CN110577956A - Soybean sHSP26 gene and application thereof - Google Patents

Abstract

the invention provides a soybean sHSP26 gene and application thereof, belonging to the field of plant genetic engineering. The nucleotide sequence of the gene is shown in SEQ ID NO. 1. According to the invention, the soybean sHSP26 is cloned by using an RT-PCR technology, bioinformatics analysis is carried out on the basic physicochemical property, the structural domain and the like of the gene, a plant over-expression vector and a CRISPR vector of the sHSP26 gene are successfully constructed for the first time, a transgenic progeny plant is obtained through genetic transformation, statistics and analysis are carried out on physiological and biochemical indexes and agronomic characters of the transgenic progeny, the result shows that the SOD activity, POD activity and PRO content of the soybean plant transformed with the over-expression vector are higher than those of a control plant, the MDA content is lower than that of the control plant, particularly, the trend is more obvious after drought stress, and the over-expression of the gene can improve the drought resistance of the transgenic soybean.

Description

soybean sHSP26 gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a soybean sHSP26 gene and application thereof.

Background

soybean (Glycine max (L.) Merr.) is an important crop used in both oil and high-protein food and feed in the world, and has wide application in the fields of food, medicine, industry, animal husbandry and the like. The soybean is extremely sensitive to drought, the yield is closely related to the number of pods, the number of grains per pod, the weight of hundreds of grains and the like, and the soybean seeds are formed by ovary development, so the development stage of the soybean ovary is a key period for determining the yield of the soybean, and the yield of the soybean can be seriously influenced if the soybean suffers from stress such as drought and the like in the period, so that the method has very important significance for finding the gene which is related to the development of the soybean ovary and can improve the drought resistance of the soybean and obtaining a new drought-resistant transgenic soybean strain. The experiment clones the screened differential expression gene sHSP26 and further researches the gene function through the overexpression and CRISPR/Cas9 technology on the basis of sequencing of a soybean drought-resistant mutant (TB18) seven-leaf stage unpolished ovary transcriptome, and lays a foundation for creating a new strain of drought-resistant transgenic soybean through a genetic engineering technology.

The generation of drought seriously affects the yield and quality of crops, and the cultivation of high-quality drought-resistant crop varieties is very important. The molecular biology approach is applied to the research of crop drought resistance mechanism, the drought resistance related gene is cloned, and the cultivation of a new drought-resistant transgenic crop variety becomes an important target in the research of crop breeding.

In 2007, wheat protein phosphatase 2A catalytic subunit genes were cloned in xu heavily yi, and bioinformatics analysis and function research were carried out on the genes. Researches show that the protein phosphatase 2A catalytic subunit gene is mainly expressed in cell nucleus and cytoplasm, and the transgenic tobacco over-expressing the protein phosphatase 2A catalytic subunit gene has higher drought resistance than a control plant under drought stress. In 2009, the national security research finds that the drought resistance and salt tolerance of transgenic plants over expressing GmUBC2 and GmPK genes are obviously higher than those of control plants. In 2010, Penghui research finds that the expression of CarNAC3 gene can improve the drought tolerance of Arabidopsis plants. In 2010, a heat shock transcription factor CarHSFB2 is separated from chickpea by a comet, and the drought resistance and heat resistance of an arabidopsis plant transformed with the gene are obviously improved. In 2015, GhCBF3 gene is separated from cotton by Mallotus philippinarum, and researches show that the drought resistance and salt tolerance of Arabidopsis with over-expression of the gene are higher than those of control plants.

Disclosure of Invention

The invention aims to provide a soybean sHSP26 gene and application thereof, and the expression of the sHSP26 gene can improve the drought resistance of soybeans.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention firstly provides a soybean sHSP26 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also provides an amino acid sequence of the soybean sHSP26 gene, which is shown in SEQ ID NO. 2.

The invention also provides a plant overexpression vector containing the soybean sHSP26 gene, and the overexpression vector is named as pCAMBIA3301-sHSP 26.

The invention also provides a plant gene editing vector containing the soybean sHSP26 gene, wherein the gene editing vector is named as a CRISPR-sHSP26 gene.

the invention also provides application of the soybean sHSP26 gene in drought resistance of plants.

