CN116789775A

CN116789775A - Application of soybean transcription factor GmGRAS487 in regulation and control of salt tolerance of plants

Info

Publication number: CN116789775A
Application number: CN202210249178.4A
Authority: CN
Inventors: 张劲松; 陶建军; 张万科; 韦伟; 阴翠翠; 陈受宜
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2023-09-22

Abstract

The application discloses an application of a soybean transcription factor GmGRAS487 in regulation and control of plant salt tolerance. The soybean transcription factor GmGRAS487 disclosed by the application is a protein with an amino acid sequence of sequence 2. The application transfers the coding gene of the transcription factor GmGRAS487 into the hairy root of the acceptor soybean to obtain the transgenic hairy root and the transgenic chimera, and compared with the transgenic empty vector soybean hairy root, the salt tolerance of the transgenic soybean hairy root is obviously improved. The transcription factor GmGRAS487 and the coding gene thereof can regulate and control the salt tolerance of plants, and have important theoretical and practical significance for cultivating high salt tolerance varieties of plants.

Description

Application of soybean transcription factor GmGRAS487 in regulation and control of salt tolerance of plants

Technical Field

The application relates to the field of biotechnology, and relates to application of a soybean transcription factor GmGRAS487 in regulation and control of plant salt tolerance.

Background

The change of physical and chemical factors in the environment, such as drought, saline-alkali, cold injury, freeze injury, waterlogging and other stress factors, is one of the reasons for serious crop yield reduction. During the period of 40 years from 1939 to 1978, the insurance industry pay statistics for crop yield reduction show that the pay proportion for yield reduction due to salt damage and drought is about 40.8%, higher than waterlogging (16.4%), low temperature (13.8%), hail (11.3%), wind (7.0%), and much higher than insect damage (4.5%), disease (2.7%), and other factors. Thus, cultivation of salt/drought tolerant crops is one of the main objectives of the planting industry. Improving the salt/drought tolerance of crops, and not only utilizing the traditional breeding method, molecular genetic breeding is one of the fields concerned by technological workers at present.

GRAS domain proteins are a plant-specific class of proteins, generally comprising 400-700 amino acid residues, and can be divided into at least 13 subfamilies. As plant transcription factors, this family of proteins is involved in many biological processes, such as gibberellin signaling, root and stem development, and the like.

Disclosure of Invention

The technical problem to be solved by the application is how to improve the salt tolerance of plants.

To solve the above technical problems, the present application provides, first, any one of the following applications of proteins or substances regulating the activity or content of the proteins:

d1 Regulating and controlling the salt tolerance of plants;

d2 Preparing a product for regulating and controlling the salt tolerance of plants;

d3 Cultivating a salt tolerance enhancing plant;

d4 Preparing and cultivating a salt tolerance enhanced plant product;

d5 Plant breeding;

the protein is derived from soybean and named GmGRAS487, which is A1), A2) or A3) as follows:

a1 A protein whose amino acid sequence is sequence 2;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate purification of the protein of A1), a tag as shown in the following table may be attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in the sequence 2 in the sequence table.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The GmGRAS487 protein in A2) has 75% or more identity with the amino acid sequence of the protein shown in the sequence 2 and has the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The GmGRAS487 protein in A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The coding gene of the GmGRAS487 protein in the A2) can be obtained by deleting one or a plurality of amino acid residues in the DNA sequence shown in the sequence 1 and/or carrying out one or a plurality of base pair missense mutations and/or connecting the coding sequences of the labels shown in the table at the 5 'end and/or the 3' end. Wherein the DNA molecule shown in the sequence 1 codes for GmGRAS487 protein shown in the sequence 2.

In the above application, the substance may be any one of the following B1) to B9):

b1 A nucleic acid molecule encoding GmGRAS 487;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

b8 A nucleic acid molecule that reduces the expression level of GmGRAS 487;

b9 An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of B8).

