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

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 regulating plant salt tolerance

Technical field

The present invention relates to the application of soybean transcription factor GmGRAS487 in the regulation of plant salt tolerance in the field of biotechnology.

Background technique

Changes in physical and chemical factors in the environment, such as drought, salinity, cold damage, freezing damage, waterlogging and other stress factors are one of the reasons for severe crop yield reduction. In the 40 years from 1939 to 1978 in the United States, the insurance industry’s compensation statistics for crop production losses show that the proportion of compensation for crop production losses due to salt damage and drought accounted for approximately 40.8%, which was higher than waterlogging (16.4%) and low temperature (13.8%). , hail (11.3%) and wind (7.0%), which are much higher than insect disasters (4.5%), diseases (2.7%) and other factors. Therefore, developing salt/drought tolerant crops is one of the main goals of the crop industry. To improve the salt/drought tolerance of crops, in addition to using traditional breeding methods, molecular genetic breeding has become one of the areas of concern for scientific and technological workers.

GRAS domain proteins are a type of protein unique to plants. They generally contain 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 shoot development, etc.

Contents of the invention

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

In order to solve the above technical problems, the present invention first provides any of the following applications of proteins or substances that regulate the activity or content of the proteins:

D1) Regulating plant salt tolerance;

D2) Prepare products for regulating plant salt tolerance;

D3) Cultivate plants with enhanced salt tolerance;

D4) Prepare and cultivate plant products with enhanced salt tolerance;

D5)Plant breeding;

The protein is derived from soybeans, and its name is GmGRAS487, which is as follows A1), A2) or A3):

A1) The amino acid sequence is a protein of sequence 2;

A2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing;

A3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of A1) or A2).

In order to facilitate the purification of the protein in A1), a tag as shown in the table below can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing.

Table: Sequence of tags

Label Residues sequence 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) above is a protein that has 75% or more identity with the amino acid sequence of the protein shown in Sequence 2 and has the same function. The said having 75% or more identity means having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% identity. .

The GmGRAS487 protein in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then biologically expressed.

The gene encoding the GmGRAS487 protein in the above A2) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in Sequence 1, and/or performing a missense mutation of one or several base pairs, and /Or obtained by connecting the coding sequence of the tag shown in the above table to its 5' end and/or 3' end. Among them, the DNA molecule shown in sequence 1 encodes the GmGRAS487 protein shown in sequence 2.

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

B1) Nucleic acid molecules encoding GmGRAS487;

B2) An expression cassette containing the nucleic acid molecule described in B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

B7) Transgenic plant organs containing the nucleic acid molecule described in B1), or transgenic plant organs containing the expression cassette described in B2);

B8) Nucleic acid molecules that reduce the expression of GmGRAS487;

B9) Expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B8).

In the above application, the nucleic acid molecule described in B1) can be the following b11) or b12) or b13) or b14):

b11) The coding sequence is the cDNA molecule or DNA molecule of sequence 1 in the sequence listing;

b12) cDNA molecule or DNA molecule of sequence 1 in the sequence listing;

b13) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes GmGRAS487;

b14) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by b11) or b12) or b13) under stringent conditions and encodes GmGRAS487.

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

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the GmGRAS487 protein of the present invention using known methods, such as directed evolution and point mutation. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the GmGRAS487 protein isolated in the present invention, as long as they encode the GmGRAS487 protein and have the function of the GmGRAS487 protein, are derived from the nucleic acid of the present invention. The nucleotide sequence is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

In the above application, the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5MNaPO ₄ and 1mM EDTA, 2×SSC, 0.1% at 50°C Rinse in SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse in 50°C, 1×SSC, 0.1% SDS; also: 50°C, Hybridize in a mixed solution of 7% SDS, 0.5M NaPO and 1mM EDTA _, rinse in 0.5×SSC, 0.1% SDS at 50°C; also: 50°C, in 7% SDS, _0.5M NaPO and 1mM Hybridization in a mixed solution of EDTA, rinsed in 0.1×SSC, 0.1% SDS at 50°C; alternatively: hybridization at 50°C, in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, at 65°C. Rinse in 0.1×SSC, 0.1% SDS; also: hybridize in 6×SSC, 0.5% SDS solution at 65°C, and then use 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS. Wash the membrane once; it can also be: hybridize and wash the membrane twice in a solution of 2×SSC, 0.1% SDS at 68°C, 5 min each time, and then in a solution of 0.5×SSC, 0.1% SDS, at 68°C. Hybridize and wash the membrane twice, 15 minutes each time; it can also be: hybridize and wash the membrane in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C.