The invention has the advantages of

The invention firstly provides a soybean sHSP26 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1. According to the invention, the soybean sHSP26 is cloned by using an RT-PCR technology, bioinformatics analysis is carried out on the basic physicochemical property, the structural domain and the like of the gene, a plant over-expression vector and a CRISPR vector of the sHSP26 gene are successfully constructed for the first time, a transgenic progeny plant is obtained through genetic transformation, statistics and analysis are carried out on physiological and biochemical indexes and agronomic characters of the transgenic progeny, the result shows that the SOD activity, POD activity and PRO content of the soybean plant transformed with the over-expression vector are higher than those of a control plant, the MDA content is lower than that of the control plant, particularly, the trend is more obvious after drought stress, and the over-expression of the gene can improve the drought resistance of the transgenic soybean.

Drawings

FIG. 1 is a schematic diagram of the T-DNA structure of plant over-expression vector pCAMBIA3301-sHSP 26;

FIG. 2 is an RT-PCR electrophoresis picture of sHSP26 gene. M: DL2000 molecular weight standards 1-4: RT-PCR products;

FIG. 3 is a PCR identification electrophoresis of cloning vectors, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR product a: bacterial liquid PCR b: carrying out plasmid PCR;

FIG. 4 is a double restriction enzyme digestion verification detection map of the cloning vector; m: DL2000 molecular weight standard 1: cloning vector plasmid 2-3: performing enzyme digestion on the product;

FIG. 5 is a comparison chart of sequencing results;

FIG. 6 is a diagram of analysis of protein conserved domains;

FIG. 7 is a graph of hydropathic and hydrophobic analysis;

FIG. 8 is a signal peptide prediction graph;

FIG. 9 is a coiled coil prediction graph;

FIG. 10 is a model of the tertiary structure of sHSP26 protein;

FIG. 11 shows the PCR identification of the overexpression vector pCAMBIA3301-sHSP26, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR products;

FIG. 12 shows the restriction enzyme identification of the overexpression vector pCAMBIA3301-sHSP26, M: DL2000 molecular weight standard 1: overexpression vector plasmids 2-3: performing enzyme digestion on the product;

FIG. 13 is a comparison of sequencing results of overexpression vectors;

Fig. 14 is an electrophoresis diagram of CRISPR-sHSP26 vector PCR identification, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR products;

FIG. 15 is a comparison graph of sequencing results of CRISPR-sHSP26 vector;

FIG. 16 is a PCR identification chart of Agrobacterium tumefaciens liquid, M: DL2000 molecular weight standard 1: negative controls 2-6: PCR product, a: the overexpression vector pCAMBIA3301-sHSP26 bacterial liquid PCR b: PCR of CRISPR-sHSP26 carrier bacterial liquid;

FIG. 17 is a PCR assay of transgenic overexpression vector pCAMBIA3301-sHSP26 plants, M: DL2000 molecular weight standard 1: positive control 2: negative controls 3-12: PCR product, a: t is₀Generation detection result b: t is₁and (c) generation detection result: t is₂generating a detection result;

Fig. 18 is a PCR detection map of a CRISPR-sHSP26 transformed vector plant, M: DL2000 molecular weight standard 1: positive control 2: negative controls 3-15: PCR product, a: t is₀generation detection result b: t is₁and (c) generation detection result: t is₂generating a detection result;

FIG. 19 is a Southern blot assay of transgenic plants, M: southern standard molecular weight +: positive control-: negative controls 1-4: t is₂Transforming plants;

FIG. 20 is a graph showing the relative expression amounts of sHSP26 gene;

FIG. 21 is a graph showing detection of superoxide dismutase activity;

FIG. 22 is a peroxidase activity assay;

FIG. 23 is a graph showing the results of detecting the malonaldehyde content;

FIG. 24 is a graph showing the results of measurement of proline content

Detailed Description

The soybean materials used were Jinong 18(JN18) and Jinong 18 drought-resistant mutant (TB18), both provided by the plant Biotechnology center of Jilin agriculture university.