In the above applications, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 1 in a sequence table;

b12 A cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b13 A cDNA or DNA molecule which has 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and which codes for GmGRAS 487;

b14 Under stringent conditions with the nucleotide sequence defined under b 11) or b 12) or b 13) and encoding a cDNA molecule or a DNA molecule of GmGRAS487.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the GmGRAS487 protein according to the application can be mutated easily by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the GmGRAS487 protein isolated by the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the GmGRAS487 protein and have the function of the GmGRAS487 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 2 of the present application. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above applications, the expression cassette (GmGRAS 487 gene expression cassette) described in B2) containing a nucleic acid molecule encoding the GmGRAS487 protein refers to a DNA capable of expressing the GmGRAS487 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the GmGRAS487 gene but also a terminator for terminating transcription of the GmGRAS487 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoter from tomato, leucine aminopeptidase ("L)AP ", chao et al (1999) Plant Physiol 120: 979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

The recombinant vector containing the GmGRAS487 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present application is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be a pROKII vector or a pZH01 vector.

B3 The recombinant vector may specifically be pROKII-GmGRAS487. The pROKAI-GmGRAS 487 is a recombinant vector obtained by replacing a DNA fragment between BamHI and KpnI recognition sequences of pROKAI vector with a GmGRAS487 gene shown in a sequence 1 in a sequence table, and can express a fusion protein formed by GmGRAS487 shown in a sequence 2 in the sequence table and NPT II.

B9 The recombinant vector can be pZH01-GmGRAS487-RNAi, and the pZH01-GmGRAS487-RNAi is a recombinant vector obtained by inserting the DNA fragments shown in 1077 th to 1363 rd positions of the sequence 1 into the multiple cloning sites of pZH01 twice, wherein the directions of the inserted fragments are opposite.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacterium may be Agrobacterium, such as Agrobacterium rhizogenes K599.

In the above applications, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

In the above application, the plant may be M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) leguminous plants;

m3) soybean.

The application also provides any one of the following methods:

x1) a method for cultivating a salt tolerance enhancing plant, comprising knocking out a coding gene of GmGRAS487 expressed in a receptor plant, or inhibiting the expression of the GmGRAS487 coding gene in the receptor plant, or reducing the content of GmGRAS487 in the receptor plant, or reducing the activity of GmGRAS487 in the receptor plant, so as to obtain a target plant with enhanced salt tolerance;

x2) a method for enhancing salt tolerance of a plant, comprising knocking out a gene encoding GmGRAS487 expressed in a recipient plant, or inhibiting expression of the gene encoding GmGRAS487 in the recipient plant, or reducing the content of GmGRAS487 in the recipient plant, or reducing the activity of GmGRAS487 in the recipient plant, to obtain a target plant with enhanced salt tolerance, and achieving enhancement of salt tolerance of the plant.

In the above method, the inhibition of expression of the gene encoding GmGRAS487 in the recipient plant in X1) and X2) may be achieved by introducing into the recipient plant a nucleic acid molecule that inhibits expression of the gene encoding GmGRAS487 or a recombinant vector that expresses the nucleic acid molecule.

In the above method, the coding gene may be the nucleic acid molecule of B1).

In the above method, the coding gene of the GmGRAS487 may be modified as follows, and then introduced into a recipient plant to achieve a better expression effect:

1) Modification and optimization are carried out according to actual needs so as to enable the genes to be expressed efficiently; for example, the codon of the encoding gene of GmGRAS487 according to the present application may be changed to conform to plant preferences while maintaining the amino acid sequence thereof according to the codon preferred by the recipient plant; during the optimization process, it is preferable to maintain a certain GC content in the optimized coding sequence to best achieve high level expression of the introduced gene in the plant, wherein the GC content may be 35%, more than 45%, more than 50% or more than about 60%;

2) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

4) The expression efficiency of the gene of the application can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present application;

5) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

The coding gene of the GmGRAS487 can be introduced into a recipient plant by using a recombinant vector containing the coding gene of the GmGRAS487. The recombinant vector can be specifically the pCAMBIA1301-GmGRAS487.

The recombinant vector may be introduced into plant cells or tissues by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, agrobacterium-mediated methods, etc., and the transformed plant tissues are cultivated into plants. The plant host to be transformed may be either a monocot or a dicot.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly by drought treatment without adding any selectable marker gene.

The plant of interest is understood to include not only the first generation plants in which the GmGRAS487 protein or its coding gene has been altered, but also their progeny. For the plant of interest, the gene may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The plants of interest include seeds, calli, whole plants and cells.

