisting of genes functionally equivalent to the gene, the Os04g0584800 gene and the second gene subgroup consisting of genes functionally equivalent to this gene More than a species of gene. Hereinafter, the first gene subgroup and the second gene subgroup will be sequentially described.

(First gene subgroup and constituent genes (first gene))
The first gene can be selected from the first gene subgroup consisting of the Os12g0150200 gene, the Os05g0445100 gene, the Os11g0151400 gene, the Os01g0858350 gene, the AT3G48520 gene and the AT2G27690 gene, and genes functionally equivalent to any of these genes. The first gene subgroup encodes a protein having jasmonic acid inactivating activity, as described below.

  Each gene product of the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, the Os12g0150200 gene, the AT3G48520 gene, and the AT2G27690 gene is presumed to belong to the CYP94 family that is the cytochrome P450 enzyme family. More specifically, as shown in the molecular phylogenetic tree and alignment of FIG. 1, the Os01g0858350, Os05g0445100, Os11g0151400, and Os12g0150200 genes are the Arabidopsis CYP94C1 protein encoded by AT2G27690 and its rice (Oryza sativa). It is thought that it is a homologue (ortholog). Os01g0858350, Os05g0445100, Os11g0151400, and Os12g0150200 are considered to be homologs (paralogs).

  The molecular phylogenetic tree and alignment shown in FIG. 1 are obtained by comparing CLUSTALW (Multiple Sequence Alignment; http: //www.Multiple Sequence Alignment; http: // www. Genome.jp/tools/clustalw/).

  In addition, the protein encoded by AT3G48520 is also estimated to belong to the CYP94 family, which is the cytochrome P450 enzyme family.

  CYP94C1 protein of Arabidopsis thaliana is activated jasmonic acid, that is, 12-hydroxy-7-isojasmono acid obtained as an inactive jasmonic acid by introducing a hydroxyl group into the 12-position carbon of 7-isojasmonoylisoleucine. It is considered that it has an activity to oxidize ylisoleucine and convert it to 12-carboxyl-7-isojasmonoylisoleucine (hereinafter also referred to as jasmonic acid inactivating activity) (Heitz, T., Widemann, E., Lugan, R., Miesch, L., Ullmann, P., Desaubry, L. et al. (2012), J. Biol. Chem. 287: 6296-6306, Kitaoka, N., Matsubara, T. , Sato, M., Takahashi, K., Wakuta, S., Kawaide, H. et al. (2011), Plant Cell Physiol. 52: 1757-1765, Koo, AJ, Cooke, TF and Howe, GA (2011 ), Proc. Natl. Acad. Sci. US A. 108: 9298-9303).

  In addition, the present inventors have confirmed that in the transformant with enhanced Os12g0150200, jasmonic acid and its active form induced by wound treatment are reduced and the inactive form is increased. Furthermore, as shown in FIG. 1, according to the alignment of the amino acid sequence of AT2G27690 with the amino acid sequences of Os01g0858350, Os05g0445100, Os11g0151400, and Os12g0150200, the substrate binding site (SRS), heme-binding region, and ERR triad in Arabidopsis CYP94C1 protein Sites, oxygen binding sites, and activation sites are all preserved. Therefore, it can be said that the proteins encoded by these genes also have jasmonic acid inactivating activity.

  Moreover, it is already known that the product of Arabidopsis AT3G48520 also has jasmonic acid inactivating activity.

  From the above, it is considered that any of the first genes encodes a protein (enzyme) having an activity of converting active jasmonic acid to inactive jasmonic acid.

  In addition, the base sequence of each coding region of the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, and the Os12g0150200 gene in the first gene has 54% to 58% identity to the base sequence of the coding region of the AT2G27690 gene. doing. The amino acid sequence encoded by the base sequence of each coding region of the Os01g0858350 gene, the Os05g0445100 gene, the Os11g0151400 gene, and the Os12g0150200 gene of the first gene is Have 53% to 57% identity.

  The first gene also includes a gene functionally equivalent to the gene specified above. Such functionally equivalent genes include homologs (paralogs, orthologs) of the genes specified above, and include genes encoding proteins having jasmonic acid inactivating activity, regardless of their origin. it can. The first gene is preferably derived mainly from the plant kingdom. It may be derived from a dicotyledonous plant including legumes such as soybean, and a monocotyledonous plant including a grassy plant including rice, corn, sugarcane and the like.

  For example, as a gene functionally equivalent to a specific gene, the nucleotide sequence of the gene or the relevant gene can be obtained using a database such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). A search is performed using the amino acid sequence of the protein encoded by the gene as a query sequence, and whether the gene having high identity is functionally equivalent to the gene. For example, in the present disclosure, the gene is activated. It can be obtained by evaluating whether or not it encodes a protein (enzyme) having an activity of converting jasmonic acid into inactive jasmonic acid.

  The first gene is appropriately selected according to the plant to be transformed. For example, genes functionally equivalent to the Os12g0150200 gene in soybean include a gene encoding cytochrome P450 94A1-like [Glycine max] (accession number, base sequence: XM — 006592251.1 (SEQ ID NO: 13), amino acid sequence: XP — 006592314 .1 (SEQ ID NO: 14)), a gene encoding cytochrome P450 94A2-like [Glycine max] (accession number, base sequence: XM_003538086.2 (SEQ ID NO: 15), amino acid sequence: XP_003538134.1 (SEQ ID NO: 16) ) And a gene encoding cytochrome P450 94A1-like [Glycine max] (accession number, base sequence: XM — 006577004.1 (SEQ ID NO: 17), amino acid sequence: XP — 006577067.1 (SEQ ID NO: 18)). Each of the above three genes has an identity with the amino acid sequence of the protein encoded by the Os12g0150200 gene and an identity with the amino acid sequence of the CYP94C1 protein of Arabidopsis thaliana, which is a protein encoded by the AT2G27690 gene, respectively. They were 63.4%, 57.8% / 59.9%, and 53.8% / 57.8%.

  In addition, for example, in maize, a gene functionally equivalent to the Os12g0150200 gene includes a gene encoding a protein with unknown function (accession number, base sequence: BT086294.1 (SEQ ID NO: 19)), amino acid sequence: ACR36647.1 ( SEQ ID NO: 20)), a gene encoding cytochrome P450 CYP94C20 (accession number, nucleotide sequence: EU956091.1 (SEQ ID NO: 21), amino acid sequence: ACG28209.1 (SEQ ID NO: 22)) and a gene encoding cytochrome P450 CYP94D27 (Accession number, base sequence: EU975752.1 (SEQ ID NO: 23), amino acid sequence: ACG47870.1 (SEQ ID NO: 24)) and the like. The above three genes have the same identity with the amino acid sequence of the protein encoded by the Os12g0150200 gene and the amino acid sequence of the CYP94C1 protein of Arabidopsis thaliana, which is a protein encoded by AT2G27690, respectively. They were 9%, 82.1% / 54.3%, and 43.8% / 42.6%.

  As long as the first gene encodes a protein having jasmonic acid inactivating activity as described above, it may be prepared from nature or artificially prepared. Therefore, in addition to the various genes described above, the gene may be artificially introduced with mutations. In addition to genomic DNA, the gene may be cDNA or the like.

The first gene can also be specified by a protein having the coding region of each gene and / or the amino acid sequence encoded by the coding region. The protein encoded by the first gene can be obtained from HP, such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). You can obtain it by accessing The protein encoded by the first gene is preferably derived mainly from the plant kingdom. It may be derived from a dicotyledonous plant or a monocotyledonous plant, and in particular may be derived from a gramineous plant. Information on such genes and proteins can be appropriately obtained by those skilled in the art by accessing HP such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). Hereinafter, the protein encoded by the first gene (hereinafter also referred to as the first protein) will be described.

  One embodiment of the first protein includes a protein comprising the amino acid sequence represented by SEQ ID NO: 2 encoded by the Os12g0150200 gene. Moreover, as long as it has jasmonic acid inactivating activity, other aspects of the present protein are consistent with known sequence information such as SEQ ID NO: 1 and SEQ ID NO: 1, 2 which are the base sequences of the coding region of the Os12g0150200 gene. A protein having the following relationship may be used.

  In another embodiment of the first protein, the amino acid sequence represented by SEQ ID NO: 2 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted, A protein having an activating activity may be mentioned.

  The amino acid sequence represented by SEQ ID NO: 2 (Os12g0150200) has two substrate recognition sites (SRS1 (21 amino acids) and SRS4 (20 amino acids)) as shown in FIG. 1B.