the used molecular biological reagent is a RNAiso plus kit and T₄Ligase, pMD-18T Vector, DL2000 Marker and the like are purchased from Bao bioengineering (Dalian) Co., Ltd., All-in-One qPCR Kit is purchased from GeneCopoeia Co., Ltd., plasmid minium extraction Kit is purchased from Kangji century Biotech Co., Ltd., southern blot digoxin Kit is purchased from Roche, CRISPR/Cas Vector construction Kit is purchased from Baige Gene technology (Jiangsu) Co., Ltd., and detection Kit of superoxide dismutase and the like is purchased from Suzhou Keming Biotechnology Ltd. Consumables such as centrifuge tube and pipette tip are supplied by Changchun Feikai biological Co Ltd, and biochemical reagents such as isopropanol are purchased from Changchun Ci Jintai Co Ltd

Example 1 cloning and identification of Soybean sHSP26 Gene

TABLE 1 PCR primer List

According to the sequencing result of a seven-leaf-stage unpolarized ovary transcriptome of a soybean drought-resistant mutant (TB18), an up-regulated expression gene GmZFCY08 in TB18 soybean is selected as a research object, the gene is determined to be a small heat shock protein gene positioned on a soybean No. 6 chromosome through Soybase database query and comparison with an NCBI database, and the molecular weight of a protein coded by the gene is predicted to be 26059.52D by using ProtParam online software at the later stage, so that the gene is named as GmsHSP 26, and the nucleotide sequence is shown as SEQID NO. 1.

Specific primers GmZFCY 08S and GmZFCY08 AS (table 1) are designed by using Primer Premier 5.0 software according to the nucleotide sequence of the soybean sHSP26 gene, and BglII and BstEII enzyme digestion recognition sequences are directly introduced into the 5' ends of the two primers for the convenience of constructing an overexpression vector. Taking cDNA generated by reverse transcription of total RNA of TB18 soybean ovary AS a template, and carrying out PCR amplification by using GmZFCY 08S and GmZFCY08 AS primers under the following amplification conditions: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing

At 55 ℃, 30s, 32 cycles; extension 72 ℃ for 30s, and post-extension 72 ℃ for 10 min. The amplification results were detected by electrophoresis on a 1% agarose gel. The results are shown in FIG. 2, from which it can be seen that there is a specific amplified band around 696bp, and the product matches with the size of sHSP26 gene.

Example 2 construction and identification of recombinant cloning vector for Soybean sHSP26 Gene

1. Construction and PCR verification of soybean sHSP26 gene cloning vector

The recovered and purified PCR product was ligated with pMD-18T cloning vector overnight at 16 ℃ to transform E.coli DH5 alpha competent cells. Then, single colonies on the transformation plate were picked, placed in a test tube to which 5mLLB + Kan liquid medium was added, and cultured overnight at 37 ℃ with shaking at 200 rpm. And (3) performing PCR amplification by taking the cultured bacterial liquid AS a template and GmZFCY 08S and GmZFCY08 AS AS primers, extracting plasmids, and performing plasmid PCR identification by taking the plasmids AS the template.

as shown in FIG. 3, it can be seen that a specific amplification band appears around 696bp after PCR amplification, and the size of the band is consistent with that of the soybean sHSP26 gene.

2. Double enzyme digestion identification of soybean sHSP26 gene cloning vector

And (3) carrying out double enzyme digestion identification on the constructed recombinant cloning vector by using two restriction enzymes BglII and BstEII according to the enzyme digestion site introduced during gene cloning by using a plasmid with the correct size detected by PCR as a template, carrying out enzyme digestion reaction at 37 ℃ for 4h, terminating the reaction at 65 ℃ for 20min, and detecting the double enzyme digestion result by 1% agarose gel electrophoresis. The result is shown in FIG. 4, which shows that a target gene band of about 696bp and a linear pMD-18T vector band can be obtained after double enzyme digestion of the recombinant cloning vector, and the success of cloning vector construction is primarily proved.

3. Sequencing identification of soybean sHSP26 gene cloning vector

the recombinant cloning vector verified by PCR and double enzyme digestion is sent to Jilin province Kuumei Biotechnology Ltd for sequencing, and the sequencing result is compared by using DNAMAN software (figure 5), and the comparison result shows that the nucleotide sequence of the sequencing plasmid is 100% identical to the nucleotide sequence of the sHSP26 gene, so that the construction success of the sHSP26 gene cloning vector is proved.

