CN107459565B - Application of soybean drought resistance-related proteins in regulating soybean drought resistance - Google Patents

Abstract

The invention discloses application of soybean drought-resistant related protein in regulation of soybean drought resistance. The soybean drought-resistant related protein (GmSQE1) disclosed by the invention is A1) or A2) or A3) as follows: A1) a protein with an amino acid sequence of sequence 1; A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 and has the same function and is derived from A1); A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that compared with plants which are not transformed with GmSQE1 gene, plants transformed with the positive GmSQE1 gene show drought-tolerant character after drought treatment; after rehydration, the plant which is not transformed with the GmSQE1 gene dies, and the plant which is transformed with the GmSQE1 gene positively can grow normally. The GmSQE1 and the coding gene thereof 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 more and more land desertification, drought has become an extremely important factor that threatens global agricultural development and food security. Drought stress can cause crop withering and stunting, resulting in reduced crop yields. It is difficult to change the general trend of global warming in a short period of time, so it is a good solution to cultivate crops with drought resistance through modern biotechnology.

Soybean (Glycine max) is one of the five major economic crops originating in China with important food security status in the world. my country is the world's largest soybean importer and consumer, and soybeans are of great strategic significance to ensuring the safety of oil and fat supply in my country. Soybeans can provide people with high-quality soybean oil and soybean vegetable 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 freshwater resources, drought has had an important impact on soybean yields. Therefore, breeding soybean varieties with good drought resistance is of great significance for soybean production. At present, soybean drought resistance breeding mainly focuses on traditional hybrid breeding and transgenic breeding technology. Due to the lack of excellent drought-resistant germplasm resources in traditional cross-breeding, and traditional cross-breeding requires continuous cross-breeding and combination of groups, and then get isolated groups to find drought-resistant progeny plants, and then continuously breed to select stable genetic drought-resistant progeny. Traditional soybean hybrid breeding is time-consuming and requires a lot of manpower and material resources. Moreover, traditional soybean cross-breeding may cause the parents to lose the original excellent traits, such as yield, plant type, disease resistance, etc., after acquiring drought resistance traits. Soybean transgenic breeding can quickly introduce drought resistance genes into parent soybean varieties with excellent trait background through plant genetic transformation technology under the premise of clearing the key drought resistance genes, and determine the homozygosity of single copy gene insertion through the screening of transgenic positive soybean plants. progeny plants. Drought-resistant breeding of transgenic soybean can easily and quickly breed new soybean varieties with clear drought resistance. At present, the domestic and international soybean genetically modified breeding mainly focuses on the cultivation of herbicide resistance, insect resistance and regulation of growth and development. There are few reports on soybean drought-resistant transgenic breeding.

SUMMARY OF THE INVENTION

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

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

A1) protein whose amino acid sequence is sequence 1;

A2) A protein derived from A1) with the same function obtained by substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1;

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

In order to facilitate purification of the protein in A1), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.

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

The GmSQE1 protein in the above A2) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The encoding gene of the GmSQE1 protein in the above A2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, and / or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The invention also provides the application of the biological material 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 described in 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) a transgenic plant tissue containing the expression cassette of B2);

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

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

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

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

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

b3) a cDNA molecule or a genomic DNA molecule that is 75% or more identical to b1) or b2) or the defined nucleotide sequence and encodes GmSQE1;

b4) Hybridizes under stringent conditions to the nucleotide sequences defined in b1) or b2) and encodes a cDNA molecule or a genomic DNA molecule of 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 of ordinary skill in the art can easily mutate the nucleotide sequence encoding the GmSQE1 protein of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of the GmSQE1 protein isolated by the present invention, as long as they encode the GmSQE1 protein and have the function of the GmSQE1 protein, are derived from the nucleus of the present invention. nucleotide sequences and are equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. 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 in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions were hybridized and washed twice at 68°C in a solution of 2×SSC, 0.1% SDS for 5 min each, and then in a solution of 0.5×SSC, 0.1% SDS, at 68°C. Hybridize and wash the membrane twice at 68°C for 15 min each; or, in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