  The jasmonic acid inactivating activity can be obtained by detecting the catalytic activity of a reaction for producing inactive jasmonic acid using active jasmonic acid as a substrate. In addition, as long as it has the said activity, it does not ask | require that it has the said jasmonic acid inactivation activity.

  The amino acid mutation relative to the amino acid sequence represented by SEQ ID NO: 2 may be any one of deletion, substitution, addition and insertion, or two or more may be combined. The total number of these mutations is not particularly limited, but is preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less. More preferably, it is 1 or more and 5 or less. More preferably, they are 1 or more and 4 or less, More preferably, they are 1 or more and 3 or less.

  As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups. (Glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

  In addition, it is preferable that the variation | mutation with respect to the amino acid sequence represented by sequence number 2 exists other than a substrate identification site | part in the amino acid sequence represented by sequence number 2. In other words, even a mutant preferably has high identity with AT2G27690 at the two substrate recognition sites. That is, the identity of the amino acid sequence at each substrate recognition site in FIG. 1B is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and even more preferably 95% or more. is there. In addition, with respect to the amino acid sequence portion (17 amino acids for SRS1, 18 amino acids for SRS4) represented by the same amino acid residue between Os12g0150200 and AT2G27690 at each substrate recognition site, 1 to 4 amino acids by conservative substitution It is preferable that 1-2 amino acids are amino acids by conservative substitution, more preferably all are the same.

  Another embodiment of the first protein includes a protein having an amino acid sequence having 60% or more identity to the amino acid sequence represented by SEQ ID NO: 2 and having jasmonic acid inactivating activity. It is done. The identity is preferably 70% or more, more preferably 80% or more, still more preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, Preferably it is 98% or more.

  As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. “Identity” in the art refers to a protein or polynucleotide sequence as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance between. Similarity is also the correlation between protein or polynucleotide sequences as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as Similarity in the BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  Note that the amino acid sequence represented by SEQ ID NO: 2 or the base sequence encoding the amino acid sequence having a certain relationship with the amino acid sequence as described above does not change the amino acid sequence of the protein according to the degeneracy of the genetic code At least one base of a base sequence encoding a predetermined amino acid sequence can be substituted with another kind of base. Therefore, this gene also includes a gene encoding a base sequence converted by substitution based on the degeneracy of the genetic code.

  The first protein is also a protein encoded by a polynucleotide comprising the base sequence represented by SEQ ID NO: 1. In yet another embodiment, jasmonic acid inactivation is encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1. Examples include proteins having activity.

  The stringent condition refers to, for example, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, the nucleic acid having high nucleotide sequence identity, that is, 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 85% or more, and still more preferably 90% with the nucleotide sequence represented by SEQ ID NO: 1. % Or more, more preferably 95% or more, and most preferably 98% or more DNA complementary strands consisting of nucleotide sequences hybridize, and nucleic acid complementary strands with lower homology do not hybridize. It is done. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Furthermore, under stringent conditions, the filter washing conditions after hybridization are usually such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC.

  More specific stringent conditions include, for example, hybridization at 45 ° C. and 6 × SSC (sodium chloride / sodium citrate), followed by 50 to 65 ° C., 0.2 to 1 × SSC, and 0.1% SDS. Washing can be mentioned, or as such conditions, hybridization at 65 to 70 ° C. and 1 × SSC, and subsequent washing at 65 to 70 ° C. and 0.3 × SSC can be mentioned. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

  From the above, as yet another embodiment, the base sequence represented by SEQ ID NO: 1 is 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, A protein encoded by a polynucleotide having a base sequence having an identity of preferably 95% or more, most preferably 98% or more, and having jasmonic acid inactivating activity is exemplified.

  Furthermore, the various aspects of the first protein in the same form as described for Os12g0150200 are applied to other first genes, namely, Os05g0445100 gene, Os11g0151400 gene, Os01g0858350 gene, AT3G48520 gene, AT2G27690 gene, and the like. Thereby, further aspects of the first gene are identified. The base sequence and amino acid sequence of the coding region of each of the Os05g0445100 gene, Os11g0151400 gene, Os01g0858350 gene, AT3G48520 gene and AT2G27690 gene are SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, 9 and 10 and SEQ ID NOS: 11 and 12.

  The first gene encoding the first protein of the various embodiments is, for example, DNA extracted from gramineous plants using primers designed based on the sequence of SEQ ID NO: 1 or the like, various cDNA libraries or genomic DNA A nucleic acid fragment can be obtained by performing PCR amplification using a nucleic acid derived from a library or the like as a template. Further, it can be obtained as a nucleic acid fragment by performing hybridization using a nucleic acid derived from the above library or the like as a template and a DNA fragment which is a part of this gene as a probe. Alternatively, this gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  In addition, the first gene encoding the first protein of the various aspects described above is, for example, DNA encoding the amino acid sequence represented by SEQ ID NO: 2 (for example, comprising the base sequence represented by SEQ ID NO: 1) Can be obtained by modification by a conventional mutagenesis method, site-directed mutagenesis method, molecular evolution method using error-prone PCR, or the like. Examples of such methods include known methods such as Kunkel method or Gapped duplex method, or similar methods. For example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant-K (TAKARA Bio) Or Mutant-G (manufactured by TAKARA Bio)), or the LA PCR in vitro Mutagenesis series kit from TAKARA Bio.

  In addition, those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, for example, based on a known sequence such as SEQ ID NO: 1 or 2, it is possible to obtain the first gene encoding the first protein of various aspects.

(Second gene subgroup and constituent genes (second gene))
The second gene can be selected from the second gene subgroup consisting of the Os04g0584800 gene and genes functionally equivalent to this gene. Although the function of the protein encoded by the Os04g0584800 gene is unknown, it is a protein having a RAS / GTP binding domain on the N-terminal side and an Adaptin binding domain (SEQ ID NO: 27) on the C-terminal side. A gene functionally equivalent to the Os04g0584800 gene is a protein having a RAS / GTP binding domain on the N-terminal side and an Adaptin binding domain on the C-terminal side, and has a certain identity or more with the amino acid sequence of the Os04g0584800 gene. A gene encoding the protein possessed.

  In addition to the above homologues, artificially introduced mutations may be used. In addition to the genomic DNA, the gene may be cDNA or the like.

  A gene functionally equivalent to the Os04g0584800 gene is a gene that can enhance the tolerance of a plant to salt stress when expression of the gene is enhanced. In other words, it is a gene that encodes a protein having an activity of enhancing the tolerance of a plant to salt stress when expression of the gene is enhanced.

  Examples of genes functionally equivalent to the Os04g0584800 gene include the Arabidopsis AT5G65960 gene, the European grape LOC100266179 gene, the poplar cottonwood POPTR_0002s17770g gene, and the soybean LOC100813911 [Glycine max] gene. Furthermore, a gene encoding a barley predicted protein [Hordeum vulgare subsp. Vulgare] gene can be mentioned.

  The base sequence of the coding region of the gene encoding the second gene, barley predicted protein, has 83% identity to the base sequence of the coding region of Os04g0584800. Monocotyledonous plants have a base sequence with such high identity, but genes of other plant species show 57-64% identity to the base sequence of the coding region of Os04g0584800. In addition, the amino acid sequence encoded by the base sequence of the coding region contained in these genes has 48% to 77% identity to the amino acid sequence encoded by the base sequence of the coding region of Os04g0584800.

  Similar to the first gene, the second gene can be identified by a protein having a coding region of each gene and / or an amino acid sequence encoded by the coding region. The second gene may be prepared from nature or artificially prepared as long as it encodes a protein having an activity of enhancing resistance to salt stress when enhanced in a plant.

  As the second gene, the base sequence of the coding region of the Os04g0584800 gene and the amino acid sequence of the protein to be coated are represented by SEQ ID NO: 25 (NM_001060207) and SEQ ID NO: 26 (NP_001053672), respectively. Similarly, Arabidopsis AT5G65960 is SEQ ID NO: 28, 29 (NM_125993, NP_569023), European grape LOC100266179 is SEQ ID NO: 30, 31 (XP_002276437, XM_002276401), respectively, and POPTR_0002s17770g of Cottonwood, which is a kind of poplar, is arranged. Nos. 32 and 33 (XP_002302663, XM_002302627), genes encoding barley predicted protein [Hordeum vulgare subsp. Vulgare] are represented by SEQ ID NOs: 34 and 35 (BAJ96675, AK365472), respectively, and LOC100813911 [Glycine max] of soybean is Are represented by SEQ ID NOs: 36 and 37 (XP_003534231, XM_003534183), respectively.

  The second gene can adopt various aspects described in the first gene according to the protein encoded by the second gene.