In the above method, the recipient plant may be M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) leguminous plants;

m3) soybean.

The application also provides a product for enhancing salt tolerance of plants, which contains GmGRAS487 or the substance for regulating the activity or content of the protein.

The product can take GmGRAS487 or the substance for regulating the activity or the content of the protein as an active ingredient, and can also combine GmGRAS487 or the substance for regulating the activity or the content of the protein with a substance with the same function as the active ingredient.

GmGRAS487 or the substances which regulate the activity or the content of the protein also belong to the scope of protection of the application.

In the present application, the salt tolerance may specifically be the tolerance of a plant to a high-salt environment simulated by NaCl. The high salt environment simulated by NaCl can be an environment with a NaCl concentration of 50mM-300mM (such as 100 mM).

Salt tolerance of a plant may be manifested by the survival rate of the plant, the leaf wilting degree, and/or the leaf relative ion permeability.

The application transfers the coding gene of the transcription factor GmGRAS487 into the hairy root of the acceptor soybean to obtain the transgenic hairy root and the transgenic chimera, and compared with the transgenic empty vector soybean hairy root, the salt tolerance of the transgenic soybean hairy root is obviously improved. The transcription factor GmGRAS487 and the coding gene thereof can regulate and control the salt tolerance of plants, and have important theoretical and practical significance for cultivating high salt tolerance varieties of plants.

Drawings

FIG. 1 is a transcription pattern of GmGRAS487 under 120mM NaCl treatment.

FIG. 2 is a schematic diagram of plant expression vectors pROKAI-GmGRAS 487 and pZH-GmGRAS 487-RNAi.

FIG. 3 is a molecular characterization of soybean hairy roots overexpressing GmGRAS487 and GmGRAS487-RNAi.

FIG. 4 shows the salt tolerance phenotype of GmGRAS 487-transformed hairy roots and chimeras.

FIG. 5 is a graph showing the statistics of physiological indexes under salt stress of GmGRAS487 transgenic hairy root chimera. * P is less than 0.05; * P < 0.01.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

DALeguminosae No. 1 (Glycine max L.Merr. Kefeng 1) in the examples described below is described in W.K.Zhang, Y.J.Wang, G.Z.Luo, J.S.Zhang, C.Y.He, X.L.Wu, J.Y.Gai, S.Y.Chen, QTL mapping of ten agronomic traits on the soybean (Glycine max L.Merr.) genetic map and their association with EST markers, theor. Appl. Genet,2004,108:1131-1139, publicly available from national academy of sciences of genetics and developmental biology research,

soybean [ Glycine max (l.) Merr ] nannong 1138-2: the soybean improvement center germplasm library of the Nanjing agricultural university is provided by the soybean improvement center of the Nanjing agricultural university.

The expression vector pROKII vector (binary expression vector) in the examples described below is described in D.C.Baulcombe, G.R.Saunders, M.W.Bevan, M.A.Mayo and B.D. Harrison, expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature321 (1986), pp.446-449, publicly available from national academy of sciences genetic and developmental biology research;

pZH 01A vector, stratagene, is described in Han Xiao, et al functional analysis of the rice AP3 homologue OsMADS16 by RNA interference, plant Molecular Biology,2003,52,957-966, and is available to the public from national academy of sciences of genetics and developmental biology.

Agrobacterium rhizogenes K599 is described in Attila Kereszt, et al Agrobacterium rhizogenes-mediaded transformation of soybean to study of root biology, nature Protocols,2007,2 (4), 549-552), and is publicly available from Peter M Gressnon professor The University of Queensland, st Lucia, queensland 4072, australia, or from the institute of genetics and developmental biology after agreement (written agreement) by Peter M Gressnon professor.