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

In above-mentioned application, B2) described expression cassette (GmSQE1 gene expression cassette) containing the nucleic acid molecule of coding GmSQE1 protein, refers to the DNA that can express GmSQE1 protein in host cell, this DNA can not only include the start that starts GmSQE1 gene transcription It can also include a terminator that terminates the transcription of the GmSQE1 gene. Further, the expression cassette may also include enhancer sequences. 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 of cauliflower mosaic virus 35S: a 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-thiol acid S-methyl ester)); tomato protease Inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Pat. No. 5,187,267); tetracycline-inducible promoter (US Pat. No. 5,057,422); Seed-specific promoters, such as foxtail 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. Human (1985) EMBO J. 4:3047-3053)). They can 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 terminators (see, eg: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; ^Guerineau et al. (1991) Mol. Gen. Genet, 262:141; 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).

A recombinant vector containing the GmSQE1 gene expression cassette can be constructed by using an existing expression vector. 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) and so on. The plant expression vector may also contain the 3' untranslated region of the foreign gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed from the 3' end of the storage protein gene) 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 initiation codons or adjacent region initiation codons, etc., but must be associated with 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 can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived 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 a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound. Gene, etc.), marker genes for antibiotics (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate) or marker genes for chemical resistance (such as herbicide resistance genes), mannose-6- which provides the ability to metabolize mannose Phosphoisomerase gene. Considering the safety of transgenic plants, the transformed plants can be directly screened under stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be the vector pGWC or the 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 a sequence of The DNA fragment shown in 2 was introduced into the recombinant vector obtained in plasmid pB2GW7.0, and the GmSQE1-pGWC could express the GmSQE1 protein shown in sequence 1.

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

In the above application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ do not include propagation material.

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 a target plant, increasing the content of GmSQE1 in the target plant, promoting the expression of a gene encoding GmSQE1, and obtaining the same target plant as the target plant. Drought tolerant plants with enhanced drought tolerance compared to .

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 plant drought resistance, the product containing GmSQE1 or the biological material.

The product can use GmSQE1 or the biomaterial as its active ingredient, and can also use GmSQE1 or the biomaterial 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 dicotyledonous plant or a monocotyledonous plant; the target plant can be a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant may be a legume. The legume 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 GmSQE1 gene into the target plant, but also its progeny. For transgenic plants, the gene can be propagated in that species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

Experiments showed that, compared with the plants without GmSQE1 gene, the plants with positive GmSQE1 gene showed drought tolerance after drought treatment, while the plants without GmSQE1 gene died after rehydration treatment, while the plants with positive GmSQE1 gene were able to survive. normal growth. It was shown that GmSQE1 and its encoding 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 shows the identification of drought resistance of transgenic soybean.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than 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. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

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

CCM liquid medium in the following examples, 1L contains B- ₅ powder 0.321g, MES 5.9g, 100*B5 organic 9ml, sucrose 30g, Na2S2O3 solution 1ml, _{L -} cys solution 4ml, DTT solution 1ml, AS solution 2ml, 6-BA solution 1.67ml and GA3 solution 100μl, the balance is water. CCM solid medium is a medium obtained by adding agar powder to CCM liquid medium, pH=5.4.

1 L of the liquid bud induction medium in the following examples 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 500 μl/500 ml of agar powder and a PPT solution to a liquid bud induction medium.

The 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.

The rooting medium in the following example, 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 organically contains: 5g of myo-lnositol, 0.05g of nicotinic acid, 0.05g of pyridoxine hydrochloride (vitamin B6), 0.5g of Thiamine hydrochloride, and the rest The amount is water.

Na ₂ S ₂ O ₃ (sodium thiosulfate) solution: 3.16g Na ₂ S ₂ O ₃ /20ml, soluble 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 dissolve in a small amount of 1M NaOH aqueous solution, then distill water to make up the volume.