Example 3 bioinformatic analysis of Soybean sHSP26 Gene

the method comprises the steps of analyzing basic physicochemical properties such as molecular weight, theoretical isoelectric point, molecular formula, extinction coefficient, instability coefficient and the like of the protein coded by the soybean sHSP26 gene by using ProtParam online software, analyzing a Conserved Domain of a protein sequence of the gene by using NCBI Conserved Domain SearchService, analyzing the hydrophile and hydrophobicity of the gene by using ProtScale, performing signal peptide prediction analysis on an amino acid sequence by using SignalP, performing coiled coil prediction analysis on the amino acid sequence by using COILS, and predicting a protein tertiary structure by using Swiss-Model. The results show that: the molecular weight of the protein coded by the gene is predicted to be 26059.52D, the theoretical isoelectric point pI is 6.98, and the molecular formula is C₁₁₃₀H₁₈₁₈N₃₃₀O₃₅₅S₁₁The extinction coefficient measured at 280nm was 22585, the instability coefficient was 36.98, indicating that the protein was stable, the fat index was 72.12, and the total average hydrophilicity (GRAVY) was-0.705, indicating that the protein was a hydrophilic protein. The method specifically comprises the following steps:

1. Conserved domain analysis of protein encoded by soybean sHSP26 gene

The protein sequence of sHSP26 gene was analyzed by using NCBI Conserved Domain Search Service, and the result showed that the protein encoded by the gene belongs to the alpha-crystallin-Hsps _ p23-like superfamily (see FIG. 6), and contains an alpha-crystallin Domain (ACD).

2. Hydrophilic and hydrophobic property analysis of soybean sHSP26 gene

The protein encoded by the sHSP26 gene is proved to be hydrophilic protein by using ProtScale to analyze the hydrophilicity and hydrophobicity of the amino acid encoded by the sHSP26 gene on line, wherein the hydrophilicity and hydrophobicity map is shown in FIG. 7a under the condition of a default window 9, the window is adjusted to 19 for the convenience of result analysis, and the result is shown in FIG. 7b, and as can be seen from the map, when the |. Score |. is ≧ 1.5 as a threshold value, both the regions belong to high-hydrophilicity regions, and the result is consistent with the result of analysis by ProtParare in 3.2.3.1.

3. amino acid sequence signal peptide prediction of soybean sHSP26 gene

The signal peptide prediction analysis of the amino acid sequence by using SignalP is shown in FIG. 8, and it can be seen that the protein encoded by the gene has no signal peptide sequence in the coding region, indicating that the protein is a non-secretory protein.

4. Prediction of the coiled coil of the protein encoded by the soybean sHSP26 Gene

The results of prediction of the coiled-coil of the protein encoded by the gene using COILS are shown in FIG. 9, and it can be seen that the protein encoded by the soybean sHSP26 gene can detect 6 coiled-coil regions in Window 14.

5. Tertiary structure model of protein encoded by soybean sHSP26 gene

The tertiary structure Model of the gene-encoded protein was constructed on-line using Swiss-Model (http:// www.swissmodel.expasy.org /), and the results are shown in FIG. 10. The amino acid sequence of the protein had a Coverage (Coverage) with the template of 0.61, a degree of Identity (Seq Identity) of 55%, no ligand, and a global model mass estimation (GMQE) of 0.56.

Example 4 construction and identification of Soybean sHSP26 Gene plant overexpression vector

Carrying out PCR amplification by using a recombinant cloning vector plasmid as a template, carrying out double enzyme digestion on a PCR amplification product and a plant expression vector pCAMBIA3301 plasmid by using BglII and BstEII, recovering the enzyme digestion product by using a DNA product gel recovery kit, and then using T₄the two double digestion products are connected by ligase at 16 ℃, and the connection product is transformed into escherichia coli DH5 alpha competent cells. The T-DNA structure of the plant overexpression vector pCAMBIA3301-sHSP2 to be constructed is shown in FIG. 1.

1. PCR identification of plant overexpression vector pCAMBIA3301-sHSP26

Selecting a single colony on a transformation plate, placing the single colony in a liquid culture medium of LB + Kan, carrying out shaking culture at 37 ℃ overnight, extracting plasmids by using a kit, and carrying out PCR amplification by using GmZFCY 08S and GmZFCY08 AS, wherein the amplification conditions are AS follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and 32 cycles; extension 72 ℃ for 30s, and post-extension 72 ℃ for 10 min. PCR identification was performed on the constructed pCAMBIA3301-sHSP26 vector. The results are shown in FIG. 11. As can be seen from the figure, the constructed recombinant plant overexpression vector pCAMBIA3301-sHSP26 can amplify a specific band about 696bp, and the success of the construction of the plant overexpression vector pCAMBIA3301-sHSP26 is preliminarily proved.

2. Double enzyme digestion identification of plant overexpression vector pCAMBIA3301-sHSP26

In order to further verify whether the construction of the plant overexpression vector pCAMBIA3301-sHSP26 is successful, a plasmid with the correct PCR amplification size is used as a template, BglII and BstEII are used for carrying out enzyme digestion identification on the constructed recombinant plant overexpression vector, and the result is shown in figure 12.

3. Sequencing identification of plant overexpression vector pCAMBIA3301-sHSP26

For the plasmid with correct PCR and enzyme digestion identification, CX1 and CX2 are used as primers (see Table 1), sequencing is carried out by Jilin province, Kuumei, Biotech limited company, and the constructed result of the over-expression vector is identified. The alignment of the sequencing results shows that the sequence is 100% consistent with the expected results, which indicates that the target gene is inserted into the plant expression vector pCAMBIA3301 and the plant over-expression vector pCAMBIA3301-sHSP26 is successfully constructed.

Example 5 construction and identification of CRISPR vector of soybean sHSP26 Gene

1. Construction of soybean CRISPR-sHSP26 vector and PCR identification

The CRISPR vector of the soybean sHSP26 gene is constructed by the steps in the CRISPR/Cas vector construction kit of the Baige creature, and Oligo primers 8UP Oligo and 8Low Oligo used in the construction of the vector are shown in Table 1. After the vector construction is finished, the reaction product is transformed into escherichia coli competent cells, and then the bacterial liquid is coated on a solid plate of LB + Kan and is inversely cultured overnight.

Picking single colony on a transformation plate, carrying out shake culture in a liquid culture medium of LB + Kan at 37 ℃ for one night, extracting plasmids by using a Wegeners plasmid miniprep extraction kit, carrying out PCR amplification on a Bar gene on a CRISPR vector by using Bar S and Bar AS primers, and carrying out ddH₂And taking the PCR product of O as a negative control, and carrying out plasmid PCR identification. The results are shown in FIG. 14, from which it can be seen that a specific band of about 428bp, corresponding to the expected size, can be obtained by PCR amplification.

2. Sequencing identification of soybean CRISPR-sHSP26 vector

and identifying the correct plasmid through PCR, sequencing the plasmid by using a sequencing primer CRI, and further identifying the construction condition of the soybean sHSP26 gene CRISPR vector. The sequencing result is consistent with 100% of the sequence to be aligned, and the success of constructing the soybean CRISPR-sHSP26 vector is proved.

EXAMPLE 6 Agrobacterium-mediated transformation of Soybean

Plasmid DNA of pCAMBIA3301-sHSP26 and CRISPR-sHSP26 vectors is extracted, and a single colony on a transformation plate is picked according to the transformed Agrobacterium competent cell EHA105, and is subjected to shake culture overnight at 28 ℃ in a YEP + Kan liquid medium. The method is characterized in that agrobacterium engineering bacteria liquid is used AS a template, PCR verification is carried out on the plant overexpression vector pCAMBIA3301-sHSP26 by using GmZFCY 08S and GmZFCY08 AS primers and on the CRISPR-sHSP26 vector by using Bar S and Bar AS primers, the result is shown in figure 16, and AS can be seen from the figure, target bands can be amplified by PCR amplification of the two bacteria liquids, the sizes of the two bacteria liquids are consistent with expectations, and the two agrobacterium engineering bacteria liquids can be used for genetic transformation of soybeans.

example 7 detection of transgenic progeny plants

1. PCR detection of transgenic over-expression vector pCAMBIA3301-sHSP26 soybean plant

Extracting leaf genome DNA of soybean plants of JN18 untransformed plants and transformation overexpression vector pCAMBIA3301-sHSP26 by using plant genome DNA extraction kit of Kangji century Biotech CoS, BarAS the extracted genomic DNA was subjected to PCR amplification using the PCR product of the overexpression vector plasmid as a positive control and water as a negative control, and the results are shown in FIG. 17. Transforming by agrobacterium-mediated method to obtain T₀Generating 23 resistant plants, detecting by PCR, obtaining 2 positive plants, and harvesting T₀Generation PCR positive seeds, additional generation planting in greenhouse with T₁Carrying out PCR detection by using the genome DNA of the leaf of the generation plant as a template, obtaining T when the detection result of 3 plants is positive₁planting seeds in Jilin forest agriculture large-scale transgenic experimental field, and breeding T₂Carrying out PCR detection on the generation plants.

2. PCR detection of soybean plant with transformed CRISPR-sHSP26 vector

Extracting genomic DNA of soybean plant leaves of JN18 untransformed and transformed with CRISPR-sHSP26 vector, performing PC detection on the soybean plant by taking the genomic DNA AS a template and Bar 1S, Bar 1AS AS a primer, taking a PCR amplification product of CRISPR-sHSP26 plasmid AS a positive control and taking water AS a negative control, and obtaining a result shown in figure 18. Transforming by agrobacterium-mediated method to obtain T₀The resistant plants were 29 plants, 3 plants were positive by PCR (FIG. 18a), and T was harvested₀Generation PCR positive seeds, additional generation planting in greenhouse, detecting, and T₁The test results for the 6 generations were positive (FIG. 18b), and T was harvested₁Seed generation, planting in transgenic experimental field of Jilin agriculture university, randomly sampling, and subjecting to T₂PCR detection of the plant generation, T₂The partial results of the generation are shown in FIG. 18 c.

3. southern blot detection of transgenic plants

Extraction of T positive in PCR identification₂Transferring genome DNA of the leaf blades of a soybean plant and a JN18 control plant of an overexpression vector, carrying out enzyme digestion overnight by using HindIII restriction enzyme, using a vector plasmid as a positive control, preparing a probe by using Bar, and carrying out Southern blot detection, wherein the result is shown in figure 19. As can be seen from the figure, the untransformed plants had no crossing signal, and the T detected₂In the plants of generation transformation over-expression vector, 2, 3 and 4 plants all have hybridization signals and are single copies, but the sites of gene integration are different, and 1 plant does not have hybridization signalsAnd the result shows that the gene is not integrated into the genome of the plant and is a false positive plant.

4. Fluorescent quantitative PCR detection of transgenic plant target gene

Extraction of T₂3 transgenic lines (transgenic OV8-1 and OV8-2) transferring the overexpression vector, 2 transgenic lines (CR8-1 and CR8-3) transferring the CRISPR vector and RNA of an ovary of a control JN18 soybean plant are generated by reverse transcription to generate cDNA. Fluorescent quantitative PCR was performed using cDNA as template, YP15 and YP16 as primers (see Table 1), soybean actin gene (. beta. -actin) as internal reference, and the results were obtained according to 2^-△△CtThe relative expression level of sHSP26 gene was calculated and analyzed, and the results are shown in FIG. 20. As can be seen from the figure, the relative expression level of the target gene in the CR8-2 strain is the lowest and is 48 percent of that of the control plant, the relative expression level of the OV8-2 strain is the highest and is 35.13, and the very significant difference (P < 0.01) is achieved among the strains.

Example 7 Studies of function of Soybean sHSP26 Gene

1. Drought-resistant physiological and biochemical index detection of transgenic plant

When T is₂After three compound leaves grow on the generation positive transformation plant and the contrast JN18 plant, taking out the whole plant, cleaning, putting the plant into a solution of MS liquid culture medium added with 400mM mannitol for continuous culture, respectively taking soybean leaves before and after 6h of mannitol treatment, detecting physiological and biochemical indexes related to plant drought resistance such as superoxide dismutase (SOD) activity, Peroxidase (POD) activity, Malondialdehyde (MDA) content, Proline (PRO) content and the like by using a kit (the specific method is shown in the test kit specification of Suzhou Keming Biotech limited), and using a DPS data processing system to arrange and analyze data. The change of drought resistance of the transgenic plant after drought stress simulation is researched, and further the function of the soybean sHSP26 gene is analyzed.

1) Detection of transgenic plant superoxide dismutase (SOD) activity

T of transgenic pCAMBIA3301-HSP26(OV8-1, OV8-2) and CRISPR-sHSP26(CR8-1, CR8-3) using superoxide dismutase kit from Suzhou Keming Biotechnology Ltd₂Generation transgenic Soybean lines and control JN18SOD activity detection is carried out before mannitol stress and 6h after mannitol stress, and the DPS software is utilized to analyze experimental data (figure 21), the result shows that the change of SOD value is obvious (P is less than 0.05) in different strains and before and after the treatment, the soybean strains transformed with the overexpression vectors show the trend that the SOD activity is greatly increased after the stress, and the higher the SOD activity is, the stronger the drought tolerance of the plants is.

2) Detection of Peroxidase (POD) activity of transgenic plants

POD activity detection is carried out on transgenic soybean strains and control JN18 before and after mannitol stress by using the kit, the result is shown in figure 22, the figure shows that the POD activity in the plants is obviously improved after the mannitol stress, the difference among the strains is obvious (P is less than 0.05), and the POD activity of over-expressed plants is more than that of the control plants and more than that of the CRISPR plants.

3) Detection of Malondialdehyde (MDA) content of transgenic plant

When plants are stressed by drought, membrane substances can undergo peroxidation, and the final decomposition product of membrane substance peroxidation is Malondialdehyde (MDA), so that the higher the content of the malondialdehyde is, the larger the damage degree of the plants caused by drought is, and conversely, the smaller the change of the content of the malondialdehyde is, the stronger the stress resistance of the plants is. MDA activity detection is performed on transgenic soybeans and control JN18 soybeans before and after mannitol stress by using a malondialdehyde content determination kit of Suzhou Keming Biotechnology Co., Ltd, and the results are shown in FIGS. 3-23. As can be seen from the figure, after stress treatment, the malondialdehyde content of the CRISPR vector-transferred strains (OV8-1 and OV8-3) is increased to the maximum extent, which indicates that the strains are seriously damaged by adversity stress.

4) Detection of Proline (PRO) content in transgenic plant

PRO content detection was performed on transgenic soybeans and control JN18 soybeans before and 6h after mannitol stress, and the results are shown in FIG. 24. The PRO content change difference between the over-expression vector strains (OV8-1 and OV8-2), the CRISPR vector strains (CR8-1 and CR8-3) and the control plants before and after stress is obvious (P is less than 0.05), and the PRO change of the over-expression vector strains is most obvious and is obviously higher than that of the control plants.

2. Agronomic character survey of transgenic plants

Plant height, node number, branch number, pod number, three-pod number, four-pod number, hundred-grain weight and single plant yield of transgenic strains and control plants are investigated, obtained data are analyzed by a DPS data processing system, and as shown in Table 2, results show that all investigation indexes do not reach a significant difference (P <0.05), but the four-pod proportion and the single plant yield of a strain (OV8-1) with a trans-overexpression vector are higher than those of a strain (CR8-3) with a JN18 control and a CRISPR vector.

TABLE 2 statistical table of agronomic traits of transgenic soybeans

Sequence listing

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Claims (5)

1. A soybean sHSP26 gene is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. An amino acid sequence encoding the soybean sHSP26 gene of claim 1, which is represented by SEQ ID No. 2.

3. The plant overexpression vector containing the soybean sHSP26 gene of claim 1, wherein the overexpression vector is named as pCAMBIA3301-sHSP 26.

4. A plant gene editing vector containing the soybean sHSP26 gene of claim 1, wherein the gene editing vector is named CRISPR-sHSP26 gene.

5. The use of the soybean sHSP26 gene of claim 1 in drought resistance of plants.