  As used herein, “the activity to enhance the tolerance to salt stress when enhanced in a plant” means, for example, that the second gene was enhanced relative to a wild-type plant as disclosed in the Examples. Occasionally, it refers to an activity that enhances resistance to salt stress at least as compared to wild-type plants. Here, the wild-type plant can typically be Osa sativa Nipponbare. The second gene can be introduced into plants via Agrobacterium to enhance expression. A promoter that controls the expression of the first and second genes is preferably a constitutive promoter. Note that the expression level of the first gene is preferably appropriately adjusted as necessary. Furthermore, tolerance to salt stress can typically be evaluated using an evaluation method disclosed in the examples as an evaluation at a practical level.

  For example, the expression level of the first gene such as the Os12g0150200 gene can be set to an expression level of 5 to 150 times the expression level in the wild type of the plant (for example, Nipponbare corresponds to rice). Such an expression level may be 10 times or more, 20 times or more, or 30 times or more. Further, the expression level may be 100 times or less, 80 times or less, 70 times or less, 60 times or less, or 50 times or less. It may be. The expression level can be evaluated by a known method, for example, the amount of mRNA that is the expression product of the first gene.

  Moreover, it is preferable that the protein encoded by the second gene is mainly derived from the plant kingdom, like the first gene. It may be derived from a dicotyledonous plant or a monocotyledonous plant, and in particular may be derived from a gramineous plant. Information on such genes and proteins can be appropriately obtained by those skilled in the art by accessing HP such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov).

  In order to impart or enhance environmental stress tolerance to a plant body, these first gene and / or second gene may be used. It may be only the first gene or only the second gene. One or more of the first genes can be used, and one or more of the second genes can also be used.

  The first gene can confer good tolerance to salt stress (the stress tolerance is described below with reference to the wild type of Nipponbare of Japanese rice O. sativa). In particular, the first gene can confer high tolerance to salt stress. Therefore, it is preferable to apply the first gene to a plant body in which a high salt stress is predicted. Furthermore, the first gene can confer good resistance to high temperature stress. Therefore, it is preferable to apply the first gene to a plant body in an environment where both salt stress and high-temperature stress are predicted.

  On the other hand, the second gene can confer good resistance to salt stress. Furthermore, the second gene can also confer good resistance to ion stress. Therefore, it is preferable to apply the second gene to a plant in which salt stress and ion stress are predicted. Furthermore, the second gene can impart good resistance to high temperature stress and / or hyperosmotic stress. Therefore, the second gene is preferably applied to a plant body in an environment where high temperature stress and / or high osmotic pressure stress is predicted in addition to salt stress and / or ion stress.

In addition, in this specification, salt stress means the salt stress evaluated in the salt water stress tolerance test (closed greenhouse) similar to the salt tolerance test of International Rice Research Institute (IRRI). That is, it refers to salt stress evaluated by conducting the following hydroponic cultivation test based on the description of Characterizing the Saltol Quantitative Trait Locus for Salinity Tolerance in Rice. Rice 3: 148-160. The germinated seedlings are floated on a netted float and grown in pure water for 3 days, followed by hydroponics in Yoshida medium. The electrical conductivity (EC) of the hydroponic medium is adjusted to 6 dS m ^-1 for the first 3 days and then adjusted to 12 dS m ^-1 by dissolving NaCl. At this time, the EC and pH of the medium are adjusted by adding water or NaOH every 2-3 days. Two weeks later, symptoms caused by salt stress are assessed using the assessment score of Thomson et al. (Rice 3: 148-160).

  In the present specification, the hyperosmotic stress refers to a plant body hydroponically cultivated with pure water for 3 days and with Yoshida medium for 15 days, a medium containing 26% polyethylene glycol (PEG4000) for 7 days and PEG4000. It can be evaluated by the survival rate when it is left in a non-culture medium for 4 days (it may be compared with the survival rate of the wild type under the same conditions).

  In this specification, the high temperature stress is a case where a plant body hydroponically cultivated at 28 ° C. for 3 days with pure water, 17 days with Yoshida medium, 7 days at 42 ° C., and 7 days after returning to 28 ° C. for 7 days. It can be evaluated by the survival rate (which may be compared with the survival rate of the wild type under the same conditions).

  In this specification, the ion stress was cut for 4 cm above the plant portion grown for 7 days in Murashige and Skoog (MS) medium, transplanted to MS medium containing 40 mM LiCl, and grown for 2 weeks. And the survival rate (which may be compared with the survival rate of the wild type under the same conditions).

  Both the first gene and the second gene can impart resistance to salt stress as well as resistance to further environmental stress by enhancing their expression in the plant body.

(Expression vector)
The expression vector disclosed herein can be used as an expression vector for expressing the first gene and / or the second gene in plant cells. The vector of the present disclosure can comprise a first gene and / or a second gene. This vector is intended to introduce this gene as exogenous DNA regardless of the presence or absence of the endogenous gene on the chromosome in the host cell (plant cell), resulting in enhanced expression of this gene. can do. It is not excluded that the present vector is intended to enhance the expression of the endogenous gene on the chromosome in plant cells by homologous recombination or the like.

  The plant cell is not particularly limited, and examples thereof include cells of Arabidopsis thaliana, soybean, rice, corn, potato, tobacco, etc., preferably monocotyledonous plants, and more preferably grasses. Examples of gramineous plants include rice, wheat, barley, corn, sorghum and sugar cane. In addition to cultured cells such as suspension cultured cells, plant cells include protoplasts and callus. Plant cells include shoot primordia, multi-buds, hairy roots and the like, as well as cells in plant bodies such as leaf sections.

  When this vector is intended to introduce and express this gene as exogenous DNA in a plant cell, it comprises a promoter that can be transcribed in the plant cell and this gene operably linked under the control of the promoter. be able to. Furthermore, a terminator containing a poly A addition signal can also be included. Examples of such a promoter include a promoter for constitutively or inducibly expressing the gene. Examples of promoters for constant expression include the cauliflower mosaic virus 35S RNA promoter (Odell et al. 1985 Nature 313: 810) and the rice actin 1 gene promoter (Zhang et al. 1991 Plant Cell 3: 1155), the promoter of corn polyubiquitin 1 gene (Cornejo et al. 1993 Plant Mol. Biol. 23: 567), and the like. In addition, promoters for inducibly expressing this gene may be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, viruses, low temperature, high temperature, drying, ultraviolet irradiation, and spraying of specific compounds. And promoters of known genes. Examples of such promoters include rice chitinase gene promoter (Xu et al. 1996 Plant Mol. Biol. 30: 387) and tobacco PR protein gene promoter (Ohshima et al. 1990 Plant Cell 2:95), Rice "lip19" promoter (Aguan et al. 1993 Mol. Gen. Genet. 240: 1), rice "hsp80" and "hsp72" promoters (Van Breusegem et al. 1994 Planta 193: 57) Arabidopsis thaliana "rab16" gene promoter (Mundy et al. 1990 Proc. Natl. Acad. Sci. USA 87: 1406), parsley chalcone synthase gene promoter (Schulze-Lefert et al. 1989 EMBO J. 8: 651), the promoter of corn alcohol dehydrogenase gene (Walker et al. 1987 Proc. Natl. Acad. Sci. USA 84: 6624).

  This vector may also be intended to produce the first protein and / or the second protein as a recombinant protein in host cells of cells such as E. coli, yeast, animal and plant cells, and insect cells. Good. In this case, this vector can be provided with this gene under the control of a promoter operable in an appropriate host cell.

This vector can be constructed by those skilled in the art using, for example, commercially available materials such as various plasmids known to those skilled in the art. For example, in addition to plasmids “pBI121”, “pBI221”, “pBI101” (all manufactured by Clontech), etc., a vector that expresses this gene in a plant cell can be constructed to produce a transformed plant body. it can.

  According to the disclosure of the present specification, host cells such as plant cells into which such an expression vector has been introduced are also provided. Moreover, the chemical | medical agent which improves the environmental stress tolerance of a plant body which uses a 1st gene and / or a 2nd gene as an active ingredient can also be provided. This drug may contain the present vector as an active ingredient as the first gene and / or the second gene.

(Plant)
The plant body disclosed in the present specification includes a transformed plant body and a plant body by crossing or mutation. The plant body disclosed in the present specification has enhanced expression of the first gene and / or the second gene. In the transformed plant body, the first gene and / or the second gene to be enhanced may vary depending on the form of the plant body, but may be a gene endogenous to the plant body or a foreign gene. May be. Both of these may be used.