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

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

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

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

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

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

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

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

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

Example 1. Soybean drought resistance-related protein GmSQE1 can regulate soybean drought resistance

This example 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 table. 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 (that is, the gene encoding GmSQE1) was 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

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

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

GmSQE1-pB2GW7.0 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria, which was named EHA105/GmSQE1-pB2GW7.0; pB2GW7.0 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria EHA105/GmSQE1 , as an empty vector control.

2. Construction of genetically modified soybeans

The transgenic soybean was obtained by transforming the soybean variety Tianlong No. 1 with the recombinant strain EHA105/GmSQE1-pB2GW7.0 by the Agrobacterium infection method, and the empty vector plant obtained by the recombinant strain EHA105/GmSQE1 was used as a control. 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), and cultured overnight at 28°C, 200rpm. During the period, the concentration of the bacterial solution was detected. When the OD600 value reached 1.2-1.5, centrifuge at 4000 rpm for 10 min to collect the bacterial cells. Resuspend in CCM liquid medium (Co-culture Medium, CCM), adjust OD600 to about 0.6, and obtain bacterial suspension for use.

2.2 Preparation of explants:

The mature seeds of soybean variety Tianlong No. 1 with full, smooth surface and no disease scars were selected and sterilized by chlorine gas for 5 hours before use. Soak the sterilized soybean seeds in sterile water overnight at room temperature, and use a scalpel to divide the soaked soybean seeds into two along the hilum from the middle to ensure that the embryos on both sides of the cotyledons are intact, remove the seed coat, and cut off the large Part of the hypocotyl, leaving only the 3-5mm hypocotyl close to the cotyledon, make 1-3 cuts at the junction of the cotyledon and the hypocotyl with a diameter of about 3mm. Half seeds with intact embryos were obtained as explants.

2.3 Agrobacterium infection of explants and co-culture:

Immerse the explants obtained in step 2.2 into the bacterial suspension obtained in step 2.1, and after infecting them for 30 minutes at 28°C and 200 rpm, take out the explants and dry them until there is no obvious bacterial liquid on the surface. The body was placed ventral side down, plated on 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 clump buds:

After dark culture, the bacterial liquid on the surface of explants was washed with sterile water and liquid bud induction medium (Shoot Induction Medium, SIM) successively, and excess water was absorbed on sterile filter paper. The grown hypocotyl was truncated again, leaving only the 3-5mm hypocotyl near the cotyledons, and scraping off the milk buds near the growth point with a knife. At an angle of about 45 degrees, insert the SIM solid medium at an angle of about 45 degrees for screening and culture, seal it, and place it in a culture room for culture at a culture temperature of 26°C and a photoperiod of 16h/8h. Subculture once every two weeks for three times in total to obtain explants that induce clump buds.

2.5 Elongation and rooting of clump buds:

The explants from which clump buds were induced were cut off, and then placed in solid shoot elongation medium (Shoot Elongation Medium, SEM) at a culture temperature of 26°C and a photoperiod of 16h/8h. Subculture once every two weeks, 3-5 times depending on the situation. At each passage, the outer layer of browned and blackened tissue was scraped off with a knife, and the inner layer of green tissue was exposed and placed in a solid SEM. When the clump shoots extended to about 5 cm, they were cut off from the explants and inserted into rooting medium (RM) for rooting.

2.6 Seedling refining 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, keep the humidity of the growth environment, remove the seedlings from the medium after 3 days, rinse the medium at the roots, and transplant the seedlings into the soil (nutrient soil: vermiculite ratio is 1:2), and cover with plastic wrap to keep moisture, remove the film after 3 days, culture temperature 25 ℃, photoperiod 16h/8h. Pay attention to the management of water and fertilizer, and wait for the seedlings to grow up a little before sampling to identify whether they are positive plants.

