CN107459565A - Application of the soybean drought resisting GAP-associated protein GAP in regulating and controlling soybean drought resistance - Google Patents

Abstract

The invention discloses the application of soybean drought-resistance-related protein in regulating soybean drought-resistance. The soybean drought resistance-related protein (GmSQE1) disclosed by the present invention is the following A1) or A2) or A3): A1) the protein whose amino acid sequence is sequence 1; A2) the amino acid sequence of sequence 1 is substituted and/or deleted and/or A protein derived from A1) with the same function obtained by adding one or several amino acid residues; A3) a fusion protein obtained by linking tags at the N-terminal or/and C-terminal of A1) or A2). Experiments have shown that, compared with plants without GmSQE1 gene transfection, plants positively transfected with GmSQE1 gene show drought-tolerant traits after drought treatment; and after rehydration treatment, plants without GmSQE1 gene transfection die, while positive GmSQE1 gene transgenic plants can normal growth. It was shown that GmSQE1 and its coding gene can improve the drought resistance of plants.

Description

Application of Soybean Drought Resistance Related Proteins in Regulating Soybean Drought Resistance

technical field

The invention relates to the application of soybean drought-resistance-related protein in regulating soybean drought-resistance in the field of biotechnology.

Background technique

With global warming and increasing land desertification, drought has become an extremely important factor threatening global agricultural development and food security. Drought stress can cause the wilting and stunting of crops, which in turn leads to crop yield reduction. It is very difficult to change the general trend of global warming in a short period of time, so cultivating crops with drought resistance through modern biotechnology is a good solution.

Soybean (Glycine max) is one of the five major economic crops that originate in China and have important food security status in the world. my country is the world's largest importer and consumer of soybeans, and soybeans are of great strategic significance to ensure the security of my country's oil supply. Soybeans can provide people with high-quality soybean oil and soybean plant protein. Soybean cake, a by-product of soybean processing, is widely used as high-quality breeding feed. However, with global warming and the gradual scarcity of fresh water resources, drought has had an important impact on soybean yield. Therefore, cultivating soybean varieties with good drought resistance is of great significance for soybean production. At present, soybean drought-resistant breeding mainly focuses on traditional hybrid breeding and transgenic breeding techniques. Due to the lack of excellent drought-resistant germplasm resources in traditional cross-breeding, traditional cross-breeding requires continuous hybridization and combination of populations, and then obtains separated populations to find drought-resistant offspring plants, and then continuously breeds to select stable genetic drought-resistant offspring. Traditional soybean hybrid breeding takes a long time and requires a lot of manpower and material resources. Moreover, traditional soybean cross-breeding may cause parents to lose their original good traits, such as yield, plant type, and disease resistance, after acquiring drought-resistant traits. Soybean transgenic breeding can quickly introduce drought resistance genes into parent soybean varieties with excellent trait background through plant genetic transformation technology on the premise of clarifying the key genes of drought resistance, and determine the homozygousity of single-copy gene insertion by screening transgenic positive soybean plants offspring plants. Drought-resistant breeding of transgenic soybeans can conveniently and quickly breed new soybean varieties with definite drought resistance. At present, domestic and international soybean transgenic breeding is mainly focused on cultivating herbicide resistance, insect resistance and regulating growth and development. So far, there are few reports on soybean drought-resistant transgenic breeding.

Contents of the invention

The technical problem to be solved by the invention is how to improve the drought resistance of plants.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of drought-resistance-related proteins in regulating plant drought resistance; the name of the drought-resistance-related proteins is GmSQE1, which is as follows A1) or A2) or A3):

A1) a protein whose amino acid sequence is sequence 1;

A2) a protein derived from A1) with the same function obtained through substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence of sequence 1;

A3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of A1) or A2).

In order to make the protein in A1) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1. 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

For the GmSQE1 protein in A2) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The GmSQE1 protein in the above A2) can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed.

The gene encoding the GmSQE1 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 2, and/or performing a missense mutation of one or several base pairs, and /or obtained by linking the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The present invention also provides the application of biological materials related to GmSQE1 in regulating the drought resistance of plants;

The biological material is any one of the following B1) to B14):

B1) a nucleic acid molecule encoding GmSQE1;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1);

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule of B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

B9) a transgenic plant cell line containing the nucleic acid molecule of B1);

B10) a transgenic plant cell line containing the expression cassette of B2);

B11) a transgenic plant tissue containing the nucleic acid molecule of B1);

B12) transgenic plant tissue containing the expression cassette described in B2);

B13) a transgenic plant organ containing the nucleic acid molecule of B1);

B14) A transgenic plant organ containing the expression cassette described in B2).

In the above application, the nucleic acid molecule described in B1) can be the gene shown in b1), b2), b3) or b4) as follows:

b1) The nucleotide sequence is a cDNA molecule or a DNA molecule of sequence 2 in the sequence listing;

b2) The nucleotide sequence is a cDNA molecule or a DNA molecule of sequence 3 in the sequence listing;

b3) It has 75% or more identity with b1) or b2) or the defined nucleotide sequence, and encodes a cDNA molecule or genomic DNA molecule of GmSQE1;

b4) a cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes GmSQE1.

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.

Wherein, the DNA molecule shown in sequence 2 encodes the GmSQE1 protein shown in sequence 1.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the GmSQE1 protein of the present invention. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the isolated GmSQE1 protein of the present invention, as long as they encode the GmSQE1 protein and have the function of the GmSQE1 protein, are all derived from the core of the present invention. Nucleotide sequence and 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 higher, of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed visually or with computer software. Using computer software, 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-mentioned application, the stringent conditions are in a solution of 2×SSC and 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC and 0.1% SDS, in Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

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

In the above-mentioned application, the expression cassette (GmSQE1 gene expression cassette) described in B2) containing the nucleic acid molecule encoding the GmSQE1 protein refers to the DNA that can express the GmSQE1 protein in the host cell, and the DNA can not only include the initiation of GmSQE1 gene transcription A terminator that terminates the transcription of the GmSQE1 gene may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used 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: Cauliflower Mosaic Virus Constitutive Promoter 35S: Wound-Inducible Promoter from Tomato, Leucine Aminopeptidase ("LAP", Chao et al. (1999) PlantPhysiol 120:979 -992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease Inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US 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, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. 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 terminators (see, e.g.: 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 Acids Res., 15:9627).

An existing expression vector can be used to construct a recombinant vector containing the expression cassette of the GmSQE1 gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or 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 (GUS gene, luciferase gene, etc.) 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 the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the above application, the vector can be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be vector pGWC or plasmid pB2GW7.0.

The recombinant vector can specifically be GmSQE1-pGWC or GmSQE1-pB2GW7.0, the GmSQE1-pGWC is a recombinant vector obtained by introducing the DNA fragment shown in Sequence 2 into the vector pGWC, and the GmSQE1-pB2GW7.0 is the sequence The recombinant vector obtained by introducing the DNA fragment shown in 2 into plasmid pB2GW7.0, said GmSQE1-pGWC can express the GmSQE1 protein shown in sequence 1.

In the above applications, the microorganisms can be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Agrobacterium, such as Agrobacterium EHA105.

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

The present invention also provides the application of GmSQE1 or the biological material in cultivating plants with enhanced drought resistance.

The present invention also provides a method for cultivating plants with enhanced drought resistance, the method comprising: increasing the activity of GmSQE1 in the target plant, increasing the content of GmSQE1 in the target plant, promoting the expression of the coding gene of GmSQE1, and obtaining the same expression as the target plant Drought-resistant plants with enhanced drought resistance.

In the above method, the drought-resistant plant may be a transgenic plant obtained by introducing a gene encoding GmSQE1 into the target plant.

In the above method, the gene encoding GmSQE1 can be the nucleic acid molecule described in B1).

The present invention also provides a product for regulating drought resistance of plants, which contains GmSQE1 or the biological material.

The product can use GmSQE1 or the biological material as its active ingredient, and can also use GmSQE1 or the biological material together with other substances with the same function as its active ingredient.

The invention also provides the application of the product in regulating the drought resistance of plants.

In the present invention, the plant can be a dicotyledon or a monocotyledon; the target plant can be a dicotyledon or a monocotyledon. The dicot may be a leguminous plant. The leguminous plant can be soybean, such as soybean variety Tianlong No. 1.

In the present invention, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the GmSQE1 gene, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

Experiments have shown that, compared with plants without GmSQE1 gene transfection, plants positively transfected with GmSQE1 gene show drought-tolerant traits after drought treatment; and after rehydration treatment, plants without GmSQE1 gene transfection die, while positive GmSQE1 gene transgenic plants can normal growth. It was shown that GmSQE1 and its coding gene can improve the drought resistance of plants.

Description of drawings

Figure 1 shows the results of screening positive transgenic soybeans with glufosinate-resistant transgenic test strips.

Figure 2 is the identification of drought resistance of transgenic soybeans.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

pGWC (Huang et al., Cloning and Expression Analysis of the Soybean CO-Like Gene GmCOL9, Plant Mol Biol Rep (2011) 29:352-359) in the following examples, the public can obtain this biological material from the applicant, The biological material is only used for repeating related experiments of the present invention, and cannot be used for other purposes.

For the CCM liquid medium in the following examples, 1L contains 0.321g of B-5 powder, 5.9g of MES, 9ml of 100*B5 organic, 30g of sucrose, 1ml of Na ₂ S ₂ O ₃ solution, 4ml of L-cys solution, and DTT solution 1ml, 2ml of AS solution, 1.67ml of 6-BA solution and 100μl of GA3 solution, the balance is water. The CCM solid medium is a medium obtained by adding agar powder to the CCM liquid medium, and the pH is 5.4.

In the liquid bud induction medium in the following examples, 1 L contains 3.21 g of B-5 powder, 0.59 g of MES, 30 g of sucrose, 1 ml of Timentin solution, 1.12 ml of 6-BA solution and 0.4 ml of Cef solution, and the balance is water. The SIM solid medium is a medium obtained by adding agar powder and PPT solution 500 μl/500ml to the liquid shoot induction medium.

Solid shoot elongation medium in the following examples, 1L contains MS 4.43g, MES 0.59g, 100*B5 organic 9ml, sucrose 30g, IAA solution 100μl, Cef solution 400μl, GA3 solution 600μl, PPT solution 400μl, Glu solution 1ml, 1ml of Timentin solution, 1ml of ZR solution, 1ml of Asp solution and agar powder, the balance is water, pH=5.6.

Rooting medium in the following examples, 1L contains MS 2.22g, MES 0.59g, 100*B5 organic 9ml, sucrose 20g, Cef solution 400μl, Timentin solution 1ml, IBA solution 1ml, Glu solution 1ml, Asp solution 1ml and agar powder, the balance is water, pH=5.7.

Among them, every 500ml of 100*B5 contains: myo-lnositol 5g, niacin (Nicotinic acid) 0.05g, pyridoxic acid hydrochloride (vitamin B6) 0.05g, thiamine hydrochloride (Thiamine hydrochloride) 0.5g, more than The amount is water.

Na ₂ S ₂ O ₃ (sodium thiosulfate) solution: 3.16g Na ₂ S ₂ O ₃ /20ml, dissolved in distilled water.

L-cys (L-cysteine) solution: 10g L-cys/100ml, dissolved in distilled water.

DTT (dithiothreitol, DL-Dithiothreitol) solution: 3.08g DTT/20ml, dissolved in 3M NaAC aqueous solution.

AS (acetosyringone) solution: 0.392g AS/20ml, dissolved in 95% ethanol aqueous solution.

6-BA (6-benzylaminoadenine) solution: 1mg6-BA/ml, first dissolved in a small amount of 1M NaOH aqueous solution, and then distilled water to make up to volume.

GA3 (gibberellin) solution: 1mg GA3/ml, dissolved in 95% ethanol aqueous solution.

Temetime solution: 80mg Temetime/ml, dissolved in distilled water.

Cef (cephalosporin) solution: 250mg Cef/ml, dissolved in distilled water.

IAA (indole acetic acid) solution: 1mg IAA/ml, dissolve in a small amount of 95% ethanol aqueous solution, and then distilled water to make it to volume.

PPT (herbicide-glufosinate-ammonium) solution: 5mg PPT/ml, dissolved in distilled water.

Glu (glutamic acid) solution: 50mg Glu/ml, dissolved in distilled water.

ZR (trans-corn nucleotide) solution: 1mg ZR/ml, dissolve in a small amount of 1M HCl solution, and then distilled water to make up to volume.

Asp (aspartic acid) solution: 50mg Asp/ml, first dissolve a small amount of 1M HCl aqueous solution, and then distilled water to make up to volume.

IBA (indole butyric acid) solution: 1mg IBA/ml, dissolved in 95% ethanol solution.

Example 1, Soybean Drought Resistance-related Protein GmSQE1 Can Regulate Soybean Drought Resistance

This embodiment provides a soybean drought-resistance-related protein derived from soybean variety willam82, which is named GmSQE1, and the sequence of GmSQE1 is sequence 1 in the sequence listing. In soybean variety willam82, the CDS sequence of GmSQE1 is sequence 2 in the sequence listing, and the genome sequence is sequence 3 in the sequence listing.

The DNA molecule shown in Sequence 2 (i.e. the GmSQE1 encoding gene) is transferred to soybean to detect the function of GmSQE1 in regulating soybean, the specific method is as follows:

1. Construction of GmSQE1 expression recombinant vector and recombinant bacteria

The primer pair consisting of upstream primer (F: AGGCTTTGACTTTAGGTC ATGATGGGTTATGAGTATATTTTGG) and downstream primer (R: GTCTAGAGACTTTAGGTC TTAATCTTCCAAATTGGTAGGG) was used to amplify the cDNA of soybean variety willam82 by PCR, and the obtained PCR product was identical to the intermediate vector pGWC by homologous recombination. Source recombination, the GmSQE1 coding gene shown in Sequence 2 was transferred to the intermediate vector pGWC, and the resulting recombinant vector containing the correct sequence was named GmSQE1-pGWC.

In the way of homologous recombination, GmSQE1-pGWC and plasmid pB2GW7.0 (Fibrillin 5Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate Synthases in Arabidopsis) were subjected to gateway reaction to obtain a recombinant vector with correct sequence, which was named GmSQE1-pB2GW7. 0, GmSQE1-pB2GW7.0 is the GmSQE1 expression recombinant vector, which can express the GmSQE1 shown in sequence 1.

Introduce GmSQE1-pB2GW7.0 into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria, and name the recombinant bacteria EHA105/GmSQE1-pB2GW7.0; introduce pB2GW7.0 into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria EHA105/GmSQE1 , as an empty vector control.

2. Construction of transgenic soybean

The recombinant strain EHA105/GmSQE1-pB2GW7.0 was used to transform the soybean variety Tianlong No. 1 through the Agrobacterium infection method to obtain transgenic soybeans, and the empty vector plants obtained by using the recombinant strain EHA105/GmSQE1 were used as controls. The specific method is as follows:

2.1 Preparation of Agrobacterium infection solution:

The recombinant bacteria were added to LB liquid medium containing rifampicin (50ng/L) and spectinomycin (50ng/L), cultivated overnight at 28°C and 200rpm. During this period, the concentration of the bacterial solution was detected, and when the OD600 value reached 1.2-1.5, it was centrifuged at 4000 rpm for 10 minutes to collect the bacterial cells. Resuspend in CCM liquid medium (Co-culture Medium, CCM), adjust OD600 to about 0.6, obtain bacterial cell suspension, set aside.

2.2 Preparation of explants:

Mature seeds of the soybean variety Tianlong No. 1, which are plump, smooth, and disease-free, were selected and sterilized by chlorine gas for 5 hours before use. Soak the sterilized soybean seeds in sterile water overnight at room temperature, use a scalpel to divide the soaked soybean seeds into two along the hilum from the middle, ensure that the embryos on both sides of the cotyledon are complete, remove the seed coat, and cut off the large For part of the hypocotyl, only the 3-5mm hypocotyl near the cotyledon is left, and 1-3 knives are drawn in the range of about 3mm in diameter at the junction of the cotyledon and hypocotyl. Half seeds with intact embryos were obtained as explants.

2.3 Agrobacterium infection of explants and co-cultivation:

Soak the explants obtained in step 2.2 into the cell suspension obtained in step 2.1, infect at 28°C and 200 rpm for 30 minutes, take out the explants, and dry them until there is no obvious bacterial liquid on the surface. The ventral side of the body was spread on the CCM solid medium with sterile filter paper, and cultured in the dark at 26°C for three days.

2.4 Cleaning of explants and induction of clustered buds:

After the dark culture, the bacterial solution on the surface of the explants was cleaned with sterile water and liquid shoot induction medium (SIM), and the excess water was absorbed on sterile filter paper, and the dark culture was removed with a scalpel. The grown hypocotyl is cut off again, leaving only the 3-5mm hypocotyl close to the cotyledons, and scraping off the milk buds near the growth point with a knife. Insert the SIM solid medium obliquely at an angle of about 45 degrees for screening culture, seal it, and place it in a culture room for culture. The culture temperature is 26°C and the photoperiod is 16h/8h. Subculture once every two weeks, a total of three subcultures, to obtain explants with induced clustered buds.

2.5 Elongation and rooting of clustered buds:

The explants with clustered shoots were cut off and placed in solid shoot elongation medium (ShootElongation Medium, SEM) at a culture temperature of 26°C and a photoperiod of 16h/8h. Subculture once every two weeks, depending on the situation 3-5 times. For each subculture, use a knife to scrape off the browned and blackened tissue in the outer layer to expose the green tissue in the inner layer and insert it into the solid SEM. When the clustered shoots stretched to about 5 cm, they were cut off from the explants and inserted into rooting medium (Rooting Medium, RM) to take root.

2.6 Hardening and transplanting:

When the root system of the seedlings in step 2.5 is developed, open the lid of the culture bottle, harden the seedlings in the tissue culture room, and keep the humidity of the growth environment. After 3 days, remove the seedlings from the medium, rinse the medium of the roots, and transplant the seedlings into the soil. (The ratio of nutrient soil: vermiculite is 1:2), and cover it with plastic wrap to keep it moist, and remove the film after 3 days. The culture temperature is 25°C, and the photoperiod is 16h/8h. Pay attention to the management of water and fertilizer, and wait for the seedlings to grow a little before taking samples to identify whether they are positive plants.

2.7 Identification of transgenic plants:

Positive transgenic soybeans were screened by using glufosinate-resistant transgenic test strips specifically identified for basta resistance, and performing PCR amplification on the soybean genome to determine whether the transgenic plants had resistance to basta herbicides. Wherein, the primers used for PCR amplification are F: 5'-CAAAACCAAGAGTGGACAAGA-3'; R: 5'-AAGGATCACCCAAGATAACGT-3'. The plants (ie, positive transgenic soybeans) with positive results in both experiments were further tested, and the detection results of the glufosinate-resistant transgenic test strips are shown in FIG. 1 .

3. Identification of drought resistance of transgenic soybean

The three independent positive transgenic soybean lines (line-1, line-2 and line-3) obtained in step 2 and the non-transgenic (CK) soybean variety Tianlong No. 1 and the empty vector plant were transferred after seed germination After 50 days, the drought stress treatment was carried out uniformly, with at least 10 plants of each kind of soybean, and the specific stress treatment was as follows:

1) Evenly water before treatment to ensure that the soil water content before treatment between each pot of seedlings (T2 generation transgenic seedlings) is basically the same;

2) Place the treated soybean seedlings in a room temperature of 26 degrees, in a constant temperature cultivation room with a humidity of 50%, with a photoperiod of 16 hours in the daytime/8 hours in the dark, without watering during the period, and carry out drought treatment for a total of 8 days;

3) Observing the phenotype after 8 days of drought treatment, then watering normally, the amount of watering is the same, and performing rehydration treatment;

4) Observe the phenotype after 8 days of rehydration treatment.

The results showed that the growth status of the plants in each medium was basically the same before treatment. After 8 days of drought treatment, the leaves of the soybean variety Tianlong No. 1 and the transgenic plants turned yellow, drooped, and withered, while only some of the lower leaves of the three independent positive transgenic soybean lines turned yellow, and all plants had No abnormal drooping and withering of leaves occurred. After 8 days of rehydration treatment, the soybean variety Tianlong No. 1 and the transgenic plants all died, while the three independent positive transgenic soybean lines could grow normally. It was shown that GmSQE1 and its coding gene can improve the drought resistance of soybean. Among them, the results of positive transgenic soybean lines and soybean variety Tianlong No. 1 (CK) are shown in FIG. 2 .

<110> Institute of Oil Crops, Chinese Academy of Agricultural Sciences

<120> Application of Soybean Drought Resistance Related Proteins in Regulating Soybean Drought Resistance

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 529

<212> PRT

<213> soybean

<400> 1

Met Met Gly Tyr Glu Tyr Ile Leu Gly Gly Ile Ile Ala Ser Ser Leu

1 5 10 15

Val Leu Val Phe Val Ile Tyr Gly Ser Val Ser Lys Arg Lys Ala Lys

20 25 30

Ser Ser Val His Ala Glu Ser Asn Gly Gly Ser Ile Ile Arg Thr Ser

35 40 45

Pro Glu Asn Gly Asn His His Gln Glu Ile Ser Glu Thr Thr Asp Val

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Ile Ile Val Gly Ala Gly Val Ala Gly Ala Ala Leu Ala Tyr Thr Leu

65 70 75 80

Gly Lys Glu Gly Arg Arg Val His Val Ile Glu Arg Asp Leu Thr Glu

85 90 95

Pro Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Gly Tyr Leu Lys

100 105 110

Leu Ile Glu Leu Gly Leu Gln Asp Cys Val Gly Glu Ile Asp Ala Gln

115 120 125

Pro Val Phe Gly Tyr Ala Leu Tyr Lys Asp Gly Lys Asn Thr Lys Leu

130 135 140

Ser Tyr Pro Leu Glu Asn Phe Ala Ser Asp Val Ser Gly Arg Ser Phe

145 150 155 160

His Asn Gly Arg Phe Ile Gln Arg Met Arg Glu Lys Ala Ser Ser Leu

165 170 175

Pro Asn Val Lys Leu Glu Gln Gly Thr Val Thr Phe Leu Leu Glu Glu

180 185 190

Asp Arg Ile Ile Lys Gly Val Asn Phe Lys Thr Lys Ser Gly Gln Glu

195 200 205

Leu Thr Ala Lys Ala Pro Leu Thr Ile Val Cys Asp Gly Cys Phe Ser

210 215 220

Asn Leu Arg Arg Ser Leu Cys Asn Pro Lys Val Asp Val Pro Ser His

225 230 235 240

Phe Val Gly Leu Val Leu Glu Asn Cys Asn Leu Pro Tyr Ala Asn His

245 250 255

Gly His Val Ile Leu Gly Asp Pro Ser Pro Ile Leu Phe Tyr Pro Ile

260 265 270

Ser Ser Thr Glu Ile Arg Cys Leu Val Asp Val Pro Gly His Lys Leu

275 280 285

Pro Ser Leu Gly Asn Gly Asp Met Ala Arg Tyr Leu Lys Thr Val Val

290 295 300

Ala Pro Gln Val Pro Pro Glu Leu Arg Asp Ser Phe Ile Ala Ala Val

305 310 315 320

Glu Lys Gly Asn Ile Arg Ser Met Pro Asn Arg Ser Met Pro Ala Ser

325 330 335

Pro Tyr Pro Thr Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met

340 345 350

Arg His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp Ile

355 360 365

Val Leu Leu Arg Asn Leu Leu Arg Pro Leu His Asp Leu His Asp Ala

370 375 380

Asn Ala Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro

385 390 395 400

Val Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe

405 410 415

Cys Ala Ser Pro Asp Pro Ala Ser Lys Glu Met Arg Gln Ala Cys Phe

420 425 430

Asp Tyr Leu Ser Leu Gly Gly Val Phe Ser Asp Gly Pro Ile Ala Leu

435 440 445

Leu Ser Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Leu His Phe Phe

450 455 460

Ala Val Ala Ile Tyr Gly Val Gly Arg Leu Leu Ile Pro Phe Pro Ser

465 470 475 480

Pro Lys Arg Met Trp Ile Gly Ala Arg Leu Ile Ser Gly Ala Ser Ala

485 490 495

Ile Ile Phe Pro Ile Ile Lys Ala Glu Gly Ile Arg Gln Met Phe Phe

500 505 510

Pro Val Thr Val Pro Ala Tyr Tyr Arg Thr Pro Pro Thr Asn Leu Glu

515 520 525

Asp

<210> 2

<211> 1590

<212>DNA

<213> soybean

<400> 2

atgatgggtt atgagtatat tttgggaggc attatagctt ctagcttggt gcttgtgttt 60

gttatatatg gttctgtatc aaagaggaag gccaaaagtt cagtacatgc agaaagtaat 120

ggtggtagta ttataaggac atcaccagaa aatggaaacc accatcaaga aatctcagaa 180

actacggacg tcatcattgt cggtgctggg gttgctggcg cagcccttgc ttacacactt 240

ggcaaggaag gaaggcgagt gcatgttatt gaaagggact tgactgaacc agacaggatt 300

gtgggggaat tgctacaacc tggggggtat cttaagttaa ttgaattggg tctccaagat 360

tgtgtgggtg agattgatgc tcagccagtc tttggctatg ctctttacaa ggacgggaaa 420

aatactaagc tttcttaccc cttggaaaat tttgcctctg atgtttctgg aagaagcttt 480

cacaatggcc gtttcataca aaggatgcgc gaaaaggctt catctcttcc aaatgtaaaa 540

ttagaacaag gaactgtcac atttctacta gaagaagata gaatcatcaa aggggtaaac 600

ttcaaaacca agagtggaca agagctcaca gctaaggctc ccctcaccat tgtatgtgat 660

ggctgttttt ccaacctgag acgttctctt tgcaacccaa aggttgatgt accatctcat 720

tttgttggtc tggtcctaga gaactgcaat cttccatatg caaaccacgg gcacgttatc 780

ttgggtgatc cttctcccat tttgttttat cccatcagta gcactgagat tcggtgtttg 840

gttgatgtgc ctggccataa attaccttcc cttggcaatg gtgacatggc ccgttatttg 900

aaaacagtag tagctcccca ggttcctcca gagctgcgtg actcttttat agcagcagtt 960

gagaaaggaa acataagaag catgccaaac agaagcatgc ccgcatctcc ttatcccaca 1020

cctggtgccc ttctcatggg agatgccttc aacatgcgtc accctttaac cggaggggga 1080

atgactgtgg ctttgtctga cattgttttg ctaaggaacc ttcttagacc cctgcatgat 1140

ctgcatgacg ctaatgctct ttgcaaatat cttgaatcat tctacaccct acgcaagcca 1200

gtggcatcta caataaacac attagctggg gcattgtaca aggtgttttg tgcatcccct 1260

gatccagcta gtaaggaaat gcgccaggca tgttttgatt atttaagcct tggaggtgtt 1320

ttctcagatg gaccaattgc tctactctct ggtctaaatc ctcgtccatt aagcttggtt 1380

ctccacttct ttgccgtggc tatatatggt gttggtcgct tactcatacc attcccttct 1440

ccaaaacgaa tgtggattgg agctagattg atttccggtg cctctgctat cattttcccc 1500

attatcaagg ccgaaggaat tagacaaatg ttcttcccag taactgtgcc agcgtattac 1560

agaacaccccc ctaccaattt ggaagattaa 1590

<210> 3

<211> 3323

<212>DNA

<213> soybean

<400> 3

atgatgggtt atgagtatat tttgggaggc attatagctt ctagcttggt gcttgtgttt 60

gttatatatg gttctgtatc aaagaggaag gccaaaagtt cagtacatgc agaaagtaat 120

ggtggtagta ttataaggac atcaccagaa aatggaaacc accatcaaga aatctcagaa 180

actacggacg tcatcattgt cggtgctggg gttgctggcg cagcccttgc ttacacactt 240

ggcaaggtag aacaaacctt ttcttgacac gactttgtga aatctaatcc atatgtgtga 300

tgttttttct tagttactac ctgcaaatat gttttgattc atagatatat gcaaaactta 360

tgttgaggta aaataataac aataacaatt atgttagaaa gtcatgtttg attgccagaa 420

agtagaggtt ggaagataga agagtagttt tctaagtgtt attctgaatg aaattataca 480

agtgatacag ttataagtta taaccttata taggcttcat agaacattta ttctaaagat 540

agatttccca tttttttttt attttcattt tcatgaatat atttttcttg caaccagaca 600

aaaacatgaa aagttaaact gtggaaagtc caagatctct tcctttccat gatctgttct 660

tagaaccaca acacaaacta catatgcagt cagattcaga cttctagtta tatataaact 720

ttatggtaat ttaaaagaat ggtctaaaat atttttacac aatgaatcat atagtttgtg 780

ttgtaaaagg atcaatgatg ttggtctatg gttcttgctt caggaaggaa ggcgagtgca 840

tgttattgaa agggacttga ctgaaccaga caggattgtg ggggaattgc tacaacctgg 900

ggggtatctt aagttaattg aattgggtct ccaaggtaac caagcaagaa acatgtcaca 960

tattatgtca cataagtaga tgtatggaag attgtgtgag aattgaagta atcaatctta 1020

ggtgtgaact cccttgcctt tgcttgtcaa attcaaggct cttattaaca gattaagttg 1080

tgagttgttt cagattgtgtgggtgagatt gatgctcagc cagtctttgg ctatgctctt 1140

tacaaggacg ggaaaaatac taagctttct taccccttgg aaaattttgc ctctgatgtt 1200

tctggaagaa gctttcacaa tggccgtttc atacaaagga tgcgcgaaaa ggcttcatct 1260

cttccaaagt acagactctt atcatccttt ttcaaaaact gtttctgaaa aggatttttt 1320

tttattaaat cctttccttc cgttacttgc tcttattgtt ccaaattttg tgcagtgtaa 1380

aattagaaca aggaactgtc aatttctac tagaagaaga tagaatcatc aaaggggtaa 1440

acttcaaaac caagagtgga caagagctca cagctaaggc tcccctcacc attgtatgtg 1500

atggctgttt ttccaacctg agacgttctc tttgcaaccc aaaggtaact atgctgtttt 1560

ttttattatt atttatgtta aaagtgtgaa atatattctg actttgttga tgattaattt 1620

ccattgaaaa attggttgac cattttagta gtcttctctt ctaatggtgt ttttttttct 1680

gtcacaaggc tccaagccaa gactttactt ataggtccga gtccaagtca agctaactta 1740

atgttggtat ctatttgtct ttgctagtac ttagatgggt ttaatttgttt atttatttat 1800

gcaggttgat gtaccatctc attttgttgg tctggtccta gagaactgca atcttccata 1860

tgcaaaccac gggcacgtta tcttgggtga tccttctccc attttgtttt atcccatcag 1920

tagcactgag attcggtgtt tggttgatgt gcctggccat aaattacctt cccttggcaa 1980

tggtgacatg gcccgttatt tgaaaacagt agtagctccc caggtacaaa tatcctagtc 2040

tttggcttgg cttaatattc aaaacatgga acatattctt caattccact aatggaggaa 2100

attgtgtttt aggttcctcc agagctgcgt gactctttta tagcagcagt tgagaaagga 2160

aacataagaa gcatgccaaa cagaagcatg cccgcatctc cttatcccac acctggtgcc 2220

cttctcatgg gagatgcctt caacatgcgt caccctttaa ccggaggggg aatgactgtg 2280

gctttgtctg acattgtttt gctaaggaac cttcttagac ccctgcatga tctgcatgac 2340

gctaatgctc tttgcaaata tcttgaatca ttctacaccc tacgcaaggt taatatatat 2400

ataatcgaaa gagtttaata gtcatgcacc ttagaataaa agtattttct ttataaacta 2460

attagaaaac atccttatattc cttagtatgc agtactatga ctttggtggt tattataaaa 2520

gtgaacgagt ttatcttca tgacagtttg taattgaata atcgtataag aaacctttac 2580

attgttttct taaccaaata ccctgtcatg ttttatcagt attggtttga gcaaatttaa 2640

taggtggttc ttgattgtgtttgcagccag tggcatctac aataaacaca ttagctgggg 2700

cattgtacaa ggtgttttgt gcatcccctg atccagctag taaggaaatg cgccaggcat 2760

gttttgatta tttaagcctt ggaggtgttt tctcagatgg accaattgct ctactctctg 2820

gtctaaatcc tcgtccatta agcttggttc tccacttctt tgccgtggct atatatggtg 2880

ttggtcgctt actcatacca ttcccttctc caaaacgaat gtggattgga gctagattga 2940

tttccgtgag tgtttcttgc atttctttat agacataatt tttcacatat taaccataac 3000

ctttgctgca acaatattct attacaaatt atgaataatt ctagcatgag tagagtgttt 3060

aatattcaaa taaattcaac acggtctata tattttgatt aattgagtct gtaaatgttg 3120

tggtcataaa agaattgttc ccaaaatatt agttaatggt acaacaaaat ttatgatttt 3180

gaaccaagtt tgttcttgac attttcaggg tgcctctgct atcattttcc ccattatcaa 3240

ggccgaagga attagacaaa tgttcttccc agtaactgtg ccagcgtatt acagaacacc 3300

ccctaccaat ttggaagatt aaa 3323

Claims (10)

1. application of the drought resistant correlative protein in plant drought resistance is regulated and controled；The drought resistant correlative protein is following A1) or A2) or A3)：

A1) amino acid sequence is the protein of sequence 1；

A2) obtained in the amino acid sequence of sequence 1 by substituting and/or lacking and/or add one or several amino acid residues Arrive have identical function as A1) derived from protein；

A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label.

2. application of the biomaterial related to drought resistant correlative protein described in claim 1 in plant drought resistance is regulated and controled；

Any of the biomaterial, it is following B1) to B14)：

B1 the nucleic acid molecules of drought resistant correlative protein described in claim 1) are encoded；

B2 B1) is contained) expression cassettes of the nucleic acid molecules；

B3 B1) is contained) recombinant vectors of the nucleic acid molecules；

B4 B2) is contained) recombinant vector of the expression cassette；

B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；

B6 B2) is contained) recombinant microorganism of the expression cassette；

B7 B3) is contained) recombinant microorganism of the recombinant vector；

B8 B4) is contained) recombinant microorganism of the recombinant vector；

B9 B1) is contained) the transgenic plant cells systems of the nucleic acid molecules；

B10 B2) is contained) the transgenic plant cells system of the expression cassette；

B11 B1) is contained) Transgenic plant tissues of the nucleic acid molecules；

B12 B2) is contained) Transgenic plant tissue of the expression cassette；

B13 B1) is contained) the genetically modified plants organs of the nucleic acid molecules；

B14 B2) is contained) the genetically modified plants organ of the expression cassette.

3. application according to claim 2, it is characterised in that：B1) nucleic acid molecules are following b1), b2), b3) or B4 the gene shown in)：

B1) nucleotide sequence is the cDNA molecules or DNA molecular of sequence 2 in sequence table；

B2) nucleotide sequence is the cDNA molecules or DNA molecular of sequence 3 in sequence table；

B3) and b1) or b2) or the nucleotide sequence that limits there is 75% or more than 75% homogeneity, and in coding claim 1 The cDNA molecules or genomic DNA molecule of the drought resistant correlative protein；

B4) under strict conditions with b1) or b2) limit nucleotide sequence hybridization, and encode claim 1 described in drought resisting phase Close the cDNA molecules or genomic DNA molecule of albumen.

4. biomaterial described in drought resistant correlative protein described in claim 1 or Claims 2 or 3 is cultivating drought resistance enhancing Application in plant.

5. a kind of method for cultivating drought resistance enhancing plant, including：Increase drought resisting correlation described in claim 1 in purpose plant The activity of albumen, increase the content of drought resistant correlative protein described in claim 1 in purpose plant, promote institute in claim 1 The expression of the encoding gene of drought resistant correlative protein is stated, obtains the drought-resistant plant that drought resistance strengthens compared with the purpose plant.

6. according to the method for claim 5, it is characterised in that：The drought-resistant plant is by being led into the purpose plant Enter the plant that the encoding gene of drought resistant correlative protein described in claim 1 obtains.

7. according to the method for claim 6, it is characterised in that：The coding base of drought resistant correlative protein described in claim 1 Because B1 in claim 3) nucleic acid molecules.

8. regulating and controlling the product of plant drought resistance, contain institute in drought resistant correlative protein described in claim 1 or Claims 2 or 3 State biomaterial.

9. application of the product described in claim 8 in plant drought resistance is regulated and controled.

10. according to any described method or claim 8 in any described applications of claim 1-4 or claim 5-7 Application described in described product or claim 9, it is characterised in that：The plant is dicotyledon or monocotyledon； The purpose plant is dicotyledon or monocotyledon.