CN110592105A - Soybean sHSP16.9 gene and application thereof - Google Patents

Abstract

The invention provides a soybean sHSP16.9 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. The invention clones soybean small heat shock protein sHSP16.9 gene by RT-PCR technology, predicts 168 amino acids of the gene code by bioinformatics analysis, at the same time, successfully constructs overexpression and CRISPR-Cas9 gene editing vector and transforms soybean, transforms soybean cotyledon node by agrobacterium-mediated method, and obtains T through PCR detection₀Positive plant 5, T transformed with super expression vector₁Generation positive plants 8, T₂Generation positive plantsStrain; t of CRISPR-transferred vector₀Generation positive plant 4, T₁Generation positive plants 9, T₂Several plants with positive generation. The expression of the sHSP16.9 gene can improve the drought resistance of soybeans.

Description

Soybean sHSP16.9 gene and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a soybean sHSP16.9 gene and application thereof.

Background

Heat Shock Proteins (HSPs) are proteins synthesized under adverse stimuli such as high temperature, ultraviolet light, salt, heavy metal ions, drought, toxic gases, and the like, which protect cells from or are less harmful to the adverse stimuli, are essential components of plants in adapting to short-term stress and alleviating the damage of adverse stresses, and can be combined with denatured proteins and Mg in order to maintain the soluble state of the denatured proteins²⁺And ATP, which refolds the denatured protein back to an active protein. Heat Shock Proteins (HSPs) also play important roles in signaling, regulating metabolism, and inhibiting apoptosis. Heat Shock Proteins (HSPs) are a ubiquitous and conserved family of proteins distributed in various organelles and one of the most abundant intracellular proteins. HSPs can be classified by molecular weight: several families of small molecules HSP (shsp), HSP60, HSP70, HSP90, HSPl00, each of which includes multiple constituent members. Wherein the heat shock protein with the molecular weight of 12-43 kD is called small heat shock protein (sHSP).

First, sHSP has a chaperone effect. In the case of drought, high temperature, cold and other adversities, sHSP can reduce the damage of the environment to the plant growth. Secondly, sHSP plays an important role in the regulation of apoptosis, and small heat shock proteins such as sHSP27, sHSP20 and sHSP16.2 have the functions. sHSP also plays an important role in the growth and stress of plants, can participate in physiological processes such as growth of vegetative tissues, storage organs and seeds, plays different roles in different growth stages of the plants, and protects the normal development of the plants through the processes of enhancing the stress resistance of embryos, regulating hormones and chromatin and the like under the stress of the stress. In addition, sHSP can act synergistically with lipid molecules to reduce fluidity of membrane lipids, thereby resisting adversity stress.

Disclosure of Invention

The invention aims to provide a soybean sHSP16.9 gene and application thereof, and the expression of the sHSP16.9 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 sHSP16.9 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also provides an amino acid sequence of the soybean sHSP16.9 gene, which is shown in SEQ ID NO. 2.

The invention also provides a plant over-expression vector containing the soybean sHSP16.9 gene, and the over-expression vector is named as pCAMBIA 3301-sHSP16.9.

The invention also provides a plant gene editing vector containing the soybean sHSP16.9 gene, which is named as CRISPR/Cas 9-sHSP16.9.

The invention also provides application of the soybean sHSP16.9 gene in drought resistance of plants.

The invention has the advantages of

The invention provides a soybean sHSP16.9 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention clones the soybean small heat shock protein sHSP16.9 gene by using RT-PCR technology, and the gene codes 168 amino acids through bioinformatics analysis and prediction, wherein the molecular weight of the protein is 16969.61D, the theoretical isoelectric point pI is 5.68, and the molecular formula is C₈₃₂H₁₃₃₂N₂₃₂O₂₅₈S₈The gene encodes a protein belonging to the alpha-crystallin-Hsps _ p23-like superfamily, which is a hydrophilic protein, and has an extinction coefficient of 12740, a destabilization coefficient of 45.50, a fat coefficient of 75.95, and a total average hydrophilicity (GRAVY) of-0.593, measured at 280 nm. Meanwhile, the invention also successfully constructs an overexpression and CRISPR-Cas9 gene editing vector and transforms soybeans, transforms soybean cotyledonary nodes by utilizing an agrobacterium-mediated method, and obtains T through PCR detection₀Positive plant 5, T transformed with super expression vector₁8 positive plants are generated; t of CRISPR-transferred vector₀Generation positive plant 4, T₁Generation positive plants 9. The genes are integrated into the genome of soybean in a single copy form by Southern blot detection.