Example 1 screening of Gene encoding Soybean transcription factor GmGRAS487 and cDNA clone thereof

The inventors screened the gene with the Locus name Glyma08g15530 (Locus name: glyma.08G146900) in the transcriptome analysis of Leguminosae No. 1 (salt sensitivity) and Nannong 1138-2 (salt tolerance) under normal and high salt stress. Glyma08g15530 showed a dramatic drop in transcription when treated with 120mM NaCl, considering that it may negatively regulate salt tolerance in plants. Culturing soybean salt-tolerant variety Nannong 1138-2 under light, growing for 2 weeks, and respectively extracting RNA from seedlings. 1g of fresh seedlings are ground in liquid nitrogen, suspended in 4mol/L guanidine thioglycolate, the mixture is extracted by acid phenol and chloroform, absolute ethyl alcohol is added into supernatant to precipitate total RNA, then the total RNA is dissolved in water, and cDNA is synthesized by reverse transcription of reverse transcriptase. The primer is as follows:

gm15530-F1: ATGAAAACGATGGATTTTGA and is provided with

Gm15530-R1：CTATTTGACCTTGTCATCCAGATAA。

Real Time-PCR identification was performed. The Real-Time PCR reaction was performed using the TOYOBO RealTime PCR Master Mix kit, as described. The soybean Tublin gene is an internal standard, and the Primer used is Primer-TF:5' -AACCTCCTCCTCATCGTACT, and Primer-TR:5' -GACAGCATCAGCCATGTTCA.

FIG. 1 shows that Glyma08g15530 drops substantially at 1 hour with 120mM NaCl, continuing to the nadir for 3 hours. Sequencing showed that Glyma08g15530 contained 1464bp, encoding 487 amino acid residues, and this protein was designated GmGRAS487. In Nannong 1138-2, the amino acid sequence of GmGRAS487 is sequence 2 in the sequence table, and the DNA coding sequence is sequence 1.

Example 2 construction of plant expression vectors for the Soybean transcription factor GmGRAS487

1. Construction of GmGRAS487 overexpression vector pROKAI-GmGRAS 487

PCR amplification is carried out by using cDNA of Nannong 1138-2 as a template and Gm 15530-pROKAI-F2 and Gm 15530-pROKAI-R2 to obtain a PCR product.

Gm15530-pROKII-F2：AGAACACGGGGGACTCTAGAATGAAAACGATGGATTTTGA；

Gm15530-pROKII-R2：GATCGGGGAAATTCGAGCTCCTATTTGACCTTGTCATCCAGATAA。

The pROKAI vector was digested with BamHI and KpnI, and the PCR recovered fragment was ligated into pROKAI vector by homologous recombination to obtain recombinant vector pROKAI-GmGRAS 487 (partial vector schematic view is shown in FIG. 2). pROKAI-GmGRAS 487 is a recombinant vector obtained by replacing a DNA fragment between BamHI and KpnI recognition sequences of pROKAI vector with GmGRAS487 gene shown in sequence 1 in a sequence table, and can express fusion protein formed by GmGRAS487 shown in sequence 2 in the sequence table and NPT II.

2. Construction of GmGRAS487 RNAi expression vector pZH01-GmGRAS487-RNAi

Firstly, extracting total RNA of Nannong 1138-2 soybean by using a Trizol method, using cDNA obtained by reverse transcription as a template, and using a vector construction primer to amplify a DNA fragment of 287bp from 1077 th position to 1363 th position of CDS region of GmGRAS487 gene to construct an RNAi vector.

The RNAi vector used was pZH01, the primers were as follows:

GmGRAS487Ri-F1：(recognition sequence singly underlined as Xba I, doubly underlined as Sac I);

GmGRAS487Ri-R1：(recognition sequence of Sal I is underlined singly and recognition sequence of Kpn I is underlined doubly).

The PCR product and pZH01 vector are subjected to double digestion by Sac I and Kpn I, the obtained large fragment of the PCR product is connected with a vector skeleton, the obtained recombinant vector with correct sequence and the PCR product are subjected to double digestion by Xba I and Sal I, the obtained large fragment of the PCR product is connected with the vector skeleton, and the obtained recombinant vector with correct sequence is recorded as pZH-GmGRAS 487-RNAi (figure 2).

The constructed vectors are sequenced, and the next experiment is carried out after the sequence is verified to be correct.