  In addition, gene expression is enhanced when the expression level of the gene (the amount of the primary transcription product of the gene and the production amount of the protein encoded by the gene) is greater than that before transformation, or It means that the activity of the protein is increased from that before transformation. As a result of enhancing the expression of the gene, the expression level of the gene may increase and the activity of the protein itself may increase. In addition, the expression level of these genes can be evaluated by a known method, that is, the amount of the transcription product or translation product of these genes.

(Transformed plant)
The transformed plant body disclosed in the present specification has enhanced expression of the first gene and / or the second gene. In the transformed plant body, the first gene and / or the second gene to be enhanced may be a gene endogenous to the plant body or a foreign gene. Both of these may be used.

  Moreover, the aspect in which gene expression is enhanced in the transformed plant body is not particularly limited. For example, a mode in which a promoter operable in a plant cell and the present gene operably linked by the promoter are retained as a foreign DNA outside or on the chromosome of the plant cell. The gene linked to the promoter may be endogenous to the plant cell or foreign. In addition, in order to improve the activity of the promoter of the endogenous gene, an embodiment in which the entire promoter region or a part of the promoter region on the chromosome is replaced, and an embodiment in which the promoter region is replaced with the endogenous gene are also included.

  A transformed plant typically includes a plant cell into which the vector disclosed herein intended to introduce and express a gene into the plant cell.

The transformed plant body can be obtained by regenerating a plant body from a plant cell transformed by introducing the vector disclosed herein.

  For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used. For example, gene transfer to protoplasts using polyethylene glycol (Datta, SK (1995) In Gene Transfer To Plants (Potrykus, I. and Spangenberg, G. Eds.) Pp66-74), gene transfer to protoplasts using electric pulses (Toki et al (1992) Plant Physiol. 100: 1503-1507), gene transfer directly into cells by particle gun method (Christou et al. (1991) Bio / Technology, 9: 957-962) and gene via Agrobacterium (Hiei et al. (1994) Plant J. 6: 271-282; Toki et al. (2006) Plant J. 47: 969-976). In addition, regeneration of plant bodies from plant transformed cells can be performed by methods known to those skilled in the art depending on the type of plant cells (Toki et al. (1992) Plant Physiol. 100: 1503-1507). reference). For example, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1985)) can be cited for rice, and the method of Shillito et al. (Bio / Technology 7: 581 (1989)) or Gorden-Kamm for maize. (Plant Cell 2: 603 (1990)), for potatoes the method of Visser et al. (Theor. Appl. Genet. 78: 594 (1989)), for tobacco, Nagata and Takebe (Planta 99:12 (1971)), and for Arabidopsis, the method of Akama et al. (Plant Cell Rep. 12: 7-11 (1992)) is used.

  Plant regeneration from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cell (see Toki et al. (1992) Plant Physiol. 100: 1503-1507). . For example, a method for producing a transgenic plant of rice is a method of regenerating a plant (indian rice varieties are suitable) by introducing a gene into protoplasts using polyethylene glycol (PEG) (Datta, SK (1995) In Gene Transfer To Plants (Potrykus, I. and Spangenberg, G. Eds.) Pp66-74), a method for regenerating plants (Japanese rice varieties are suitable) by introducing genes into protoplasts by electric pulses (Toki et al. al. (1992) Plant Physiol. 100: 1503-1507), a method of regenerating plants by directly introducing genes into cells by the particle gun method (Christou et al. (1991) Bio / technology, 9: 957-962). .) And Agrobacterium-mediated gene transfer to regenerate plants (Hiei et al. (1994) Plant J. 6: 271-282; Toki et al. (2006) Plant J. 47: 969 -976) has already been established and is widely used in the technical field of the present invention. It has been used. In the present invention, these methods can be suitably used.

  If a transformed plant in which the present gene is integrated on the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The disclosure of the present specification includes (1) a plant cell into which the present gene has been introduced, (2) a plant containing the cell, (3) progeny and clones of the plant, and (4) The plant body, its progeny, and clonal propagation material are included.

  Note that the first gene and the second gene used for transformation can be derived from various sources, and are not limited to being derived from the same type of plant as the transformation target plant. Application beyond this is possible. However, when the target plant is a monocotyledonous plant, a gene derived from a monocotyledonous plant is preferable, and a gene derived from the same family or the same species is more preferable. The same applies to dicotyledonous plants.

(Plants by mating and mutation)
Another form of the plant disclosed in this specification is a plant by crossing or mutation. The plant body by crossing can hold | maintain a 1st gene and / or a 2nd gene in the locus | region corresponding to the locus on the original chromosome of these genes, or the said locus. Here, the original locus on the chromosome of the gene is a locus on the chromosome on which the gene sits when the plant body originally has the gene or a homologue thereof. In addition, the position corresponding to the locus on the original chromosome does not completely coincide with the locus, but from the base sequence before and after the gene, the position of the gene of the second DNA is located near the locus. The case where it is located without interfering with expression increasing activity is applicable. Such a plant body is preferably a plant body having the endogenous gene on the chromosome in advance.

  In general, a plant in which expression of the first gene and / or the second gene is enhanced by mating or mutation is used in the breeding process such as mating or mutation in the first gene and / or the second gene. Can be obtained by mutation such as polymorphism due to mutation, epigenetic mutation (including methylation) of coding region or control region, and the like. Alternatively, it can be obtained by using such a parent plant (which may be a transformant disclosed in the present specification).

  Plants with enhanced expression of the first gene and / or the second gene are screened from a normal breeding process using the expression level of the first gene and / or the second gene as an index, and are further resistant to environmental stress. Can be obtained by evaluating In the breeding process, whether the plant body has the first gene and / or the second gene is determined by performing PCR on the DNA region containing them and detecting the presence or absence of the amplified product, its length, etc. Can be confirmed. Whether such a DNA region is present at the locus can be confirmed by a known nucleotide sequencing method.

  For example, by crossing, for example, by the homologous recombination at the time of fertilization between the parent plant body in which the expression of the first gene and / or the second gene is enhanced and another plant body, The F1 generation carrying these genes can be obtained at a position on the chromosome or a position corresponding to the position. By using F2 generation, backcrossing, or the like, a homozygote can be obtained for the first gene and / or the allele (allele) of the second gene. The plant body may be heterogeneous with respect to the first and / or second gene and with respect to the allele including the present DNA region, but is preferably homozygous.

  The plant to which the first gene and / or the second gene disclosed in the present specification is applied, in other words, the plant that improves environmental stress tolerance is not particularly limited. That is, by enhancing the expression of the gene, it is possible to expect an effect of improving environmental stress tolerance for any plant. Examples of the target plant include dicotyledonous plants and monocotyledonous plants, for example, plants belonging to the family Brassicaceae, Gramineae, Eggplant, Legume, Willow, etc. (see below), but are limited to these plants. Is not to be done.

Brassicaceae: Arabidopsis thaliana, Brassica rapa, Brassica napus, Brassica campestris, Cabbage (Brassica oleracea var. Pekinensis), Chinese cabbage (Brassica rapa var. Pekinensis), Chingensai, Pakchoi varpa Turnip (Brassica rapa var. Rapa), Nozawana (Brassica rapa var. Hakabura), Mizuna (Brassica rapa var. Lancinifolia), Komatsuna (Brassica rapa var. Peruviridis), Japanese radish (Raphanus sativus), Wasabi (Wasabia japonica).
Solanum: Nicotiana tabacum, eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato (Solanum lycopersicum), capsicum (Capsicum annuum), petunia hybrida, etc.
Legumes: Soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), groundnut (Arachis hypogaea), Lotus corniculatus var. Japonicus, common bean (Phaseolus vulgaris), azuki bean (Phaseolus vulgaris) Vigna angularis), Acacia, etc.
Asteraceae: Chrysanthemum morifolium, sunflower (Helianthus annuus), etc.
Palms: oil palm (Elaeis guineensis, Elaeis oleifera), coconut (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia), etc.
Ursiaceae: Rhis succedanea, Cashew nutcrest (Anacardium occidentale), Urushi (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera), etc.
Cucurbitaceae: Pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), crow cucumber (Trichosanthes cucumeroides), gourd (Lagenaria siceraria var. Gourda), etc.
Rosaceae: Almond (Amygdalus communis), Rose (Rosa), Strawberry (Fragaria), Sakura (Prunus), Apple (Malus pumila var. Domestica), etc.
Dianthus: Carnation (Dianthus caryophyllus).
Willow: Poplar (Populus trichocarpa, Populus nigra, Populus tremula), etc.
Gramineae: corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), bamboo (Phyllostachys), sugar cane (Saccharum officinarum), napiergrass (Pennisetum purpureum), Elianus rav (Erianus rav) ), Miscanthus virgatum, Sorghum switch glass (Panicum), etc.
Lily family: Tulip (Tulipa), Lily (Lilium), etc.