The invention confirms the drought-resistant function of the gene through the research of transgenic offspring, and the result shows that the contents of SOD, POD and PRO after drought stress are as follows: overexpression vector plants > control plants > CRISPR plants, and the content of malondialdehyde: the over-expression vector plant is less than the control plant and less than the CRISPR plant, which shows that the expression of the soybean sHSP16.9 gene improves the drought resistance of the soybean.

Drawings

FIG. 1 is a schematic diagram of the T-DNA structure of plant over-expression vector pCAMBIA3301-sHSP 16.9;

FIG. 2 is a diagram of RNA extraction gel electrophoresis, 1-4: RNA extracted from ovaries;

FIG. 3 is the RT-PCR electrophoresis of sHSP16.9 gene, M: DL2000 molecular weight standard, 1-4: RT-PCR products;

FIG. 4 shows PCR electrophoresis of bacterial suspension, M: DL2000 molecular weight standards 1-6: PCR products;

FIG. 5 is a PCR electrophoretogram of plasmid, M: DL2000 molecular weight Standard 1-2: PCR products;

FIG. 6 is a double restriction enzyme validation assay of the cloning vector, M: DL2000 molecular weight standard 1: cloning vector plasmid 2: double enzyme digestion products;

FIG. 7 is a comparison chart of sequencing results;

FIG. 8 is a diagram of analysis of protein conserved domains;

FIG. 9 is a graph of hydropathic and hydrophobic analysis;

FIG. 10 is a signal peptide prediction diagram;

FIG. 11 is a coiled coil prediction graph;

FIG. 12 is a structural diagram of a model of the tertiary structure of the sHSP16.9 protein;

FIG. 13 shows the electrophoresis of the overexpression vector in PCR identification, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR product, a: bacterial liquid PCR b: carrying out plasmid PCR;

FIG. 14 is a diagram showing the double restriction enzyme identification of the overexpression vector pCAMBIA3301-sHSP 16.9; m: DL2000 molecular weight standard 1: overexpression vector plasmid 2: performing enzyme digestion on the product;

FIG. 15 is a comparison of sequencing results of overexpression vectors;

FIG. 16 is PCR identification electrophoresis diagram of CRISPR-sHSP16.9 vector; m: DL2000 molecular weight standard 1: negative controls 2-8: PCR product, a: bacterial liquid PCR b: carrying out plasmid PCR;

FIG. 17 is a comparison chart of sequencing results of CRISPR-sHSP16.9 vector;

fig. 18 is a PCR identification detection diagram of agrobacterium tumefaciens bacterial solution, M: DL2000 molecular weight standard 1: negative controls 2-7: PCR product, a: the over-expression vector pCAMBIA3301-sHSP16.9 bacterial liquid PCR b: PCR of CRISPR-sHSP16.9 carrier bacterial liquid;

FIG. 19 is a diagram of Agrobacterium-mediated transformation of soybean cotyledonary nodes,

a. germinating b, pre-culturing c.d, infecting e, co-culturing f, screening g, extending h, rooting i, hardening seedlings j, and transplanting;

FIG. 20 is a PCR detection map of transgenic over-expression vector pCAMBIA3301-sHSP16.9 plant,

m: DL2000 molecular weight standard +: positive control-: negative controls 1-13: PCR product a: t0 generation test result b: detection result c of T1 generation: the detection result of the T2 generation;

fig. 21 is a PCR detection map of CRISPR-shsp16.9 vector transformed plants, M: DL2000 molecular weight standard +: positive control-: negative controls 1-13: PCR product, a: t0 generation test result b: detection result c of T1 generation: the detection result of the T2 generation;

FIG. 22 shows Southern blot analysis of T2 generation pCAMBIA3301-sHSP16.9 vector plants, M: southern standard molecular weight +: positive control-: negative controls 1-4: t2 generation transformed plants;

FIG. 23 shows relative expression levels of sHSP16.9 gene;

FIG. 24 is a graph showing superoxide dismutase activity;

FIG. 25 is a peroxidase activity assay;

FIG. 26 is a graph showing the results of measurement of malondialdehyde content;

FIG. 27 is a graph of proline content.

Detailed Description

The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials and reagents used in the following examples were all commercially available from a commercial source unless otherwise specified.

Plant material the Jinong 18 soybean mutant (TB18) was provided by the plant Biotechnology center of Jilin university of agriculture. The used molecular biological reagent is a RNAiso plus kit and T₄Ligase, pMD-18T Vector, DL2000 Marker, etc. were purchased fromIn Baobioengineering (Dalian) Co., Ltd., All-in-One qPCR Kit was purchased from GeneCopoeia Co., Ltd., plasmid miniprep Kit was purchased from Kangji Biotech Co., Ltd., Southern blot digoxin Kit was purchased from Roche Co., Ltd., CRISPR/Cas vector construction Kit was purchased from Baige Gene technology (Jiangsu) Co., Ltd., and detection Kit for superoxide dismutase was purchased from Suzhou Keming Biotechnology Co., Ltd. Consumables such as a centrifuge tube and a pipette tip are supplied by Henkel Feika biological Limited, and biochemical reagents such as isopropanol are purchased from Jintai Limited in Changchun city.

Example 1 cloning and identification of Soybean sHSP16.9 Gene

TABLE 1 primer List

Specific primers GmZFCY02S and GmZFCY02AS (see table 1) are designed by using Primer Premier 5.0 software according to the nucleotide sequence of the sHSP16.9 gene, and BglII and BstEII enzyme cutting recognition sequences are directly introduced into the 5' ends of the two primers for the convenience of constructing an overexpression vector. Extracting total RNA of TB18 soybean ovary, purifying and reverse transcribing to generate cDNA, performing PCR amplification by using the cDNA as a template and GmZFCY02S and GmZFCY02AS as primers under the following amplification conditions: 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. And the amplification result was electrophoretically detected using 1% agarose gel.

The total RNA of the seven-leaf stage TB18 soybean ovary is extracted by using a Triol method, 2 mu L of the total RNA is detected by electrophoresis, and the result is shown in figure 2. As can be seen from the figure, three bands of 28s, 18s and 5s rRNA can be seen in the extracted RNA, and the ratios of OD260/OD280 are all 1.9-2.0 through detection of the NanoDrop1000, which indicates that the extracted RNA has good quality and can be used for subsequent experiments. cDNA generated by reverse transcription of RNA is taken as a template, specific primers GmZFCY02S and GmZFCY02AS are designed according to the nucleotide sequence of the sHSP16.9 gene, the gene is amplified by using RT-PCR technology, PCR amplification products are detected by using 1% agarose gel electrophoresis, and the result is shown in figure 3, wherein a specific amplification band exists at 507bp, and the product conforms to the size of the sHSP16.9 gene. The success of cloning the target gene is proved, and the nucleotide sequence of the soybean sHSP16.9 gene is shown in SEQ ID NO. 1.

Example 2 construction and identification of Soybean sHSP16.9 Gene cloning vector

Connecting the recovered and purified RT-PCR product with a pMD-18T cloning vector, placing the mixed reaction solution into an incubator at 16 ℃ for reacting overnight, then transforming the reaction solution into escherichia coli DH5 alpha competent cells, culturing overnight, and observing colonies.

Single colonies on the plates were picked and placed in tubes of LB + Kan broth and cultured overnight at 37 ℃ with shaking at 200 rpm. And (3) performing PCR identification by taking the cultured bacterial liquid as a template and GmZFCY02S and GmZFCY02AS as primers, extracting plasmid DNA (deoxyribonucleic acid) to perform plasmid PCR, double enzyme digestion and sequencing identification, wherein the identification result is correct.

PCR amplification of the sHSP16.9 gene was performed using the bacterial suspension as a template, and the results are shown in FIG. 4. As can be seen from FIG. 4, the sizes of the PCR products of the bacterial solutions 1, 2, 5 and 6 are 507bp, which is expected, and the PCR products of the bacterial solutions 3 and 4 have no result, and plasmids of the bacterial solutions 1, 2, 5 and 6 are extracted for carrying out the plasmid PCR verification. The result of the plasmid PCR amplification is shown in FIG. 5, and it can be seen from the figure that the specific product with the size of about 507bp can be obtained by the plasmid PCR amplification and is consistent with the size of the soybean sHSP16.9 gene.

Plasmid with correct size detected by plasmid PCR is used as a template, and the constructed cloning vector is subjected to double enzyme digestion identification by using BglII and BstEII restriction enzymes. The results are shown in FIG. 6, and it can be seen that after the recombinant cloning vector is digested, a target gene band of about 507bp and a linear vector band can be obtained, and the results are in line with the expectation.

Example 3 sequencing identification of Soybean sHSP16.9 Gene cloning vector

The recombinant cloning vector verified by PCR and double enzyme digestion is sent to Jilin province Customi biotechnology and Limited company to be sequenced by using a T vector universal primer, and the sequencing result is compared by using DNAMAN software (figure 7), wherein the comparison result shows that the nucleotide sequence of the sequencing plasmid is 99.8 percent identical to the nucleotide sequence of the sHSP16.9 gene, only one base is different, but the amino acids coded by the nucleotides are completely identical, and the successful cloning of the sHSP16.9 gene is proved.

Example 4 analysis of the basic physicochemical Properties of the protein encoded by the Soybean sHSP16.9 Gene

The molecular weight, theoretical isoelectric point, molecular formula, extinction coefficient, instability coefficient and other basic physicochemical properties of the protein coded by the soybean sHSP16.9 gene are analyzed by ProtParam online software (http:// web. expasy. org/ProtParam /), and the prediction result shows that the gene codes 168 amino acids, the molecular weight of the protein is 16969.61D, the theoretical isoelectric point pI is 5.68, and the molecular formula is C₈₃₂H₁₃₃₂N₂₃₂O₂₅₈S₈The extinction coefficient measured at 280nm was 12740 and the instability coefficient was 45.50, which indicates that the protein is unstable, the fat coefficient was 75.95, and the overall average hydrophilicity (GRAVY) was-0.593, indicating that the protein is a hydrophilic protein. The amino acid sequence is shown as SEQ ID NO. 2.

Example 5 conservative Domain analysis of protein encoded by Soybean sHSP16.9 Gene

The protein sequence of the soybean sHSP16.9 gene was subjected to Conserved Domain analysis using the on-line software NCBI Conserved Domain Search Service (http:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi), and it was shown that the protein encoded by the gene belongs to the alpha-crystallin-Hsps _ p23-like superfamily (see FIG. 8), the members of which all have an alpha-crystallin Domain (ACD) comprising an alpha-crystallin type small heat shock protein and similar domains found in the p23-lik protein. The most typical feature of small heat shock proteins is the presence of an ACD domain. Small heat shock proteins are usually oligomers composed of multiple subunits, which are generally considered ATP-independent chaperones that prevent polymerization and play an important role in binding to other heat shock proteins, protein folding. p23 is a synergistic chaperone in the Hsp90 signaling pathway, which can confine Hsp90 protein and participate in the folding process of Hsp90 protein, which contains the internal portion of the progesterone receptor, and at the same time, it can also act as a cellular prostaglandin E2 synthase.

Example 6 hydrophilic and hydrophobic analysis of Soybean sHSP16.9 Gene

The protein encoded by the sHSP16.9 gene is analyzed for hydrophilicity and hydrophobicity on line by using a ProtScale program (http:// web. expasyy. org/ProtScale /), in the case of a default window 9, the hydrophilicity and hydrophobicity are shown in FIG. 9a, and in order to facilitate result analysis, the window is adjusted to 19, in FIG. 9b, it can be seen that when the threshold value of |. Score ≧ 1.5, two peaks with high scores are present, both peaks are located in a high hydrophilicity region, and the result is consistent with the result of analysis by using ProtParam in 2.2.3.1, and the protein encoded by the soybean sHSP16.9 gene is further determined to be a hydrophilic protein.

Example 7 prediction of amino acid sequence Signal peptide of Soybean sHSP16.9 Gene

The signal peptide prediction analysis of the amino acid sequence of the soybean sHSP16.9 gene is carried out by using a SignalP online program (http:// www.cbs.dtu.dk/services/SignalP /), and the result is shown in FIG. 10, and the signal peptide sequence of the protein coded by the gene is absent in a coding region, which indicates that the sHSP16.9 protein is not a secretory protein.

Example 8 prediction of the coiled coil of protein encoded by the Soybean sHSP16.9 Gene

The results of predicting the coiled-coil of the protein encoded by the gene by using COILS (http:// www.ch.embnet.org/software/COILS _ form. html) are shown in FIG. 11, and it can be seen that the protein encoded by the soybean sHSP16.9 gene detects 2 coiled-coil regions in windows Window14 and 21 and detects 1 coiled-coil region in Window Window 28.

Example 9 prediction of the tertiary Structure of protein encoded by the Soybean sHSP16.9 Gene

A tertiary structure Model of the PROTEIN encoded by the gene was constructed using Swiss-Model (http:// www.swissmodel.expasy.org /), and as a result, as shown in FIG. 12, the amino acid sequence of the PROTEIN had a Coverage (Coverage) with the template of 0.82, a degree of Identity (Seq Identity) of 46.38%, no ligand, and a global Model mass estimation (GMQE) of 0.62, which is described as HEAT SHOCK PROTEIN 16.9B.

Example 10 construction and identification of Soybean sHSP16.9 Gene plant overexpression vector

Extracting the correctly sequenced cloning vector plasmid, performing PCR amplification by using the correctly sequenced cloning vector plasmid as a template, performing double enzyme digestion on a gel recovery product amplified by the PCR and a plant expression vector pCAMBIA3301 plasmid by using two restriction enzymes BglII and BstEII, recovering a double enzyme digestion product, and performing double enzyme digestion by using T₄The target gene after double enzyme digestion is connected with a linearized plant expression vector pCAMBIA3301 by ligase, and the T-DNA structure of the plant over-expression vector pCAMBIA3301-sHSP16.9 to be constructed is shown in figure 1. Then, the ligation product is transformed into escherichia coli DH5 alpha competent cells, and a monoclonal colony is obtained through primary screening of an LB + Kan screening culture medium.

Single colonies on the plates were picked and cultured overnight at 37 ℃ with shaking in liquid medium containing 5mL of LB + Kan, and confirmed by PCR of the culture broth (FIG. 13a) and plasmid PCR (FIG. 13 b). As can be seen from the figure, a specificity strip of about 507bp can be amplified by the constructed recombinant plant overexpression vector, and the success of constructing the plant overexpression vector pCAMBIA3301-sHSP16.9 is preliminarily proved.

In order to further verify whether the construction of the plant over-expression vector pCAMBIA3301-sHSP16.9 is successful, plasmids are extracted and used as templates for carrying out plasmid PCR identification. The plasmid which is correctly identified by PCR is taken as a template, the constructed recombinant plant overexpression vector is subjected to double enzyme digestion identification by utilizing BglII and BstEII restriction enzymes, the constructed recombinant plant overexpression vector is subjected to double enzyme digestion identification according to a double enzyme digestion system at 37 ℃ for 3 hours, the result is shown in figure 14, and a linear vector band and a target gene band of about 507bp can be seen through double enzyme digestion, which is the same as expected.

For the plasmid with correct PCR and double enzyme digestion identification, CX1 and CX2 are used as primers (Table 1), sequencing is carried out by Jilin province Kuumei biotechnology and technology limited company, and the constructed result of the over-expression vector is identified. As shown in FIG. 15, the alignment of the sequencing results shows that the sequence is 100% consistent with the expected results, which indicates that the target gene has been inserted into the plant expression vector pCAMBIA3301 and the recombinant plant expression vector pCAMBIA3301-sHSP16.9 is successfully constructed.

Example 11 construction and identification of Soybean sHSP16.9 Gene CRISPR-Cas9 Gene editing vector

According to the nucleotide sequence of the sHSP16.9 gene, guide RNA (sgRNA) and DNA (pagr RNA) are designed through an online program of CRISPR-P (http:// CRISPR. hzau. edu. cn/CRISPR2/), and Oligo primers GmZFCY02 UP Oligo and GmZFCY02 Low Oligo (shown in Table 1) are designed through a Baige organism website (http:// www.biogle.cn /), and a CRISPR vector construction kit of the sHSP16.9 gene of soybean is utilized to construct a CRISPR vector of the Baige organism.

Picking single colony on the plate, shaking and culturing overnight at 37 ℃ in a liquid culture medium added with 5mLLB + Kan, taking bacterial liquid AS a template, utilizing Bar S and Bar AS primers to PCR amplify Bar gene on the vector, carrying out bacterial liquid PCR verification (figure 16a), extracting plasmid, taking the plasmid AS the template to carry out plasmid PCR identification (figure 16b), and AS can be seen from the figure, both the bacterial liquid PCR and the plasmid PCR can amplify specific bands of about 428bp to preliminarily prove the success of vector construction.

For the plasmid with correct PCR identification, sequencing primer CRI is utilized to send the sequencing primer CRI to Jilin province-Cumei biotechnology limited company for sequencing, as shown in figure 17, the sequencing result is completely consistent with the sequence to be compared, and the success of construction of the CRISPR-sHSP16.9 vector of the soybean sHSP16.9 gene is proved.

EXAMPLE 12 Agrobacterium-mediated transformation of Soybean

Soybean cotyledonary node is transformed by agrobacterium-mediated transformation, soybean transformed plants of the sHSP16.9 gene overexpression vector and the CRISPR vector are obtained through seed germination, pre-culture, infection, screening, seedling hardening, transplanting and the like, and the culture medium and the specific method used in the experiment are disclosed in Qujing paper.

1. PCR verification of bacterial liquid of agrobacterium engineering bacteria

A single colony on a YEP + Kan plate is picked and inoculated into a liquid culture medium containing 5mL of YEP + Kan, the liquid culture medium is subjected to shaking culture at the temperature of 28 ℃ and the speed of 210rpm overnight, a bacterial liquid is used as a template, and a GmZFCY02S primer and a GmZFCY02AS primer (shown in table 1) are used for carrying out bacterial liquid PCR verification on a plant over-expression vector pCAMBIA3301-sHSP16.9, a BarS primer and a BarAS primer (shown in table 1) on a CRISPR-sHSP16.9 vector, and the result is shown in fig. 18.

2. Agrobacterium transformed soybean cotyledon node

The constructed over-expression vector pCAMBIA3301-sHSP16.9 and CRISPR-sHSP16.9 are transformed into JN18 soybean cotyledonary node by adopting agrobacterium-mediated transformation method, and 56 soybean regenerated plants are obtained by the processes of seed germination, pre-culture, infection, co-culture, screening culture, elongation culture, rooting culture, hardening seedling transplantation and the like (see figure 19), wherein 31 strains of the over-expression vector pCAMBIA3301-sHSP16.9 and 25 strains of the CRISPR-sHSP16.9 are transformed.

Example 13 testing of transgenic progeny plants

1. PCR detection of transgenic over-expression vector pCAMBIA3301-sHSP16.9 soybean plant

Extracting the genomic DNA of leaves of untransformed and transformed soybean plants by using a plant genomic DNA extraction kit of the Kangji Biotechnology Ltd, performing PCR amplification by using Bar S and Bar AS primers (see Table 1) with the extracted genomic DNA AS a template, and detecting the amplification result by using 1% agarose gel electrophoresis.

Extracting leaf genome DNA of a soybean plant of a JN18 untransformed plant and a transformation overexpression vector pCAMBIA3301-sHSP16.9, respectively carrying out PCR amplification on the extracted genome DNA by utilizing Bar S and Bar AS, taking a PCR product of an overexpression vector plasmid AS a positive control, taking a PCR product of a JN18 untransformed plant AS a negative control, and detecting an amplification result by using 1% agarose gel electrophoresis, wherein the result is shown in figure 20. Transforming by agrobacterium-mediated method to obtain T₀Generating 52 resistant plants, detecting 5 positive plants through PCR detection, and harvesting T₀Generation PCR positive seeds, additional generation planting in greenhouse with T₁Taking the genome DNA of the generation plant leaves as a template, carrying out PCR detection, obtaining T when the detection result of 8 plants is positive₁Seed generation, planting in transgenic experimental field of Jilin agriculture university, randomly sampling, and subjecting to T₂PCR detection of plant leaves, FIG. 20c is T₂And substituting part of detection results.

2. PCR detection of soybean plant with CRISPR-sHSP16.9 vector

PCR amplification products of CRISPR-sHSP16.9 are used as positive control, PCR amplification products of JN18 untransformed plants are used as negative control, Bar gene PCR amplification is carried out on soybeans of the CRISPR-sHSP16.9 vector, the amplification result is detected by 1% agarose gel electrophoresis, and the result is shown in figure 21. Transforming by agrobacterium-mediated method to obtain T₀Generating 38 resistant plants, obtaining 4 positive plants through PCR detection, and harvesting T₀Planting PCR positive seeds in greenhouse, detecting, and performing T₁The detection result of 9 strains in the generation is positive, and T is harvested₁Generation of seeds, planting in transgenic experimental field of Jilin agriculture university, randomly sampling and testing T₂Carrying out PCR detection on the generation plants.

3. Southern blot detection of transgenic plants

Extracting positive T identified by PCR₂The genomic DNA of the leaves of soybean plants and JN18 control plants transformed with pCAMBIA3301-sHSP16.9 vector was digested overnight with HindIII restriction enzyme, the vector plasmid was used as a positive control, Bar was used to prepare a probe, and Southern blot detection was performed, and the results are shown in FIG. 22. As can be seen from the figure, the untransformed plants had no crossing signal, and the T detected₂In the plants of the generation transformation over-expression vector, the plants 1, 3 and 4 all have hybridization signals and are single copies, but the sites for gene integration are different, and the plant 2 does not have the hybridization signals, which indicates that the genes are not integrated into the genome of the soybean.

4. Fluorescent quantitative PCR detection of transgenic plant sHSP16.9 gene

Separately extracting T₂The RNA of the soybean plant and the non-pollinated ovary of the non-transformed plant is transformed by the overexpression vector with positive PCR and Southern blot detection, and the cDNA is generated by reverse transcription. Using cDNA generated by reverse transcription as a template, YP3 and YP4 as primers (shown in table 1), soybean actin gene (beta-actin) as an internal reference, using untransformed JN18 plants as a reference, and rotating 3T of an overexpression vector₂Generation transgenic lines (OV2-2, OV2-3, OV2-5) and 3T of CRISPR vector₂Generation transgenic strains (CR2-1, CR2-3, CR2-4) sHSP16.9 relative expression of the Gene fluorescent quantitative PCR assay was performed according to 2^-△△CtAnd calculating and analyzing the relative expression quantity of the gene in the transgenic plant. The data were collated to produce a bar chart, see FIG. 23. As can be seen from the figure, the relative expression levels of the sHSP16.9 gene of the plant with the trans-overexpression vector are improved by 22.19-25.63%, and the expression levels of the sHSP16.9 gene of the plant with the trans-CRISPR vector are obviously reduced (39-65%) and reach extremely obvious difference (P is less than 0.01).

Example 14 Studies of Soybean sHSP16.9 Gene function

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 in a solution of MS liquid culture medium added with 400mM mannitol for simulating drought conditions for continuous culture, and respectively taking soybean leaves before and after the mannitol treatment for 6 hours for carrying out detection on drought-resistant related physiological and biochemical indexes. The detection comprises detecting superoxide dismutase (SOD) activity, Peroxidase (POD) activity, Malondialdehyde (MDA) content and Proline (PRO) content, and the specific method is described in the specification of detection kit of Suzhou Keming Biotechnology Co., Ltd. The change of drought resistance of the transgenic plant after drought stress simulation is researched, and further the function of the soybean sHSP16.9 gene is analyzed.

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

Use of JN18 untransformed plants as control for T₂The transgenic lines of the transgenic pCAMBIA3301-sHSP16.9(OV2-2, OV2-3 and OV2-5) and CRISPR-sHSP16.9(CR2-1, CR2-3 and CR2-4) vectors which are positive through molecular detection are detected to detect the SOD activity before and after mannitol stress by using a superoxide dismutase activity measuring kit of Suzhou Keming biotechnology limited company, and the result is shown in figure 24, after mannitol stress treatment, the SOD activity of the transgenic pCAMBIA3301-sHSP16.9 vector plant is increased to the maximum extent, and the difference between the transgenic plant with the CRISPR-sHSP16.9 vector and a control plant is obvious (P is shown in figure 24) (the P)<0.05), the plant of the trans-overexpression vector has higher drought resistance.

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

POD activity detection is carried out on transgenic soybeans (OV2-2, OV2-3 and OV2-5) of a transgenic overexpression vector, transgenic soybeans (CR2-1, CR2-3 and CR2-4) of a transgenic CRISPR vector and control JN18 soybeans before and after mannitol stress by using a superoxide dismutase activity detection kit. As shown in fig. 25, it can be seen from the figure that the activity of POD in the leaves of the plants is increased after mannitol simulated stress, the difference between the trans-overexpression vector, the CRISPR strain and CK is significant (P < 0.05), the increase amplitude of the transgenic soybean of the trans-CRISPR vector is smaller and lower than that of the control plant, and the increase amplitude of the POD activity of the over-expression plant is the largest, because POD mainly plays a role in eliminating hydrogen peroxide in the plant, the activity of POD is strong, and the stress resistance of the plant is strong.

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

The kit is used for carrying out MDA content detection on transgenic soybeans and contrast JN18 soybeans before and after mannitol stress, the result is shown in figure 26, and the figure shows that the MDA content difference between strains before and after mannitol stress is obvious, the MDA content rise amplitude in the strains of CRISPR vector transformation after drought stress is the largest, which indicates that the plant membranous peroxidation degree is high, the plant damage is serious, the plant rise amplitude of the transformation overexpression vector is smaller, and the stress resistance of the plant is stronger when stress occurs.

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

The proline content detection kit is used for detecting the PRO content of transgenic soybean strains with overexpression and CRISPR vectors and control JN18 soybeans before 400mM mannitol stress and 6h after stress, the result is shown in figure 27, and the PRO content difference of each strain before and after stress is obvious (P is less than 0.05), wherein the PRO content increase amplitude of the strains with the overexpression vectors is maximum after stress.

2. Agronomic character survey of transgenic plants

The plant height, node number, branch number, pod number, three pod number, four pod number, hundred grain weight, and individual plant yield of 10 each of the transgenic lines that transferred the overexpression vector (OV2-3) and the CRISPR vector (CR2-1) and 10 untransformed control plants were investigated, and the data obtained were analyzed using the DPS data processing system, and the results are shown in Table 2. As can be seen from the table, no significant difference was achieved between the survey data, but as can be seen from the table, the lines OV2-3 exhibited higher grain weight per hundred, four pod numbers and individual yield than the control plants.

TABLE 2 statistical table of agronomic traits of transgenic soybeans

Sequence listing

<110> Jilin university of agriculture

<120> soybean sHSP16.9 gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 507

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtctctgt ttgcacccct gttgttgaac cagagcgacc ccttcgacca tttccgagcc 60

ttactcggtg gaaattcaga atcattggat ctcggagcgt acacccaaat ggattggaag 120

gaaacccttg acgcccatgt gttcgaaatc gatcttcccg ggttcgccaa agaggatgtg 180

aagcttggag tgaaagaaaa cagagtgctc tgcatcaaag cagagaaaaa agcagaacaa 240

gaagaacaag aagagaagac aaagctgaaa tggcattgca gggagagaag gagcagtggc 300

gtggtctcta gggagtttag gttgcctgag aattccaagg ttgatggtgt cagagcttca 360

atgtgtgatg gggtgttgac agtaacagtg cctaaggatg agagtgagac cctgaagaag 420

cacaagaagg aggtgcagat ttttgaagag gatggtgaag gggttgctcc aaaaggaatt 480

ggtcgttttg tttgctgcaa atcttag 507

<210> 2

<211> 168

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Leu Phe Ala Pro Leu Leu Leu Asn Gln Ser Asp Pro Phe Asp

1 5 10 15

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

20 25 30

Ala Tyr Thr Gln Met Asp Trp Lys Glu Thr Leu Asp Ala His Val Phe

35 40 45

Glu Ile Asp Leu Pro Gly Phe Ala Lys Glu Asp Val Lys Leu Gly Val

50 55 60

Lys Glu Asn Arg Val Leu Cys Ile Lys Ala Glu Lys Lys Ala Glu Gln

65 70 75 80

Glu Glu Gln Glu Glu Lys Thr Lys Leu Lys Trp His Cys Arg Glu Arg

85 90 95

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

100 105 110

Lys Val Asp Gly Val Arg Ala Ser Met Cys Asp Gly Val Leu Thr Val

115 120 125

Thr Val Pro Lys Asp Glu Ser Glu Thr Leu Lys Lys His Lys Lys Glu

130 135 140

Val Gln Ile Phe Glu Glu Asp Gly Glu Gly Val Ala Pro Lys Gly Ile

145 150 155 160

Gly Arg Phe Val Cys Cys Lys Ser

165

Claims (5)

1. A soybean sHSP16.9 gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. An amino acid sequence encoding the soybean shsp16.9 gene of claim 1, which is as set forth in SEQ ID No. 2.

3. A plant over-expression vector containing the soybean shsp16.9 gene of claim 1, wherein the over-expression vector is named pCAMBIA3301-shsp 16.9.

4. A plant gene editing vector containing the soybean shsps 16.9 gene of claim 1, wherein said gene editing vector is named CRISPR/Cas 9-shsps 16.9.

5. The use of the soybean shsps 16.9 gene of claim 1 in drought resistance of plants.