Example 3 obtaining of soybean hairy root transformed with GmGRAS487 Gene

Agrobacterium rhizogenes infection is slightly modified according to the methods of Attila Kereszt et al (Attila Kereszt, et al Agrobacterium rhizogenes-mediaded transformation of soybean to study of root biology, nature Protocols,2007,2 (4), 549-552) according to the literature "Wang, fang; chen, hao-Wei; li, qing-Tian; wei, wei; li, wei; zhang, wan-Ke; ma, biao; bi, ying-Dong; lai, yong-Cai; liu, xin-Lei; man, wei-Qun; zhang, jin-Song; chen, shou-Yi, gmWRKY27 interacts with GmMYB to reduce expression of GmNAC29 for stress tolerance in soybean plants,2015,The Plant Journal,83,224-236", or patent" Chen Shouyi "or the like, plant stress tolerance-related transcription factor GmWRKY78, and encoding gene and application thereof, patent No.: the agrobacterium rhizogenes-mediated transgenic root system method in ZL2011 0053083.7, authorized day 2013.10.09 ".

Preparation of overexpression GmGRAS487 and GmGRAS487-RNAi hairy root:

1) Acquisition of recombinant Agrobacterium

Recombinant expression vectors pROKII-GmGRAS487 and pZH-GmGRAS 487-RNAi obtained in example 2 were introduced into Agrobacterium rhizogenes K599 by electric shock method, respectively, to obtain recombinant Agrobacterium. Recombinant Agrobacterium containing the above plasmid was designated K599/pROKII-GmGRAS 487 and K599/pZH01-GmGRAS487-RNAi, respectively.

2) Hairy root transformation

Inoculating and growing the recombinant agrobacterium K599/pROKII-GmGRAS 487 and K599/pZH01-GmGRAS487-RNAi respectively for 6 days by using a syringe, wherein the specific method is as described in the introduction, and the moisturizing and growing are as follows: the mixture is irradiated for 16 hours at the temperature of 25 ℃ and the humidity of 50 percent. After 2 weeks, the growing hairy roots were the transgenic hairy roots. The over-expression chimeric of 121-turn K599/pROKII-GmGRAS 487 (GmGRAS 487-OE) and 120K 599/pZH-GmGRAS 487-RNAi chimeric plants (GmGRAS 487-RNAi) are obtained respectively, and can be further used for transgene identification and salt tolerance detection.

Agrobacterium rhizogenes K599/pROKII containing empty vector pROKII is transferred into DaLeguminosae Feng 1 by the same method to obtain 124 empty vector hairy root systems to be used as empty vector control.

3) Molecular identification of transgenic hairy roots

Total RNA from transgenic hairy roots and empty vector controls was extracted and reverse transcribed into cDNA. Using cDNA as a template, gm15530-F1: GGTGTGGGAGGAAGGGTTTT and Gm15530-R1: GCCATTGGTTCCCAGATTGAAG the GmGRAS487 gene expression level was analyzed. The soybean GmTubulin gene is an internal standard, and the Primer used is Primer-TF:5' -AACCTCCTCCTCATCGTACT, and Primer-TR:5' -GACAGCATCAGCCATGTTCA. Experiments were repeated three times and the results averaged ± standard deviation.

FIG. 3 shows that the expression level of GmGRAS487 in the empty vector control, gmGRAS487-OE and GmGRAS487-RNAi hairy root was about 0.024, 2.35 and 0.013, respectively, the expression level of GmGRAS487 in GmGRAS487-OE was significantly higher than that in the empty vector control, and the expression level of GmGRAS487 in GmGRAS487-RNAi hairy root was significantly lower than that in the control (FIG. 3).

EXAMPLE 4 salt tolerance identification of GmGRAS487 and GmGRAS487-RNAi hairy roots

The experimental samples were the empty vector control obtained in example 3 and GmGRAS487-OE, gmGRAS487-RNAi hairy roots and plants.

Dividing the above 3 experimental samples into 2 groups, taking about 12 each, and treating one group with 100mM NaCl aqueous solution for 3 days, namely immersing in 100mM NaCl solution, and treating at 25deg.C for 3 days; the second group was immersed in water as a control. Experiments were repeated three times and the results averaged ± standard deviation.