  Of these, food crops such as gramineous and leguminous are preferred. These food crops can be cultivated and harvested stably without being affected by climate change, so that a stable food supply becomes possible. It is also preferable to target energy crops that can be used as raw materials for biofuels such as sugarcane, corn, rapeseed, and sunflower. By improving the environmental stress tolerance of an energy crop, the cultivation range and cultivation conditions of the energy crop can be greatly expanded. In other words, energy crops can be cultivated even under land and environmental factors that could not be cultivated in the wild type (for example, average temperature, salt concentration in soil, etc.). This is because the cost of biofuels such as biomethanol, bio DME, bio GTL (BTL) and biobutanol can be reduced.

  As described above, the plant body disclosed in the present specification can be obtained not only by artificial genetic modification but also by breeding by conventional mating. The plant body disclosed in the present specification has improved resistance to one or more environmental stresses and has resistance to environmental stresses at a more practical level.

(Method for imparting environmental stress resistance to plant body and method for producing plant body)
The method for imparting environmental stress tolerance to a plant and the method for producing a plant imparted with environmental stress tolerance disclosed in the present specification are the first gene and / or the second gene, which are endogenous or foreign. A step of enhancing the gene of As described above, this enhancement step can be performed by combining transformation and / or breeding processes such as mating and mutation with respect to the target plant. In particular, in producing a plant body by crossing, it is preferable to include a step of selecting a plant body by crossing using the expression of the first gene and / or the second gene as an index. In addition to resistance to stress by various evaluations, efficient breeding and production are possible by using the expression status of these genes as an index.

(Crop production method)
The crop production method disclosed in the present specification can include a step of cultivating a crop that is a plant disclosed in the present specification. According to this production method, various useful crops such as food crops and energy crops can be cultivated stably, avoiding or suppressing the adverse effects of climate change, even in the environment where various environmental stresses may occur in addition to salt stress. Can be harvested.

  The cultivation process in this production method can be carried out by appropriately selecting conditions and the like by those skilled in the art according to the type of crop.

(Plant screening method)
The plant screening method disclosed in the present specification provides an indication of the expression of one or more genes selected from a gene group consisting of a first gene subgroup and a second gene subgroup. And a step of screening one or more plants, and a step of evaluating the salt tolerance of the plant having a high expression level of the one or more genes. According to this screening method, by using the gene disclosed in the present specification, such as the Os12g0150200 gene, as an index, a plant body that may be excellent in salt tolerance can be easily primary screened. Furthermore, by evaluating the salt tolerance of a possible plant body, a plant body having excellent salt tolerance can be obtained. In particular, the above-mentioned genes including the Os12g0150200 gene tend to be highly expressed even under non-stress conditions (ie, non-stress conditions) such as high salt (NaCl) conditions, and are easily possible even under non-stress conditions. Sexual plants can be screened.

  In the screening process, the expression level of the above-mentioned gene such as Os12g0150200 gene is used as an index. For example, a general variety of the plant body, typically, a wild type commonly used in the plant body, a typical cultivation In addition to varieties, select plants with higher expression levels than model plants. The screening conditions may generally be high salt conditions used for salt tolerance evaluation, or may be normal conditions for salt concentration. Since the above-mentioned genes including the Os12g0150200 gene tend to be highly expressed even under salt stress conditions as described above, screening can also be performed under non-salt stress conditions.

  Although the plant body to be subjected to screening is not particularly limited, it can be a young plant body or a part thereof in consideration of screening efficiency. For example, a part of a leaf or the like of a young plant can be mentioned.

  Further, in order to screen using the expression of the above genes including the Os12g0150200 gene as an index, the amount of mRNA or protein that is the expression product of these genes can be used as an index. Methods for evaluating these are well known to those skilled in the art. In the evaluation of the gene expression level, as described above, the expression level of a gene of a wild type, a variety, or a variety to be modified in a plant to be screened can be used as a control. For example, Nipponbare can be used when the plant body is rice. For example, when Nipponbare is used as a control, a plant having an expression level of about several to 100 times, preferably about 10 to 100 times that of the control can be selected. In addition, for example, a plant having an expression level of about 10 times to 80 times can be selected. Furthermore, for example, the lower limit of the expression level may be 10 times or more, 20 times or more, or 30 times or more. The upper limit of the expression level may be 70 times or less, 60 times or less, and 50 times or less.

  For plants having a high expression level of the Os12g0150200 gene, salt tolerance under salt stress conditions can be further evaluated. By further evaluating the salt tolerance, it is possible to surely screen for plants having excellent salt tolerance. For the evaluation of salt tolerance, a known salt tolerance evaluation method as disclosed in Examples described later can be appropriately selected and used according to the purpose.

  Plants with excellent salt tolerance that can be screened in this way are useful per se. That is, in a coding region of the gene such as the Os12g0150200 gene in the plant body and a region involved in regulation of expression upstream and / or downstream of the coding region, a mutation or the like that enhances the expression of the gene such as the Os12g0150200 gene Other modifications may be provided. By identifying factors that contribute to the enhancement of the expression level of the above genes such as the Os12g0150200 gene, such as chromosomal mutations, etc., it is useful for the creation of plants with excellent salt tolerance. That is, a DNA region containing these factors can be used for improving salt tolerance in other varieties and plants. For applying these DNA regions to other plants and varieties, known plant breeding methods such as crossing and transformation can be used.

  As described above, the first gene and / or the second gene disclosed in the present specification can impart practical environmental stress resistance to a plant body.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to these Examples.

  Selection of salt-tolerant lines using rice full-length cDNA overexpression (FOX) rice lines (Nakamura et al. 2007; Hakata et al. 2010; Tsuchida-Mayama et al. 2010) distributed by the National Institute of Agrobiological Sciences Went. Murashige and Skoog (MS) prepared in mayonnaise bottles 13 cm in height after removing the rice husks of wild-type rice (variety: Nipponbare; Oryza sativa cv. Nipponbare) and selected FOX rice seeds. ) Placed on basic solid medium [containing 1% sucrose, 0.25% gellan gum, 0.05% MES-KOH (pH 5.8)], germinated and grown aseptically. The seedlings were grown under conditions of 25 ° C, 14 hours of light (4000 lux of white light)-10 hours of darkness. The above-ground base (4 cm length) of a one-week-old seedling was aseptically cut and transplanted to a new MS basic solid medium, and an equal volume of 600 mM NaCl aqueous solution (final concentration 300 mM) was added aseptically. . After growing for 5 weeks under this salt stress condition, the plants were soaked in tap water and acclimated for 1 week, and the survival rate was examined. The survival rate of each overexpression line is shown in FIG. 2A. Nipponbare: Wild type (Nihonbare).

  As shown in FIG. 2A, two lines out of many FOX rice lines exhibited good scores. The full-length cDNAs encoded by the genes Os12g0150200 and Os04g0584800 introduced into these two strains were identified by the method of literature (Nakamura et al. 2007; Hakata et al. 2010; Tsuchida-Mayama et al. 2010).

  The full-length cDNA (accession number AK064287 encoded by Os12g0150200) introduced into the FOX rice line that showed salt tolerance was amplified by PCR using the primers shown in Table 1, and pENTR / D-TOPO After subcloning into vector (Invitrogen), it was inserted downstream of the rice actin promoter of binary vector pSMAHdN637L-GateA. The resulting construct was used to transform rice (Nipponbare) via Agrobacterium to obtain a new Os12g0150200 cDNA overexpression line (BBC105 lineage group).

  A salt tolerance test similar to that described above was performed using a progeny seed of a wild type, Os12g0150200 cDNA overexpression line (FE047 line and BBC105 line group). After growing for 5 weeks under salt stress conditions, the plants were soaked in tap water and acclimated for 1 week, and the survival rate was examined. FIG. 2B shows the results obtained by testing the wild type, FE047, BBC105_2, BBC105_6, and BBC105_9. The number of test individuals (n) is 10 for WT and FE047, 4 for BBC105_2, and 5 for BBC105_6 and BBC105_9.

  As shown in FIG. 2B, the Os12g0150200 cDNA overexpression lines FE047, BBC105_2, BBC105_6, and BBC105_9 all exhibited high survival rates relative to the wild type.

  Next, wild type rice and the FE047 line were cultivated under salt treatment in a closed greenhouse, and their salt tolerance was compared.