2.7 Identification of transgenic plants:

Positive transgenic soybeans were screened by PCR amplification of soybean genome using glufosinate-resistant transgenic test strips specifically identified for basta resistance to determine whether transgenic plants were resistant to basta herbicides. The primers used for PCR amplification were F: 5'-CAAAACCAAGAGTGGACAAGA-3'; R: 5'-AAGGATCACCCAAGATAACGT-3'. The plants whose two experimental results were positive (ie, positive transgenic soybean) were subjected to further experiments, and the detection results of the glufosinate-resistant transgenic test strips were shown in Figure 1 .

3. Identification of drought resistance of transgenic soybeans

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

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

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

3) Observe the phenotype after 8 days of drought treatment, and then water normally, the watering amount is the same, and carry out the rehydration treatment;

4) The phenotype was observed after 8 days of rehydration treatment.

The results showed that the growth conditions of the plants were basically the same before treatment. After 8 days of drought treatment, the leaves of soybean variety Tianlong No. 1 and the transgenic vector plants turned yellow, drooped and withered, while only the lowermost leaves of some plants of the three independent positive transgenic soybean lines turned yellow, and all plants had all the leaves. No abnormal drooping and drying of leaves occurred. After 8 days of rehydration treatment, both the soybean variety Tianlong No. 1 and the transgenic vector plants died, while the three independent positive transgenic soybean lines could grow normally. It was shown that GmSQE1 and its encoding 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 Figure 2.

<110> Oil Crops Research Institute, 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> Soybeans

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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> Soybeans

<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

agaacacccc ctaccaattt ggaagattaa 1590

<210> 3

<211> 3323

<212> DNA

<213> Soybeans

<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 cagattgtgt gggtgagatt 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 acatttctac 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 ttatttgttt 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 atccttattc cttagtatgc agtactatga ctttggtggt tattataaaa 2520

gtgaacgagt ttatcttaca tgacagtttg taattgaata atcgtataag aaacctttac 2580

attgttttct taaccaaata ccctgtcatg ttttatcagt attggtttga gcaaatttaa 2640

taggtggttc ttgattgtgt ttgcagccag 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 (13)

1. The drought-resistant related protein is a protein shown in a sequence 1, and the drought resistance of the plant is improved by increasing the content of the drought-resistant related protein in the plant.

2. Use of a biological material associated with said drought-resistant related protein of claim 1 to increase drought resistance in a plant by increasing the content of said drought-resistant related protein in said plant,

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

B1) a nucleic acid molecule encoding the drought-resistant associated protein of claim 1;

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

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

B4) a recombinant vector comprising the expression cassette of B2);

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

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

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

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

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

B12) transgenic plant tissue comprising the expression cassette of B2);

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

B14) a transgenic plant organ containing the expression cassette according to B2).

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is b1) or b2) as follows:

b1) the nucleotide sequence is a DNA molecule of a sequence 2 in a sequence table;

b2) the nucleotide sequence is a DNA molecule of a sequence 3 in a sequence table.

4. Use of the drought-resistant related protein of claim 1 or the biological material of claim 2 or 3 for breeding drought-resistant enhanced plants.

5. Use according to any one of claims 1 to 4, characterized in that: the plant is a dicotyledonous plant or a monocotyledonous plant.

6. A method of breeding a drought-resistant-enhanced plant, comprising: increasing the content of the drought-resistant related protein in the claim 1 in the target plant or promoting the expression of the coding gene of the drought-resistant related protein in the claim 1 to obtain the drought-resistant plant with enhanced drought resistance compared with the target plant.

7. The method of claim 6, wherein: the drought-resistant plant is obtained by introducing the gene encoding the drought-resistant related protein according to claim 1 into the target plant.

8. The method of claim 7, wherein: the gene encoding the drought-resistant related protein according to claim 1 is the nucleic acid molecule according to B1) in claim 3.

9. The method according to any one of claims 6-8, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant.

10. A product for regulating drought resistance of a plant, comprising the biological material of any one of B1) to B8) of claim 2 or 3.

11. The product of claim 10, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant.

12. Use of the product of claim 10 for modulating drought resistance in plants.

13. Use according to claim 12, characterized in that: the plant is a dicotyledonous plant or a monocotyledonous plant.

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