After 3 days of treatment with 100mM NaCl aqueous solution, the mixture was observed by photographing. FIG. 4 shows that plants and leaf phenotypes of the transgenic hairy roots and 2 transgenic hairy roots showed no significant difference in growth of the water-treated (normal condition) transgenic (pROKII) hairy roots and the transgenic GmGRAS487 gene and the transgenic GmGRAS487-RNAi hairy root chimera, and that the transgenic GmGRAS487-RNAi hairy root chimera and leaf (GmGRAS-RNAi) were significantly less wilting than the control chimera and leaf, whereas the overexpressed GmGRAS487 hairy root chimera (GmGRAS-OE) and leaf wilting significantly higher than the control.

Survival rate detection:

the right side of fig. 5 shows that in the case of hydroponics, all plants survived 100% and after 3 days of 100mM NaCl treatment, the survival rates of the control, gmGRAS-RNAi and GmGRAS-OE chimeras were about 40%, 63% and 18%, respectively, with the differences between the overexpressing chimeras and RNAi chimeras from the control being significant and very significant. It is shown that the overexpression of GmGRAS487 reduces the salt tolerance of the plants, and the reduction of the expression quantity of GmGRAS487 increases the salt tolerance of the plants.

Ion permeability detection in NaCl treatment:

when plant tissues are damaged by adversity stress, cell membrane functions are damaged or structures are destroyed, permeability is increased, and various water-soluble substances in cells including electrolytes are extravasated. The plant tissue is immersed in deionized water, and the electrical conductivity of the water is increased by the extravasation of the electrolyte. The heavier the injury, the more severe the cell membrane destruction, the more extravasation and the greater the conductivity of the water. Therefore, the conductivity change of the extravasation liquid can be measured by a conductivity meter, and the damage degree of plant tissues can be reflected indirectly. The conductivity measurements thus allow calculation of the relative ion permeability, which is indicative of the extent to which the plant cell membrane is damaged.

The measurement method comprises cutting soybean leaf, placing into a clean screw glass bottle, and rinsing with deionized water for 3 times. Then 80mL deionized water is added to fully soak the leaves, and vacuum is pumped for 45min. After standing at room temperature for 30min, the conductivity E1 was measured by a conductivity meter (DDC-308A type, shanghai Boeing instruments Co., ltd.). Boiling the leaves for 15min, cooling to room temperature, mixing, and measuring the conductivity E2 by using a conductivity meter.

Relative ion permeability EL (%) =e1/E2X 100, where E1 and E2 are conductivity.

The left panel of fig. 5 shows that in the case of hydroponics, the ion permeability of all plants was very low, about 6%, and the survival rates of the control, gmGRAS-RNAi and GmGRAS-OE chimeras were about 47%, 62% and 17%, respectively, after 3 days of 100mM NaCl treatment, indicating that the GmGRAS487 overexpressing chimeras had the most damaged leaf cell membranes under salt stress, and the control, while reducing the expression of GmGRAS487, extremely significantly protecting the cell membranes from damage under salt stress.

The results show that in the plant, gmGRAS487 negatively regulates and controls salt tolerance, reduces the expression of GmGRAS487, and obviously improves the salt tolerance of the plant.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of genetic and developmental biology of national academy of sciences

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1464

<212> DNA

<213> Glycine max Merrill

<400> 1

atgaaaacga tggattttga gcaattttat tactcatttg gccctcctta catgaatcaa 60

ctccatgagt gtatctcaga aaatgcattt ccatttcaaa cagaaaatct cttatctcca 120

aatactttcc tagatgaaat gtttgatcaa gagtactcca tggaaggatt gctgcagcaa 180

catgcaaaca accaggagga ctttggtttc ttgaagcatg atgatccact agagactgaa 240

ttttgtcatg gatttagccc tagtgctgag gaaaatatgc atgtttcaat ggaagaaggg 300

gattcttgtt tgaagggaat ccaagcagag ctaatggaag agactagttt agctgatctg 360

ttgctaacag gagctgaagc tgttgaagca caaaactggc cccttgcttc agatataatt 420

gagaaactta acaatgcctc atctttagaa aatggtgatg gtttattgaa caggttggct 480

cttttcttta ctcagagtct ctattataaa agcacaaatg cccctgaatt gctacagtgt 540

ggtgctgttt ctacgcacac aaatgctttc tgtgtgtttc aggttctcca agaactctct 600

ccctatgtaa aatttgctca tttcactgca aaccaagcaa tcttagaggc cacagaaggt 660

gctgaagatc ttcacatcat tgattttgat atcatggagg ggattcagtg gccacccttg 720

atggttgacc ttgcaatgaa gaaaagtgtt aattccctta gagtaacagc catcacagtg 780

aaccaaagag gtgcagattc tgttcaacaa acaggaagaa ggctcaaaga gtttgcagct 840

tctatcaact ttccattcat gtttgaccag ttaatgatgg aaagggaaga agattttcaa 900

ggaattgaac ttggtcaaac actcatagtc aactgcatga tacaccagtg gatgcctaat 960

aggagcttct cattggtcaa aacattcttg gatggtgtga ccaaattgtc cccaaggctt 1020

gttgttttag tggaagaaga actatttaat tttcctaggc tcaagtccat gtcctttgtg 1080

gagttcttct gtgaggcttt gcatcactac actgcacttt gtgattcact tgctagtaat 1140

ctatggggta gccacaagat ggagttgagc ctgatagaaa aagaggttat tgggctcaga 1200

atattggaca gtgtgaggca gtttccttgt gagagaaagg agagaatggt gtgggaggaa 1260

gggttttatt ccttgaaagg gtttaaacgt gtacctatga gtacatgtaa catttcacaa 1320

gccaaattct tggtaagcct ctttggtgga gggtattggg tccaatacga gaagggtagg 1380

ttggccttgt gttggaagtc aaggcctttg actgtggctt caatctggga accaatggct 1440

tatctggatg acaaggtcaa atag 1464

<210> 2

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<213> Glycine max Merrill

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Met Lys Thr Met Asp Phe Glu Gln Phe Tyr Tyr Ser Phe Gly Pro Pro