The salt tolerance test and salt tolerance evaluation basically followed the method of Thomson et al. (2010). Rice seeds with rice husks treated with benrate were placed on moist filter paper and allowed to germinate by incubating at 30 ° C for 2 days in the dark. Young seedlings were transferred onto a float in a greenhouse and grown in deionized water for 3 days. Thereafter, 240 seedlings per float were grown on 10 liters of Yoshida medium. The electrical conductivity (EC) of the hydroponic solution was 6 dSm ^-1 for the first 3 days, and then 12 dSm ^-1 by adding sodium chloride. EC and pH were adjusted by adding water and NaOH every 2-3 days. Two weeks later, damage caused by salt stress was assessed by the salinity evaluation score by Thomson et al. (2010). The obtained score is shown in FIG. 2C. The box plot shows the interquartile range (IQR). Values outside the range of 1.5 × IQR are shown as Outliers outside the first and third quartiles. Whiskers indicates the range from the minimum value to the maximum value. Median values are shown as thick horizontal bars. n = 38 (WT), n = 20 (FE047).

  As shown in FIG. 2C, in the selected FE047 line, damage due to salt treatment was suppressed as compared to the wild type.

  Furthermore, wild-type rice and FE047 lines were grown in salt-damaged soil and the salt tolerance was compared. Four-week-old plants were transplanted and cultivated in a 23 cm diameter Wagner pot containing 10 liters of soil. The maximum number of transplanted seedlings was 8 per pot. The pot was placed in a tank and filled with water, and after the plant body was grown for 2 weeks, the water was replaced with 100 mM NaCl or 150 mM NaCl. EC and pH were adjusted by adding water and NaOH, respectively, every 2-3 days. The survival rate of rice in a salt-damaged soil experiment containing 100 mM NaCl and 150 mM NaCl is shown in FIG. 2D, and the number of seeds per individual is shown in FIG. 2E. n = 17 (WT), n = 4 (FE047, 100 mM), n = 3 (FE047, 150 mM). Photographs of plants of wild-type rice and FE047 lines 40 days after addition of 150 mM NaCl are shown in FIG. 2F. Shown in

  As shown in FIGS. 2D to 2F, the FE047 line showed better survival rate, fertility, and vigorous growth than the wild type. From the above, it was found that the Os12g0150200 cDNA overexpression line has extremely practical resistance to salt stress.

  In this example, the induction of accumulation of jasmonic acid (JA), active jasmonic acid (JA-Ile) and inactive jasmonic acid (12OH-JA-Ile) after wound treatment to wild type and FE047 strains was evaluated. .

Half of the third leaf blade of rice seedlings was cut and immediately frozen in liquid nitrogen. The wound was treated by pinching the remaining half of the third leaf blade with tweezers 20 times, and then the whole seedling was incubated at room temperature in a humid chamber, and the wound leaf was collected and frozen in liquid nitrogen. Using a Mini-BeadBeater-8 (BioSpec), the frozen leaf tissue was crushed in a microcentrifuge tube containing stainless beads. Extraction of JA and its derivatives from tissues with 99.5% ethanol was performed overnight at 4 ° C in the dark. The residue was removed by centrifugation (20,000 × g) at room temperature for 5 minutes, and the supernatant ethanol solution was stored at 4 ° C.
Before the measurement, the ethanol solution was dried using nitrogen gas, and then the sample was dissolved in water. The residue was further removed by centrifugation (15,000 × g) at room temperature for 20 minutes, and the supernatant fraction was used for measurement by UPLC / TOFMS. The amounts of JA, JA-Ile and 12OH-JA-Ile (average value ± standard deviation value; n ≧ 6) per living body weight of the leaf tissue before and after the wound treatment are shown in FIG. In the figure, Asterisks shows significant differences between the wild type and the FE047 line (** p-value <0.01; * p-value <0.05, Student's t-test).

  As shown in FIG. 3, in the wild type, the wound treatment increased JA and its active form with time, and the inactive form also slowly increased. In contrast, in the FE047 line, JA and its active form initially increased but then decreased. Moreover, the tendency which an inactive type increases with these fall was seen.

  From the above, it was considered that the protein encoded by Os12g0150200 has an activity to convert JA from an active form to an inactive form.

  In this example, the response of wild type and FE047 strains to JA and coronatine (COR) was evaluated. Note that, like activated jasmonic acid, COR is known to activate the JA response by binding to the COI1 receptor.

  After removing rice hulls from seeds of wild type and FE047 lines, surface sterilized, seeded in MS basic solid medium containing various concentrations of JA or COR prepared in 13 cm high mayonnaise bottles and grown aseptically I let you. FIG. 4A shows the shoot length of a 3-day-old seedling grown in the presence of JA, and FIG. 4C shows the shoot length of a 3-day-old seedling grown in the presence of COR. In addition, FIG. 4B shows root elongation from 2 to 3 days after sowing in the presence of JA, and FIG. 4D shows root elongation from 3 to 4 days after sowing in the presence of COR. Mean values ± standard deviation values (n> 4) are shown for each. A significant difference between the wild type and the FE047 strain is shown by Asterisks (** p-value <0.01; * p-value <0.05, Student's t-test).

  As shown in FIG. 4A and FIG. 4B, it was observed that the FE047 line had a smaller degree of suppression of shoot length and root elongation than the wild type in the presence of JA, and the JA response was weakened. In contrast, as shown in FIG. 4C and FIG. 4D, no difference was detected in the shoot length and root elongation between the wild type and the FE047 line in the presence of COR.

  In addition, the response to injury of wild-type and FE047 strains was examined using JAmyb and JAZ11 showing wound-inducible expression as gene markers. A 7-day-old seedling was pinched with tweezers 20 times for mechanical wound treatment, incubated for 16 hours in a humid chamber, and then the leaf tissue was frozen in liquid nitrogen and stored at -80 ° C. Total RNA was extracted and purified using RNeasy Plant Mini kit (Qiagen). Reverse transcription from RNA using QuantiTect Rev Transcription kit (Qiagen), and PCR of the resulting single-stranded cDNA using PrimeStar HS DNA polymerase (Takara Bio) and GeneAmp PCR System 9700 (Applied Biosystems) Alternatively, amplification was performed by real-time PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) and Mx3000P (Agilent). The primers used are shown in the table below. The expression level of JAmyb mRNA and JAZ11 mRNA was normalized by the expression level of UBC mRNA, and the expression level in the wound treated leaves was set as a relative value with respect to the level of untreated leaves. The results are shown in FIGS. 4E and 4F. The average value ± standard deviation value (n = 5) is shown. Statistical significance test was performed by multiple comparisons by Tukey-Kramer test. In the figure, a-c indicates a significant difference between groups (p-value <0.01).

  As shown in FIGS. 4E and 4F, expression of JA-responsive genes JAmyb and JAZ11 increased in the wild type by wound treatment, but both were strongly suppressed in the FE047 strain.

  From the above, it was found that the protein encoded by Os12g0150200 converts active JA into inactive form in response to JA, but does not inactivate coronatine. Based on the above results and the molecular phylogenetic tree and alignment based on the amino acid sequence of the protein encoded by Os12g0150200 shown in FIG. 1, it is considered that Os12g0150200 encodes a protein having an activity to convert active jasmonic acid into an inactive form. It was.

  In this example, yellowing of leaves was evaluated under the salt stress environment of wild type and FE047 lines.

  FIG. 5A shows a plant body after removing rice hulls from rice seeds and sterilizing the surface and then sown in an MS basic solid medium containing 300 mM NaCl prepared in a 13 cm high mayonnaise bottle and grown for 33 days. The left 5 individuals are wild type and the right 5 individuals are FE047. The bar is 2 cm.

  A similar experiment was performed using an MS basic solid medium containing 275 mM NaCl. The degree of leaf yellowing when each salt concentration was used was shown as a percentage of leaf color. The percentage of yellow or brown leaves (yellow box), the percentage of yellow-green leaves (light green box) and the percentage of green leaves (dark green box) are shown in FIG. 5B. The average value ± standard deviation value (n = 10) was used.

  Similarly, wild-type and FE047 lines were grown for 33 days using MS basic solid medium containing 250 mM NaCl, and yellowed leaves (mixed from 1st to 3rd leaves) and young leaves (4th and 5th leaves) ) Expression of the aging marker gene STAYGREEN (SGR) was investigated. The expression level of SGR was examined by qRT-PCR, normalized by the expression level of UBC, and expressed as a relative value to the expression level in the wild-type fourth leaf. The SGR gene encodes a rate-limiting enzyme that degrades chlorophyll. Expression analysis was performed in the same manner as in Example 3.