The above-mentioned 75% or above identity may be 80%, 85%, 90% or 95% or above identity.

In the above application, the expression cassette containing the nucleic acid molecule encoding the GmGRAS487 protein (GmGRAS487 gene expression cassette) described in B2) refers to the DNA that can express the GmGRAS487 protein in the host cell. The DNA can not only include the promoter for initiating the transcription of the GmGRAS487 gene. The terminator may also include a terminator that terminates the transcription of the GmGRAS487 gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention 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: the constitutive promoter 35S from cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter, pathogenesis-related 1 (PR1) from tobacco (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline inducible promoter (U.S. Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited 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, for example: Odell et al. ( ¹⁹⁸⁵ ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (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).

Existing expression vectors can be used to construct a recombinant vector containing the GmGRAS487 gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' untranslated region of the foreign gene, that is, containing the poly(A) signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide poly(A) to be added to the 3′ end of the mRNA precursor, such as the Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed at the 3' end of storage protein genes all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be the same as the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The translation control signals and initiation codons come from a wide range of sources, and may be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or from a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes (GUS genes, luciferase genes) that can be expressed in plants. genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or resistance to chemical reagent marker genes (such as herbicide resistance genes), mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of transgenic plants, the transformed plants can be directly screened by stress without adding any selective marker genes.

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

B3) The recombinant vector may specifically be pROKII-GmGRAS487. The pROKII-GmGRAS487 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and KpnI recognition sequences of the pROKII vector with the GmGRAS487 gene shown in Sequence 1 in the Sequence Listing, and can express GmGRAS487 shown in Sequence 2 in the Sequence Listing and NPTⅡ. The fusion protein formed.

B9) The recombinant vector can specifically be pZH01-GmGRAS487-RNAi. pZH01-GmGRAS487-RNAi is a recombinant vector obtained by inserting the DNA fragment shown at positions 1077 to 1363 of Sequence 1 twice into the multiple cloning site of pZH01. , the two inserted fragments are in opposite directions.

In the above applications, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes K599.

In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

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

M1) Dicotyledonous plants or monocotyledonous plants;

M2) Legumes;

M3) Soybeans.

The present invention also provides any of the following methods:

X1) Methods for cultivating plants with enhanced salt tolerance, including knocking out the coding gene expressing GmGRAS487 in the recipient plant, or inhibiting the expression of the GmGRAS487 coding gene in the recipient plant, or reducing the content of GmGRAS487 in the recipient plant, or reducing the receptor The activity of GmGRAS487 in plants can lead to target plants with enhanced salt tolerance;

X2) Methods for enhancing plant salt tolerance, including knocking out the gene encoding GmGRAS487 in the recipient plant, or inhibiting the expression of the gene encoding GmGRAS487 in the recipient plant, or reducing the content of GmGRAS487 in the recipient plant, or reducing the amount of GmGRAS487 in the recipient plant. The activity of GmGRAS487 can be used to obtain target plants with enhanced salt tolerance, thus achieving the enhancement of plant salt tolerance.

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

In the above method, the encoding gene may be the nucleic acid molecule described in B1).

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

1) Modify and optimize according to actual needs to enable efficient expression of the gene; for example, according to the codons preferred by the recipient plant, the codons of the GmGRAS487 encoding gene of the present invention can be changed while maintaining the amino acid sequence to conform to the codons preferred by the recipient plant. Plant preference; during the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to best achieve high-level expression of the introduced genes in plants, where the GC content can be 35% or more than 45% , more than 50% or more than about 60%;

2) Modify the gene sequence adjacent to the starting methionine to enable efficient initiation of translation; for example, use a known effective sequence in plants for modification;

3) Connect to promoters expressed in various plants to facilitate their expression in plants; the promoters may include constitutive, inducible, temporal regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters ;The choice of promoter will vary with the temporal and spatial requirements for expression, and will also depend on the target species; e.g. tissue or organ specific expression promoters, depending on at what stage of development the receptor is required; although proven sources Many promoters for dicots will work in monocots and vice versa, but ideally, a dicot promoter is selected for expression in dicots and a monocot promoter for Expression in monocots;

4) Connecting with a suitable transcription terminator can also improve the expression efficiency of the gene of the invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to work in plants can be combined with The genes of the present invention are connected;

5) Introducing enhancer sequences such as intron sequences (eg derived from Adhl and bronzel) and viral leader sequences (eg derived from TMV, MCMV and AMV).

The GmGRAS487 encoding gene can be introduced into the recipient plant using a recombinant vector containing the GmGRAS487 encoding gene. The recombinant vector may specifically be the pCAMBIA1301-GmGRAS487.

The recombinant vector can be introduced into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated and other conventional biological methods, and the transformed plant tissue can be cultivated into plant. The transformed plant host can be either a monocot or a dicot.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes encoding enzymes or luminescent compounds that can be expressed in plants to produce color changes (GUS genes, luciferase genes etc.), resistance antibiotic markers (gentamicin markers, kanamycin markers, etc.) or resistance to chemical reagent marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, the transformed plants can be screened directly by drought treatment without adding any selective marker genes.

The target plant is understood to include not only the first generation plants in which the GmGRAS487 protein or its encoding gene has been altered, but also its progeny. For the plant of interest, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The target plants include seeds, calli, intact plants and cells.

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

M1) Dicotyledonous plants or monocotyledonous plants;

M2) Legumes;

M3) Soybeans.

The present invention also provides products for enhancing plant salt tolerance, which products contain GmGRAS487 or the substance that regulates the activity or content of the protein.

The product may have GmGRAS487 or the substance that regulates the activity or content of the protein as its active ingredient. GmGRAS487 or the substance that regulates the activity or content of the protein may also be combined with a substance with the same function as its active ingredient. Element.

GmGRAS487 or the substances that regulate the activity or content of the protein also belong to the protection scope of the present invention.

In the present invention, the salt tolerance may specifically be the plant's tolerance 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 100mM).

The salt tolerance of plants can be reflected by plant survival rate, leaf wilting degree and/or leaf relative ion permeability.

The present invention transfers the coding gene of transcription factor GmGRAS487 into the hairy roots of recipient soybeans to obtain transgenic hairy roots and transgenic chimeras. Compared with the hairy roots of soybeans transformed into empty vectors, the transgenic soybean hairy roots are more salt-tolerant. Sex has improved significantly. This shows that the transcription factor GmGRAS487 and its encoding gene can regulate plant salt tolerance and have important theoretical and practical significance for cultivating plant varieties with high salt tolerance.

Description of the drawings

Figure 1 shows the transcription pattern of GmGRAS487 under 120mM NaCl treatment.

Figure 2 is a schematic diagram of the plant expression vectors pROKII-GmGRAS487 and pZH01-GmGRAS487-RNAi.

Figure 3 shows the molecular identification of overexpressed GmGRAS487 and GmGRAS487-RNAi soybean hairy roots.

Figure 4 shows the salt-tolerant phenotype of GmGRAS487 hairy roots and chimeras.

Figure 5 shows the statistics of physiological indicators of GmGRAS487 transgenic hairy root chimeras under salt stress. *, p<0.05; **, p<0.01.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5' terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal core of the corresponding DNA/RNA. glycosides.

The soybean Glycine max L.Merr.Kefeng 1 in the following examples 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, QTLmapping of ten agronomic traits on the soybean(Glycine max L.Merr.) geneticmap and their association with EST markers,Theor.Appl.Genet,2004,108:1131-1139, which is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,

Soybean [Glycine max (L.) Merr] Nannong 1138-2: Germplasm bank of the National Soybean Improvement Center of Nanjing Agricultural University, provided by the National Soybean Improvement Center of Nanjing Agricultural University.

The expression vector pROKII vector (binary expression vector) in the following examples 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. Nature 321 (1986) , pp.446–449, available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences;

pZH01 vector, Stratagene Company, documented 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 also available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences .

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), which can be obtained from Professor Peter M Gressnon , The University of Queensland, St Lucia, Queensland 4072, Australia, or obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Professor Peter M Gressnon (written consent).

Example 1. Screening of the gene encoding soybean transcription factor GmGRAS487 and its cDNA cloning

In the transcriptome analysis of soybeans Kefeng 1 (salt-sensitive) and Nannong 1138-2 (salt-tolerant) under normal and high-salt stress, the inventor screened a gene with the locus name Glyma08g15530 (Locus name: Glyma.08G146900) . When Glyma08g15530 was treated with 120mM NaCl, its transcription amount decreased sharply, considering that it may negatively regulate salt tolerance in plants. The salt-tolerant soybean variety Nannong 1138-2 was cultured under light, and after 2 weeks of growth, the seedlings were taken to extract RNA. Grind 1g of fresh seedlings in liquid nitrogen and suspend in 4mol/L guanidine sulfate. The mixture is extracted with acidic phenol and chloroform. Absolute ethanol is added to the supernatant to precipitate total RNA. Then, it is dissolved in water to obtain total RNA. , use reverse transcriptase to reverse transcribe to synthesize cDNA. The primers are:

Gm15530-F1: ATGAAAACGATGGATTTTGA and

Gm15530-R1: CTATTTGACCTTGTCATCCAGATAA.

Perform Real Time-PCR identification. Real-Time PCR reaction uses TOYOBO's RealTime PCRMaster Mix kit and operates according to the instructions. The soybean Tublin gene is the internal standard, and the primers used are Primer-TF: 5’-AACCTCCTCCTCATCGTACT, and Primer-TR: 5’-GACAGCATCAGCCATGTTCA.

Figure 1 shows that Glyma08g15530 decreased significantly when treated with 120mM NaCl for 1 hour, and continued to reach the lowest point at 3 hours. Sequencing showed that Glyma08g15530 contains 1464 bp, encoding 487 amino acid residues, and the protein was named GmGRAS487. In Nannong 1138-2, the amino acid sequence of GmGRAS487 is sequence 2 in the sequence listing, and the DNA coding sequence is sequence 1.

Example 2. Construction of plant expression vector for soybean transcription factor GmGRAS487

1. Construction of GmGRAS487 overexpression vector pROKII-GmGRAS487

Using the cDNA of Nannong 1138-2 as the template, Gm15530-pROKII-F2 and Gm15530-pROKII-R2 were used for PCR amplification to obtain PCR products.

Gm15530-pROKII-F2: AGAACACGGGGGACTCTAGAATGAAAACGATGGATTTTGA;

Gm15530-pROKII-R2:GATCGGGGAAATTCGAGCTCCTATTTGACCTTGTCATCCAGATAA.

The pROKII vector was double-digested with restriction endonucleases BamHI and KpnI, and the PCR recovery fragment was ligated into the pROKII vector using homologous recombination to obtain the recombinant vector pROKII-GmGRAS487 (part of the vector schematic is shown in Figure 2). pROKII-GmGRAS487 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and KpnI recognition sequences of the pROKII vector with the GmGRAS487 gene shown in Sequence 1 in the Sequence Listing. It can express the gene formed by GmGRAS487 shown in Sequence 2 in the Sequence Listing and NPTⅡ. Fusion proteins.

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

First, the total RNA of Nannong 1138-2 soybean was extracted using the Trizol method. The cDNA obtained by reverse transcription was used as a template, and the vector construction primer was used to amplify the 287 bp DNA fragment from positions 1077 to 1363 of the CDS region of the GmGRAS487 gene, which was used to construct the RNAi vector.

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

GmGRAS487Ri-F1: (Single underline is the recognition sequence of Xba I, double underline is the recognition sequence of Sac I);

GmGRAS487Ri-R1: (Single underline is the recognition sequence of Sal I, double underline is the recognition sequence of Kpn I).

First, double-digest the PCR product and the pZH01 vector with Sac I and Kpn I respectively, connect the resulting large fragment of the PCR product to the vector backbone, and use Xba I and Sal I to recombine the vector and PCR product with the correct sequence. Double-enzyme digestion was performed, and the large fragment of the obtained PCR product was connected to the vector backbone, and the recombinant vector with correct sequence was recorded as pZH01-GmGRAS487-RNAi (Figure 2).

The vectors constructed above have been sequenced, and the next step of the experiment is carried out after verifying that the sequence is correct.

Example 3. Obtaining hairy roots of soybeans transgenic with GmGRAS487 gene

The Agrobacterium rhizogenes infection method is slightly modified based on the method of Attila Kereszt and others (Attila Kereszt, et al., Agrobacterium rhizogenes-mediaded transformation of soybean to study of rootbiology, 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, GmWRKY27interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants, 2015, The Plant Journal, 83, 224–236", or patent " Chen Shouyi et al., Plant stress tolerance-related transcription factor GmWRKY78 and its encoding gene and application, patent number: ZL2011 1 0053083.7, grant date 2013.10.09" were carried out using Agrobacterium rhizogenes-mediated transgenic root method.

Preparation of hairy roots overexpressing GmGRAS487 and GmGRAS487-RNAi:

1) Obtaining recombinant Agrobacterium

The recombinant expression vectors pROKⅡ-GmGRAS487 and pZH01-GmGRAS487-RNAi obtained in Example 2 were introduced into Agrobacterium rhizogenes K599 by electroporation method to obtain recombinant Agrobacterium. The recombinant Agrobacterium containing the above plasmids were named K599/pROKⅡ-GmGRAS487 and K599/pZH01-GmGRAS487-RNAi respectively.

2) Hairy root transformation

Use a syringe to inoculate the above-mentioned recombinant Agrobacterium K599/pROKⅡ-GmGRAS487 and K599/pZH01-GmGRAS487-RNAi into soybean Kefeng No. 1 seedlings containing two true leaves that have grown for 6 days. For specific methods, see the above citation. Growth under moisturizing conditions: 16 hours of light. , temperature 25℃, humidity 50%. After 2 weeks, the hairy roots that grow are transgenic hairy roots. 121 K599/pROKII-GmGRAS487 overexpression chimeras (GmGRAS487-OE) and 120 K599/pZH01-GmGRAS487-RNAi chimera plants (GmGRAS487-RNAi) were obtained respectively, which can be used for further transgene identification and salt tolerance testing.

Agrobacterium rhizogenes K599/pROKII containing the empty vector pROKⅡ was transferred into soybean Kefeng No. 1 using the same method, and 124 hairy root systems transformed into the empty vector were obtained, which were used as empty vector controls.

3) Molecular identification of transgenic hairy roots

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

Figure 3 shows that the expression levels of GmGRAS487 in the empty vector control, GmGRAS487-OE and GmGRAS487-RNAi hairy roots are approximately 0.024, 2.35 and 0.013 respectively. The expression level of GmGRAS487 in GmGRAS487-OE is extremely significantly higher than the empty vector control, while The expression of GmGRAS487 in GmGRAS487-RNAi hairy roots was significantly lower than the control (Figure 3).

Example 4. Identification of salt tolerance of GmGRAS487 and GmGRAS487-RNAi hairy roots

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

Divide the above three experimental samples into 2 groups, with about 12 samples each. One group is treated with 100mM NaCl aqueous solution for 3 days, that is, immersed in 100mM NaCl solution and treated at 25°C for 3 days; the second group is immersed in water as a control. The experiment was repeated three times, and the results were averaged ± standard deviation.

After treatment with 100mM NaCl aqueous solution for 3 days, take pictures and observe. Figure 4 shows the plant and leaf phenotypes of the hairy roots transformed into the empty vector and two transgenic hairy roots. It can be seen that the hairy roots transformed into the empty vector (pROKⅡ) and the GmGRAS487 gene and GmGRAS487-RNAi were transformed under water treatment (normal conditions). There was no significant difference in the growth of hairy root chimeras. After 100mM NaCl treatment for 3 days, the degree of wilting of hairy root chimeras and leaves (GmGRAS-RNAi) transfected with GmGRAS487-RNAi was significantly lower than that of control chimeras and leaves, while overexpression of GmGRAS487 hairy root chimeras The wilting degree of root chimera (GmGRAS-OE) and leaves was significantly higher than that of the control.

Survival rate test:

The right side of Figure 5 shows that under hydroponic conditions, the survival rate of all plants was 100%. After 100mM NaCl treatment for 3 days, the survival rates of the control, GmGRAS-RNAi and GmGRAS-OE chimeras were approximately 40%, 63% and 18, respectively. %, the differences between the overexpression chimera and RNAi chimera and the control were significant and extremely significant. It shows that overexpression of GmGRAS487 reduces the salt tolerance of plants, while reducing its expression increases the salt tolerance of plants.

NaCl treatment ion permeability detection:

When plant tissue is damaged by adverse stress, the function of the cell membrane is damaged or the structure is destroyed, and the permeability increases, causing various water-soluble substances in the cell, including electrolytes, to leak out. When plant tissue is immersed in ion-free water, the conductance of the water increases due to extravasation of electrolytes. The more serious the injury, the more severe the damage to the cell membrane, the greater the extravasation, and the greater the conductivity of water. Therefore, a conductivity meter can be used to measure changes in the conductivity of the exudate, which indirectly reflects the degree of damage to plant tissues. Therefore, the detection of electrical conductivity can calculate the relative ion permeability, which indicates the degree of damage to the plant cell membrane.

The measurement method is to cut off the soybean leaves, place them in a clean screw-top glass bottle, and rinse them three times with deionized water. Then add 80 mL of deionized water to completely soak the leaves, and vacuum for 45 minutes. After standing at room temperature for 30 minutes, conductivity E1 was measured with a conductivity meter (DDC-308A model, Shanghai Bochu Instrument Co., Ltd.). Boil the leaves for 15 minutes. After the temperature drops to room temperature, mix well and measure the conductivity E2 with a conductivity meter.

Relative ion permeability EL (%) = E1/E2 X 100, where E1 and E2 are conductivities.

The left picture of Figure 5 shows that under hydroponics, the ion permeability of all plants is very low, about 6%. After 100mM NaCl treatment for 3 days, the survival rates of the control, GmGRAS-RNAi and GmGRAS-OE chimeras were about 47, respectively. %, 62% and 17%, indicating that the GmGRAS487 overexpression chimera suffered the most serious damage to the leaf cell membrane under salt stress, followed by the control, while reducing the expression of GmGRAS487 significantly protected the cell membrane from damage under salt stress.

The above results show that in plants, GmGRAS487 negatively regulates salt tolerance, reduces the expression of GmGRAS487, and significantly improves the salt tolerance of plants.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

sequence list

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> Application of soybean transcription factor GmGRAS487 in the regulation of plant salt tolerance

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<170> PatentIn version 3.5

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Claims (10)

1. Any of the following applications of proteins or substances that regulate the activity or content of said proteins: D1) Regulating plant salt tolerance; D2) Prepare products for regulating plant salt tolerance; D3) Cultivate plants with enhanced salt tolerance; D4) Prepare and cultivate plant products with enhanced salt tolerance; D5)Plant breeding; The protein is the following A1), A2) or A3): A1) The amino acid sequence is a protein of sequence 2; A2) A protein that has the same function by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing; A3) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of A1) or A2). 2. Application according to claim 1, characterized in that: the substance is any one of the following B1) to B9): B1) Nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecule described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) Nucleic acid molecules that reduce the expression level of the protein described in claim 1; B9) Expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B8). 3. The application according to claim 2, characterized in that: B1) the nucleic acid molecule is as follows b11) or b12) or b13) or b14): b11) The coding sequence is the cDNA molecule or DNA molecule of sequence 1 in the sequence listing; b12) cDNA molecule or DNA molecule of sequence 1 in the sequence listing; b13) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes the protein described in claim 1; b14) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by b11) or b12) or b13) under stringent conditions and encodes the protein of claim 1. 4. The application according to any one of claims 1-3, characterized in that: the plant is M1) or M2) or M3): M1) Dicotyledonous plants or monocotyledonous plants; M2) Legumes; M3) Soybeans. 5. Any of the following methods: X1) A method for cultivating plants with enhanced salt tolerance, comprising knocking out the gene encoding the protein described in claim 1 in the recipient plant, or inhibiting the expression of the protein encoding gene described in claim 1 in the recipient plant, or reducing the The content of the protein described in claim 1 in the recipient plant, or reducing the activity of the protein described in claim 1 in the recipient plant, to obtain a target plant with enhanced salt tolerance; X2) A method for enhancing plant salt tolerance, including knocking out the gene encoding the protein described in claim 1 in the recipient plant, or inhibiting the expression of the protein encoding gene described in claim 1 in the recipient plant, or reducing the expression of the protein encoding gene in the recipient plant. The content of the protein described in claim 1 in the body plant, or the activity of the protein described in claim 1 in the recipient plant is reduced, so as to obtain the target plant with enhanced salt tolerance and achieve the enhancement of the salt tolerance of the plant. 6. The method according to claim 5, characterized in that: X1) and The expression of the protein coding gene is achieved by a nucleic acid molecule or a recombinant vector expressing the nucleic acid molecule. 7. The method according to claim 6, characterized in that the encoding gene is the nucleic acid molecule described in B1) of claim 2 or 3. 8. The method according to any one of claims 5-7, characterized in that: the recipient plant is M1) or M2) or M3): M1) Dicotyledonous plants or monocotyledonous plants; M2) Legumes; M3) Soybeans. 9. A product for enhancing plant salt tolerance, containing the protein of any one of claims 1-3 or the substance that regulates the activity or content of the protein. 10. The protein of any one of claims 1-3 or the substance that regulates the activity or content of the protein.