1 5 10 15

Tyr Met Asn Gln Leu His Glu Cys Ile Ser Glu Asn Ala Phe Pro Phe

20 25 30

Gln Thr Glu Asn Leu Leu Ser Pro Asn Thr Phe Leu Asp Glu Met Phe

35 40 45

Asp Gln Glu Tyr Ser Met Glu Gly Leu Leu Gln Gln His Ala Asn Asn

50 55 60

Gln Glu Asp Phe Gly Phe Leu Lys His Asp Asp Pro Leu Glu Thr Glu

65 70 75 80

Phe Cys His Gly Phe Ser Pro Ser Ala Glu Glu Asn Met His Val Ser

85 90 95

Met Glu Glu Gly Asp Ser Cys Leu Lys Gly Ile Gln Ala Glu Leu Met

100 105 110

Glu Glu Thr Ser Leu Ala Asp Leu Leu Leu Thr Gly Ala Glu Ala Val

115 120 125

Glu Ala Gln Asn Trp Pro Leu Ala Ser Asp Ile Ile Glu Lys Leu Asn

130 135 140

Asn Ala Ser Ser Leu Glu Asn Gly Asp Gly Leu Leu Asn Arg Leu Ala

145 150 155 160

Leu Phe Phe Thr Gln Ser Leu Tyr Tyr Lys Ser Thr Asn Ala Pro Glu

165 170 175

Leu Leu Gln Cys Gly Ala Val Ser Thr His Thr Asn Ala Phe Cys Val

180 185 190

Phe Gln Val Leu Gln Glu Leu Ser Pro Tyr Val Lys Phe Ala His Phe

195 200 205

Thr Ala Asn Gln Ala Ile Leu Glu Ala Thr Glu Gly Ala Glu Asp Leu

210 215 220

His Ile Ile Asp Phe Asp Ile Met Glu Gly Ile Gln Trp Pro Pro Leu

225 230 235 240

Met Val Asp Leu Ala Met Lys Lys Ser Val Asn Ser Leu Arg Val Thr

245 250 255

Ala Ile Thr Val Asn Gln Arg Gly Ala Asp Ser Val Gln Gln Thr Gly

260 265 270

Arg Arg Leu Lys Glu Phe Ala Ala Ser Ile Asn Phe Pro Phe Met Phe

275 280 285

Asp Gln Leu Met Met Glu Arg Glu Glu Asp Phe Gln Gly Ile Glu Leu

290 295 300

Gly Gln Thr Leu Ile Val Asn Cys Met Ile His Gln Trp Met Pro Asn

305 310 315 320

Arg Ser Phe Ser Leu Val Lys Thr Phe Leu Asp Gly Val Thr Lys Leu

325 330 335

Ser Pro Arg Leu Val Val Leu Val Glu Glu Glu Leu Phe Asn Phe Pro

340 345 350

Arg Leu Lys Ser Met Ser Phe Val Glu Phe Phe Cys Glu Ala Leu His

355 360 365

His Tyr Thr Ala Leu Cys Asp Ser Leu Ala Ser Asn Leu Trp Gly Ser

370 375 380

His Lys Met Glu Leu Ser Leu Ile Glu Lys Glu Val Ile Gly Leu Arg

385 390 395 400

Ile Leu Asp Ser Val Arg Gln Phe Pro Cys Glu Arg Lys Glu Arg Met

405 410 415

Val Trp Glu Glu Gly Phe Tyr Ser Leu Lys Gly Phe Lys Arg Val Pro

420 425 430

Met Ser Thr Cys Asn Ile Ser Gln Ala Lys Phe Leu Val Ser Leu Phe

435 440 445

Gly Gly Gly Tyr Trp Val Gln Tyr Glu Lys Gly Arg Leu Ala Leu Cys

450 455 460

Trp Lys Ser Arg Pro Leu Thr Val Ala Ser Ile Trp Glu Pro Met Ala

465 470 475 480

Tyr Leu Asp Asp Lys Val Lys

485

Claims

1. Any of the following uses of a protein or a substance that modulates the activity or content of said protein:

d1 Regulating and controlling the salt tolerance of plants;

d3 Cultivating a salt tolerance enhancing plant;

d4 Preparing and cultivating a salt tolerance enhanced plant product;

d5 Plant breeding;

the protein is A1), A2) or A3) as follows:

a1 A protein whose amino acid sequence is sequence 2;

2. The use according to claim 1, characterized in that: the substance is any one of the following B1) to B9):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b8 A nucleic acid molecule which reduces the expression of the protein of claim 1;

3. The use according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) or b 14) as follows:

b12 A cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b13 A cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding the protein according to claim 1;

b14 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 11) or b 12) or b 13) and which codes for the protein according to claim 1.

4. A use according to any one of claims 1-3, characterized in that: the plant is M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) leguminous plants;

m3) soybean.

5. The method comprises the following steps:

x1) a method for growing a salt tolerance-enhancing plant comprising knocking out a gene encoding a protein according to claim 1 expressed in a recipient plant, or inhibiting the expression of a gene encoding a protein according to claim 1 in a recipient plant, or reducing the amount of a protein according to claim 1 in a recipient plant, or reducing the activity of a protein according to claim 1 in a recipient plant, to obtain a salt tolerance-enhancing plant of interest;

x2) a method for enhancing salt tolerance in a plant, comprising knocking out a gene encoding a protein according to claim 1 expressed in a recipient plant, or inhibiting expression of a gene encoding a protein according to claim 1 in a recipient plant, or reducing the amount of a protein according to claim 1 in a recipient plant, or reducing the activity of a protein according to claim 1 in a recipient plant, to obtain a plant of interest having enhanced salt tolerance, to achieve enhancement of salt tolerance in a plant.

6. The method according to claim 5, wherein: inhibiting the expression of the protein-encoding gene according to claim 1 in a recipient plant in X1) and X2) is achieved by introducing into said recipient plant a nucleic acid molecule which inhibits the expression of the protein-encoding gene according to claim 1 or a recombinant vector expressing said nucleic acid molecule.

7. The method according to claim 6, wherein: the coding gene is the nucleic acid molecule of B1) in claim 2 or 3.

8. The method according to any one of claims 5-7, wherein: the recipient plant is M1) or M2) or M3):

m1) dicotyledonous or monocotyledonous plants;

m2) leguminous plants;

m3) soybean.

9. A plant salt tolerance enhancing product comprising a protein according to any one of claims 1 to 3 or said substance modulating the activity or content of said protein.

10. A protein according to any one of claims 1 to 3 or said substance which modulates the activity or content of said protein.