  The expression pattern of OsRR10, which is a cytokinin-responsive marker gene, was also displayed in the same manner. Cytokinin is a hormone that suppresses leaf aging.

  The results for the SGR marker are shown in FIG. 5C (top) and the results for the OsRR10 marker are shown in FIG. 5C (bottom). The average value ± standard deviation value is used. The leaves were numbered in order from the oldest.

  As shown in FIG. 5A and FIG. 5B, the FE047 line had fewer yellow leaves than the wild type under salt stress, and there was less growth inhibition. In addition, as shown in FIG. 5C, in the FE047 line, the expression of the senescence marker was greatly suppressed as compared to the wild type, but the activation of the cytokinin responsive marker serving as an index for the suppression of leaf senescence was not observed. It was.

  From the above, it was found that FE047 strain does not suppress yellowing by activating the function of cytokinin.

  In this example, CYP94C2b expression level and salt tolerance level using wild type, FE047 line (T2 generation), and the independent Os12g0150200 (CYP94C2b) cDNA overexpression line (BBC105 line group; T1) prepared in Example 1 were used. And evaluated.

  RNA was extracted from the leaves of 7-day-old seedlings cultivated under conditions not subjected to salt stress, and the expression level of CYP94C2b was quantified by qRT-PCR using the primers shown in Table 1. FIG. 6A shows the CYP94C2b expression level normalized by the expression level of 25S rRNA and expressed as a relative value to the average value of the wild type of 5 individuals. The presence or absence of a transgene (trangene; Os12g0150200) in the tested plant and the individual survival rate in the presence of salt stress similar to Example 1 are indicated by “+/−” below the graph. The presence or absence of the transgene was examined by PCR using the primers shown in Table 1 (for genotyping) and genomic DNA as a template. n. d. Indicates not detected.

  Furthermore, using the CYP94C2b expression level as an index, the rice of all 35 individuals in FIG. 6A was ranked. FIG. 6B shows the survival rate in the presence of salt stress for each rank (every 7 individuals) when the expression level is divided into five categories. Ranks 1 to 7 were individuals showing the lowest expression level, and included wild type (5 individuals) and individuals without the transgene (2 individuals). Conversely, individuals with ranks 29-35 showed the highest expression level, about 150 times higher than wild type or higher.

  From the above results, it is desirable that the expression level of CYP94C2b is higher than that of the wild type, which enhances environmental stress tolerance against salt stress and the like, but it is desirable that the degree of enhancement not exceed 150 times that of the wild type. all right. Preferably, it is 10 times or more and 150 times or less of the wild type, more preferably 10 times or more and 100 times or less.

  After inserting the cDNAs of the selected salt-tolerant lines of the transgenes Os12g0150200 and Os04g0584800 into the overexpression vector, they are introduced into Nipponbare and a new transformed line is created according to Example 1, and the salt tolerance test is performed using the next generation seeds. Went. Five individuals of each of the 4 lines independently for each gene were subjected to the test.

  MS basic solid medium [1% sucrose, 0.25% gellan gum, 0.05% MES-KOH (pH 5.8) prepared in a mayonnaise bottle with a height of 13 cm after surface sterilization of wild-type (Nipponbare) and transformed rice seeds ), And germinated and grown aseptically. The seedlings were grown under conditions of 25 ° C, 14 hours of light (4000 lux of white light), and 10 hours of darkness. The above-ground base (4 cm length) of a one-week-old seedling was cut out, transplanted to a new MS basic solid medium, and further a 600 mM NaCl aqueous solution (final concentration 300 mM) equivalent to the medium was aseptically added. The survival rate at the time of growing for 5 weeks under this salt stress condition was evaluated. Then, the plant body was immersed in tap water and acclimated for 1 week, and the survival rate was further evaluated. The results are shown in FIG. In addition, the survival rate at the time of salt stress is shown by a gray bar, and the survival rate at the time of subsequent non-salt stress is shown by a black bar.

  As shown in FIG. 7, the Os12g0150200-introduced line was able to confirm salt stress tolerance in all lines, and the Os04g0584800-introduced line was also found to exhibit salt stress tolerance.

  In this example, environmental stress tolerance under other conditions was evaluated for the Os12g0150200 overexpression line (FE047) and the Os04g0584800 overexpression line (CU099) prepared in Example 1.

(1) Salt stress tolerance test (closed greenhouse)
A hydroponic test similar to the salt tolerance test of the International Rice Research Institute (IRRI) was conducted (Thomson et al., 2010). After the germination treatment, NaCl was administered stepwise on the 3rd and 6th days, and the damage of the subsequent plants was scored. In the salt-tolerance test of closed greenhouses, Nipponbare and Kyudai Asahi No. 3 which is one of the salt-tolerant lines were used as comparative controls and evaluated using the evaluation scores described in the literature. Furthermore, the salt stress tolerance degree of each overexpression line was calculated by the formula of (Nipponbare evaluation score-Evaluation score of each overexpression line) / (Nipponbare evaluation score-Kyudai Asahi No.3 evaluation score). That is, the resistance value of Nipponbare was expressed as a relative value when the resistance level was “0” and the resistance level of Kyudai Asahi No.3 was “1”. The result is shown in FIG. 8A. In these graphs, plots indicate values obtained by repeated tests, and boxes indicate median values (the same applies to FIGS. 8B and 8C).

  As shown in FIG. 8A, the Os12g0150200 overexpressing line shows high salt stress tolerance in the salt-tolerance test in a closed greenhouse, and the Os04g0584800 overexpressing line is lower than the Os12g0150200 overexpressing line but has excellent salt stress tolerance. showed that.

(2) High-temperature stress tolerance test Rice seeds were hydroponically cultivated at 28 ° C for 3 days in ultrapure water and 17 days in Yoshida medium. The obtained plant body was cultivated for 7 days at 42 ° C and returned to 28 ° C for 7 days, and the survival rate was compared with that of the wild type. The resistance to high temperature stress imparted by overexpression of each gene was expressed as a value obtained by subtracting the survival rate (0.44 to 0.75) of the wild type (Nipponbare) from the survival rate of each overexpression line. The result is shown in FIG. 8B.

  As shown in FIG. 8B, it was found that both the Os12g0150200 overexpressing line and the Os04g0584800 overexpressing line have higher temperature stress tolerance than the wild type.

(3) High osmotic stress tolerance test
The Os04g0584800 overexpression line (CU099) and wild-type rice seeds were hydroponically cultivated in ultrapure water for 3 days and in Yoshida medium for 15 days. The obtained plant was cultivated in a medium containing 26% polyethylene glycol (PEG: average molecular weight 4,000) for 7 days and further in a medium not containing PEG for 4 days, and the survival rate was compared. The degree of hyperosmotic stress tolerance given by overexpression of the Os04g0584800 gene was expressed as a value obtained by subtracting the survival rate (0.21 to 0.69) of the wild type (Nipponbare) from the survival rate of the Os04g0584800 overexpression line (CU099). . The result is shown in FIG. 8C.

(4) Ion stress tolerance test
Os04g0584800 overexpression line (CU099) and wild type sterilized rice seeds were sown in MS medium and grown for 7 days. The above-ground part (4 cm) of the obtained plant was cut out, transplanted to an MS medium containing 40 mM LiCl, grown for 2 weeks, and the survival rate was compared. The degree of imparting ion stress tolerance due to overexpression of the Os04g0584800 gene was expressed as a value obtained by subtracting the survival rate of the wild type (Nipponbare) (all 0) from the survival rate of the Os04g0584800 overexpression line (CU099). The results are also shown in FIG. 8C.

  As shown in FIG. 8C, the Os04g0584800 overexpression line (CU099) was found to have excellent resistance to hyperosmotic stress and ionic stress.

  From the above, it was found that the Os12g0150200 overexpressing line has excellent salt stress resistance and high temperature stress resistance, and has excellent resistance to a plurality of stresses including salt stress. In addition, salt stress and high temperature stress are stress tolerances often required of plants in outdoor environments. Therefore, enhanced expression of the Os12g0150200 gene or a gene equivalent to the gene can impart extremely practical stress tolerance to the plant body.

  It was also found that the Os04g0584800 gene can exhibit resistance to salt stress as well as high temperature stress, as well as hyperosmotic stress and ionic stress. Therefore, the enhanced expression of the Os04g0584800 gene and a gene equivalent to the gene can impart extremely practical stress resistance to the plant body.

  In this example, the standard rice varieties (Nipponbare) and salt-tolerant rice varieties (Heitai, Pokkali) were subjected to a salt tolerance test (laboratory, in a greenhouse) according to Example 1, and Os12g0150200 in the leaf tissue of these seedlings. The expression level of the (Cyp94C2b) gene was evaluated.

  That is, in the salt tolerance test (in the laboratory), after transplanting the above-ground base of one-week-old seedlings of each variety of rice to MS basic solid medium, a 600 mM NaCl aqueous solution (final concentration 300 mM) equivalent to the medium Was added and grown for 5 weeks, and then the survival rate was evaluated when the plant body was soaked in tap water and acclimated for 1 week. The results are shown in FIG. The number of test individuals (n) was 10 for Nipponbare, 10 for Heitai, and 10 for Pokkali.

For salt tolerance tests (in the greenhouse), 20 individual rice varieties were evaluated using the salinity evaluation score by Thomson et al. (2010), and then the relative values were calculated using the median values shown in the graph. It was. The results are shown in FIG. For the calculation of the relative value, the median value of the evaluation values of the varieties subjected to the test (Nipponbare and Shinriki 7) and the following formula were used as comparison criteria.
Relative value = (Median of Nipponbare-Median of sample) / (Median of Nipponbare-Median of Shinriki 7)

  Furthermore, RNeasy Plant Mini Kit (Qiagen) was used to extract and purify the total RNA of 1-week-old seedling leaf tissues (under non-stress conditions) of rice varieties frozen in liquid nitrogen and stored at -80 ° C. After that, reverse transcription from RNA was performed using QuantiTect Rev Transcription Kit (Qiagen). The obtained single-stranded cDNA was amplified by real-time PCR using SsoAdvanced Universal SYBR Green Supermix (BIO-RAD). The primers used are shown in Table 1. The expression level of Cyp94C2b mRNA was normalized by the expression level of 25S ribosomal RNA and expressed as a relative value to the expression level in Nipponbare. The results are shown in FIG. In addition, the graph showed with the average value +/- standard deviation (n = 3).

  As shown in FIGS. 9A and 9B, the existing salt-tolerant rice varieties Heitai and Pokkali have higher survival rates than Nipponbare in salt tolerance tests under two conditions in the laboratory and in the greenhouse. Was presented. In addition, as shown in Fig. 9C, the expression level of Os12g0150200 (CYP94C2b) gene in the leaves of plants grown under non-stress conditions in Heitai and Pokkal is higher than that in the standard variety (Nipponbare; Nipponbare) Was confirmed. Heitai was equivalent to 5 to 80 times the expression level of Nipponbare, and Pokkali was equivalent to 10 to 45 times. On the other hand, as shown in FIG. 6A, in the Os12g0150200 (Cyp94C2b) gene overexpressing body with improved salt tolerance, the expression level of CYP94C2b reached about 5 to 150 times that of the wild type (Nipponbare).

  From the above, it was found that high expression of CYP94C2b can contribute to the high salt tolerance of existing salt tolerance varieties Heitai and Pokkali. In addition, the high expression of CYP94C2b in existing salt-tolerant rice varieties strongly supports that improvement of salt tolerance by enhancing expression of CYP94C2b can be realized. For example, it is considered possible to improve salt tolerance by screening cultivars with high CYP94C2b gene expression and introducing a coding region and a gene containing an expression regulatory region into the cultivar.

  In this specification, the following papers are further referred to. These documents, as well as those already referenced in this specification, are hereby incorporated by reference in their entirety.

(1) Nakamura, H., Hakata, M., Amano, K., Miyao, A., Toki, N., Kajikawa, M. et al. (2007) A genome-wide gain-of function analysis of rice genes using the FOX-hunting system. Plant Mol. Biol. 65: 357-371.
(2) Hakata, M., Nakamura, H., Iida-Okada, K., Miyao, A., Kajikawa, M., Imai-Toki, N. et al. (2010) Production and characterization of a large population of cDNA-overexpressing transgenic rice plants using Gateway-based full-length cDNA expression libraries. Breed. Sci. 60: 575-585.
(3) Tsuchida-Mayama, T., Nakamura, H., Hakata, M. and Ichikawa, H. (2010) Rice transgenic resources with gain-of-function phenotypes. Breeding Sci. 60: 493-501.
(4) Thomson, MJ, de Ocampo, M., Egdane, J., Rahman MA, Sajise, AG, Adorada, DL et al. (2010) Characterizing the Saltol Quantitative Trait Locus for Salinity Tolerance in Rice. Rice 3: 148 -160.

Claims (25)

  Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes, Os04g0584800 gene and this gene functionally A plant in which expression of one or more genes selected from the group consisting of a second gene selected from a gene group consisting of a subgroup of second genes consisting of equivalent genes is enhanced.   The plant according to claim 1, which has enhanced environmental stress tolerance.   The plant according to claim 1, wherein the salt stress tolerance is enhanced.   Furthermore, the plant body of Claim 3 with which other environmental stress tolerance was reinforced.   The plant according to any one of claims 1 to 4, wherein expression of the first gene is enhanced.   The plant according to claim 5, wherein the first gene is selected from the group consisting of at least Os12g0150200 and a gene functionally equivalent to this gene. The plant according to any one of claims 1 to 6, wherein the first gene encodes any one of the following proteins (a) to (f).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of converting activated jasmonic acid into inactive jasmonic acid (c) comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and inactivating activated jasmonic acid Protein having activity of converting to type jasmonic acid (d) Protein coated with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 (e) 90% or more identity with the base sequence represented by SEQ ID NO: 1 Encoded by a polynucleotide having a nucleotide sequence having an activity and having an activity of converting active jasmonic acid to inactive jasmonic acid Protein (f) encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 or a complementary nucleotide sequence, and converts activated jasmonic acid into inactive jasmon Protein with activity to convert to acid   The plant according to any one of claims 1 to 7, wherein expression of the second gene is enhanced.   The plant according to claim 8, wherein the expression of at least the Os04g0584800 gene is enhanced. The plant according to any one of claims 1 to 9, wherein the second gene encodes any one of the following proteins (a) to (f).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 26 (b) comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 26; A protein having an activity of enhancing the resistance to salt stress (c) comprising the amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 26, and being enhanced in the plant A protein having an activity of enhancing resistance to salt stress (d) and a protein coated with a polynucleotide consisting of the base sequence represented by SEQ ID NO: 25 (e) 90% of the base sequence represented by SEQ ID NO: 25 It is encoded by a polynucleotide comprising a base sequence having the above identity and is enhanced in a plant body. A protein having an activity of enhancing resistance to salt stress (f) encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the base sequence represented by SEQ ID NO: 25 or a complementary base sequence Protein having an activity to enhance tolerance to salt stress when enhanced in a plant body   The plant body according to claim 1, wherein the plant body is a dicotyledonous plant.   The plant according to claim 11, wherein the plant is soybean.   The plant according to claim 1, which is a monocotyledonous plant.   The plant body according to claim 13, which is a gramineous plant.   The plant according to claim 14, which is rice.   The plant according to claim 14, which is a sugarcane.   The plant according to claim 14, which is corn. An expression vector for imparting environmental stress resistance to a plant body,
Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes and Os04g0584800 gene and this gene functionally A vector comprising one or more genes selected from a gene group consisting of a second gene subgroup consisting of equivalent genes.   A transformant comprising the expression vector according to claim 18.   A transformed plant comprising the expression vector according to claim 18. Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 and AT2G27690 genes, and a first group of genes functionally equivalent to any of these genes, Os04g0584800 gene and functionally equivalent to this gene A step of enhancing an endogenous or exogenous gene which is one or more genes selected from a gene group consisting of a subgroup of second genes consisting of
A method for imparting environmental stress tolerance to a plant body. Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes and Os04g0584800 gene and this gene functionally A step of enhancing an endogenous or exogenous gene which is one or more genes selected from a gene group consisting of a subgroup of second genes consisting of equivalent genes,
A method for producing a plant body. Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes and Os04g0584800 gene and this gene functionally A step of selecting a plant by mating using as an index the expression of one or more genes selected from a gene group consisting of a subgroup of second genes consisting of equivalent genes;
A method for producing a plant body. A method for producing crops,
A method comprising cultivating a crop that is a plant according to any one of claims 1 to 17. Os04g0584800 gene, Os01g0858350 gene, Os05g0445100 gene, Os11g0151400 gene, AT3G48520 gene and AT2G27690 gene and a first group of genes functionally equivalent to any of these genes and Os04g0584800 gene and this gene functionally Screening one or more plants using as an index the expression of one or more genes selected from a gene group consisting of a subgroup of second genes consisting of equivalent genes;
Evaluating the salt tolerance of the plant body having a high expression level of the one or more genes,
A plant body screening method comprising: