CN115927393A - GsHSP gene and application thereof in improving salt tolerance of soybeans and content of isoflavone in soybean seedling vegetables - Google Patents

Abstract

The invention discloses a GsHSP gene and application thereof in improving salt tolerance of soybeans and content of isoflavone in soybean sprout vegetables, wherein the sequence of the GsHSP gene is shown as SEQ ID NO.1, the application also discloses a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene and application thereof, the GsHSP can improve salt tolerance of soybean root systems, overexpression can promote root system growth of soybeans under a salt and alkali condition and yield of the soybean sprout vegetables, and can be applied to improve content of isoflavone in the soybean sprout vegetables under salt stress, can be applied to culture high-quality salt-tolerant GsHSP recombinant soybean plants, and has good market prospect.

Description

GsHSP gene and application thereof in improving salt tolerance of soybeans and content of isoflavone in soybean seedling vegetables

Technical Field

The invention relates to the technical field of soybean functional genes, in particular to a GsHSP gene and application thereof in improving salt tolerance of soybeans and improving the content of isoflavone in bean sprouts.

Background

As an important agricultural crop in China, soybeans are rich in high-quality vegetable proteins, unsaturated fatty acids, calcium, B vitamins, isoflavone and other substances, and play an important role in national nutrition. In recent years, with the improvement of the living standard of people and the improvement of the requirement on materials, the yield of soybeans in China cannot meet the ever-increasing market demand of people. Therefore, improving the yield and quality of soybean in China is a great civil problem which needs to be solved urgently at present.

Stress factors such as salt stress are main factors influencing the yield and quality of soybeans, and great loss is caused to soybean production, so that the key for solving the problems is to improve the quality and stress resistance of the soybeans. The traditional breeding method is adopted to improve the poor traits of the soybeans, which is limited by the shortage of soybean gene resources; the resistance problem and environmental protection problem of plants caused by the indiscriminate use and overuse of chemical agents also limit the application of chemical agents.

Soybean, an important crop, contains abundant nutrients. Soybeans contain sufficient high-quality protein, unsaturated fatty acid, calcium, isoflavone, various vitamins and the like, occupy an important position in the diet of residents in China and are an important source of high-quality vegetable protein.

Soy isoflavone is a pale yellow powdery substance, which is slightly bitter in smell and accompanied by slight astringency. Isoflavones are compounds that are structurally and biologically similar to human estrogens. After the soybeans and the products thereof enter a human body, the contained soybean isoflavone is mainly metabolized and absorbed in intestinal tracts and then converted into equol and other substances, and the equol and other substances enter a blood circulation system of the human body through capillaries. Soy isoflavones are well known as phytoestrogens, a widely accepted natural replacement for HRT (hormone replacement therapy) or HRT supplement.

Soybean isoflavone as a physiologically active substance plays an important physiological function in the life activities of human bodies, and plays an important role in relieving female aging, improving climacteric symptoms, protecting cardiovascular system, resisting tumor, preventing osteoporosis, protecting nervous system, reducing blood sugar, protecting liver, regulating immune system and the like. Isoflavone is more and more accepted by people due to various nutrition and health care physiological functions. The daidzin is an important component of soybean isoflavone, and has pharmacological effects in preventing and treating cardiovascular diseases, resisting tumor, etc. Daidzin, as another important component of soybean isoflavone, plays an important role in anti-allergy and anti-thrombosis.

The improvement of the salt tolerance of the soybeans is beneficial to expanding the planting area of the soybeans and improving the yield of related products of the soybeans, and the important concerns of researchers at present are how to improve the content of isoflavones (such as daidzin and daidzin) in the soybean foods such as the bean sprouts and the like and improve the nutritional and health-care quality of the soybean foods. Therefore, the prior art is in need of further improvement.

Disclosure of Invention

Aiming at the problems, the invention provides a GsHSP gene and application thereof in improving salt tolerance of soybeans and increasing the contents of daidzin and daidzin in a bean sprout vegetable.

In order to solve the above problems, the technical solution provided by the present application is as follows:

in a first aspect, the invention provides a GsHSP gene, the sequence of which is shown in SEQ ID NO.1.

The applicant firstly excavates a salt stress response gene GsHSP from a very salt-tolerant wild soybean variety, clones and analyzes bioinformatics of the salt stress response gene GsHSP, analyzes the tissue expression characteristic and the salt stress expression mode of the gene through real-time fluorescent quantitative PCR, and discovers that the GsHSP can improve the salt tolerance in a soybean root system and improve the isoflavone metabolite content of soybean seedlings in the soybean seedlings under salt stress by constructing a gene overexpression vector GsHSP and an overexpression soybean strain, and the GsHSP can be used for cultivating high-quality soybean strains.

For example, the gene can be used for constructing GsHSP overexpression recombinant soybean, experiments prove that the overexpression recombinant soybean of the gene has excellent milk salt capacity, the root growth vigor of the overexpression recombinant soybean is superior to that of a wild type under salt stress, the yield of the bean seedling vegetables is increased, the content of daidzin and daidzin bodies is remarkably higher than that of the wild type, and the overexpression recombinant soybean has higher nutritive value.

In a second aspect, the invention provides a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene.

The recombinant vector is a vector such as an existing cloning plasmid or an expression plasmid. Preferably, the recombinant bacterium is agrobacterium.

In a third aspect, the application also provides the application of the GsHSP gene, and a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene in improving the content of isoflavone (especially daidzin and daidzin) in the bean sprout vegetables.

Experiments prove that the contents of daidzin and daidzin in the GsHSP overexpression bean seedling vegetable are obviously improved, especially the contents of the daidzin and the daidzin in the bean seedling vegetable under salt stress can be further improved by the GsHSP overexpression, and the overexpression of the gene can be used for cultivating high-quality bean seedling vegetable. Experiments prove that the content of daidzin and daidzin in the bean sprout vegetables under salt stress can be improved by over-expression of GsHSP; and the content of genistin and daidzein of soybeans knocked out by the gene of GsHSP is obviously improved.

In a fourth aspect, the application provides an application of the GsHSP gene in improving the salt tolerance of soybean. Experiments prove that under the salt stress condition, the root growth vigor, the lateral root length and the number of the GsHSP overexpression group are higher than those of the WT group, which shows that the GsHSP can improve the salt tolerance of the soybean root system and promote the growth of the soybean root system under the salt and alkali condition.

In a fifth aspect, the application also provides an application of the GsHSP gene in improving the yield of the bean seedling vegetable under saline-alkali conditions. The overexpression of GsHSP is found to promote the root growth of the bean seedling vegetable under the condition of salt stress, so that the yield of the bean seedling vegetable under the condition of salt stress is improved.

In a sixth aspect, the present application also provides a method for cultivating a GsHSP recombinant soybean plant, which comprises the following steps:

(1) Designing a first primer pair to amplify the gene sequence of the GsHSP, adding enzyme cutting sites at two ends of the gene sequence of the GsHSP, carrying out enzyme cutting on an amplification product, and connecting the amplification product to a plant overexpression vector to obtain a GsHSP plant overexpression vector;

(2) Transforming the constructed GsHSP plant overexpression vector into agrobacterium rhizogenes K599, and infecting soybean root systems to obtain a GsHSP recombinant soybean plant (GsHSP overexpression strain).

Preferably, the expression vector adopts pSuper1300.

Preferably, the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO. 2; the sequence of the downstream primer of the first primer pair is shown as SEQ ID NO. 3.

The GsHSP recombinant soybean plant is a GsHSP overexpression recombinant soybean plant, has excellent properties and good salt tolerance, has high root growth vigor, side root length and quantity and wild type under the salt stress condition, has higher content of daidzin and daidzin bodies than the wild type, has better quality and has better application prospect.

In a seventh aspect, the present application further provides a method for breeding a soybean plant with a knockout GsHSP gene, which comprises the following steps:

(1) Performing mixed reaction on the designed GsHSP gene knockout target site sequence primer pair to form an oligo dimer, inserting the oligo dimer into a Cas9/gRNA vector, converting escherichia coli competent cells, picking positive single colonies, sequencing, and selecting a vector with correct sequencing to obtain a CRISPR/Cas9-GsHSP gene knockout vector;

(2) And (3) transforming the constructed CRISPR/Cas9-GsHSP gene knockout vector into agrobacterium rhizogenes K599, and infecting soybean root systems to obtain a GsHSP gene knockout soybean plant.

Preferably, the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO. 4; the sequence of the downstream primer of the second primer pair is shown as SEQ ID NO. 5.

Experiments prove that compared with wild soybeans, the GsHSP gene knockout soybean plant has the genistin content far higher than that of wild soybeans, and can be used as high-quality soybeans with high-content genistin or used as a high-quality resource for further cultivation of high-quality soybeans rich in genistin.

On the basis of the above, in an eighth aspect, the invention also provides the application of the above GsHSP recombinant soybean plant (overexpression plant) and GsHSP gene knockout soybean plant in high-quality soybean cultivation. The GsHSP recombinant soybean plant and the GsHSP gene knockout soybean plant can be used as high-quality germplasm resources to be directly used for cultivating high-quality soybeans or used as improved raw materials, such as high-quality soybeans which are rich in isoflavone and are salt-tolerant.

The invention has the following beneficial effects:

1. the invention provides a GsHSP gene and application thereof, and an applicant discovers for the first time that the GsHSP can improve the salt tolerance of soybean root systems by reducing lipid membrane peroxidation, improving in vivo osmotic pressure, enhancing antioxidant enzyme activity and activating expression of stress-related Marker genes, wherein the GsHSP overexpression can promote the root system growth of soybeans (bean sprouts) under a salt and alkali condition, and can further improve the contents of daidzin and daidzin in the bean sprouts under the salt stress; the content of genistin and daidzein of soybeans with GsHSP gene knockout is obviously improved, which shows that the GsHSP can be applied to culturing salt-tolerant recombinant soybean plants, improves the yield and quality of the bean seedling vegetables, and has good application value and market prospect.

2. The GsHSP gene can be applied to salt-tolerant molecular breeding, and provides gene resources and technical reserve for improving the yield and quality of salinized crops.

Drawings

FIG. 1 shows the result of GsHSP high fidelity PCR electrophoresis;

FIG. 2 is the predicted secondary and tertiary structure of the GsHSP protein;

FIG. 3 is a relative expression level analysis of GsHSP in soybean rhizomes, leaves and cotyledons;

FIG. 4 is a GsHSP expression profile under GsHSP salt stress;

the expression level of A GsHSP in roots stressed by 100mM NaCl for 0,1,3,6 and 12 h; b GsHSP is stressed by 100mM NaCl and treated in the leaves of 0,1,3,6 and 12 h;

FIG. 5 is an electrophoresis diagram of a pSuper1300-GsHSP vector identified by double enzyme digestion;

FIG. 6 is a phenotype of root growth and development of soybean of each line under normal treatment and 50mM NaCl for 7 days;

FIG. 7 shows the results of transgenic plant identification;

FIG. 8 shows the results of the measurement of the relative growth of soybean roots and the number of lateral roots in different strains under salt stress;

a, the relative growth amount of soybean roots of each strain under normal treatment and 50mM NaCl treatment; b, the number of lateral roots of each strain of soybean under normal treatment and 50mM NaCl treatment;

FIG. 9 shows MDA, proline content and SOD activity and POD activity in different strains under normal conditions and salt stress;

POD activity in soybean roots under normal treatment and 50mM NaCl treatment; b, SOD activity in the soybean roots under normal treatment and 50mM NaCl treatment;

FIG. 10 is the time of peak production of each metabolite of isoflavones; 1-daidzin; 2-daidzin; 3-genistin; 4-daidzein; 5-genistein;

FIG. 11 shows the measurement results of the daidzin content in the bean sprout vegetables;

FIG. 12 shows the measurement results of daidzin content in the bean sprout vegetables;

FIG. 13 is a graph showing the measurement of daidzein content in a bean sprout;

FIG. 14 is a CRISPR/Cas9-GsHSP vector map;

fig. 15 is a measurement of genistin content in the bean sprout vegetables.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example 1 acquisition of Soybean GsHSP protein and Gene encoding the same

1.1 Experimental materials

The salt-tolerant variety G07256' of wild soybean in northeast China is a gift offered by the plant bioengineering research laboratory of the northeast agricultural university.

1.2 reagents

pMD19-T vector and

HS DNA Polymerase high fidelity PCR enzyme was purchased from Bao bioengineering (Dalian) Co., ltd.

1/2MS solid medium: 39.45g/L, pH5.8, 116 ℃ sterilization for 30min.

LB culture medium: 25g/L LB broth (liquid), 15g/L agar (solid), sterilized at 121 ℃ for 20min.

Ampicillin storage solution: prepared into mother liquor with the concentration of 50mg/mL, filtered and sterilized, and stored at-20 ℃.

1.3 test methods

1.3.1 treatment of plant Material

Immersing the seeds of intact and disease-free wild soybean G07256' in concentrated H ₂ SO ₄ Neutralizing for 8-10 min to remove mud film and pouring out concentrated H ₂ SO ₄ Washing with sterile water for 3-4 times, inoculating to 1/2MS solid culture medium (pH5.8), dark culturing at 25 deg.C, germinating, and culturing under light. When the seedlings grow to 3 weeks, rapidly collecting the root, stem, leaf and cotyledon of wild soybean, and storing at-80 deg.C.

Collecting 3-week-old wild soybean seedlings, treating with 100mM NaCl for 0,1,3,6, and 12 hr, rapidly shearing tender leaves and roots, and storing at-80 deg.C.

1.3.2RNA extraction

Placing the mortar and various glassware used in the test operation in an oven at 180 ℃ for baking for 3h; the centrifugal tube, the pipette tip and the like are sterilization and enzyme deactivation products. The wild soybean RNA Extraction was performed by the TaKaRa MiniBEST Plant RNA Extraction Kit (Baozi physician's technology, beijing, ltd.).

1.3.3 reverse transcription of RNA into cDNA

(1) RNA extension

The reagents in the reaction system in Table 1 were mixed well, centrifuged for 5s, reacted at 70 ℃ for 5min, and immediately placed on ice for at least 5min.

TABLE 1 reaction System

(2) Reverse transcription reaction

The reagent in the reaction system shown in the table 2 is mixed uniformly, centrifuged for 5s, reacted at 25 ℃ for 5min, reacted at 42 ℃ for 1h, and stored at-20 ℃.

TABLE 2 reaction System

1.3.4 high fidelity amplification of full-length sequences of genes of interest

(1) The method comprises the following steps:

designing a homologous specific primer for amplifying the full-length sequence of a target gene by taking a soybean genome sequence as a reference, and using PrimeSTAR ^TM HS DNA Polymerase high fidelity enzyme, using wild soybean total cDNA as template and gene specific primer to proceed high fidelity PCR amplification.

The PCR reaction system is as follows: 10 μ L of 5 XPrimeSTAR ^TM HS PCR Buffer, 4. Mu.L dNTP mix, 2. Mu.L 5'PCR primer, 2. Mu.L 3' PCR primer, 1. Mu.L 5-fold diluted cDNA, 0.5. Mu.L PrimeStar ^TM HS DNA Polymerase，ddH ₂ O make up to 50. Mu.L.

The conditions of the PCR reaction were: 94 ℃ 5min → [94 ℃ 30s → 60 ℃ 30s → 72 ℃ 30s ] × 35 → 72 ℃ 10min → 4 ℃.

(2) The experimental results are as follows:

the electrophoresis results are shown in FIG. 1, from which it can be seen that the band of interest appeared at 500bp, whose size is consistent with the expected band of interest.

1.3.5 glue recovery of target genes

The concrete test operation steps refer to the instructions of the gelatin recovery kit (

SV gel recovery and PCR product purification systems were purchased from Promega, beijing Biotechnology Ltd.). And storing the target gene recovered from the glue in a refrigerator at the temperature of 20 ℃ below zero for later use. And an a tail was added after the sequence.

1.3.6 ligation of pMD19-T vector

The reaction was mixed as in Table 3 and ligated overnight at 16 ℃.

TABLE 3 reaction System

1.3.7CaCl ₂ Method for preparing competent cells of Escherichia coli

The specific preparation method of competent Escherichia coli cells is described in the third edition of molecular cloning, A laboratory Manual.

1.3.8 transformation of Large intestine competence

After colony PCR, positive clones were identified, plasmids of the positive clones were extracted and sent to sequencing.

1.3.9 processing and analysis of sequencing results

According to the sequencing result, removing the sequencing low-quality region and the vector sequence, and analyzing the repeatability of a plurality of sequencing results through DNAMAN multiple sequence comparison, thereby determining the full-length gene sequence. The CDS region of the GsHSP gene is 504bp, 167 amino acids are coded, 3 conserved structural domains are provided, the conserved structural domains are an N-terminal conserved structural domain, a DnaJ _ zferficially structural domain and a C-terminal conserved structural domain respectively, and the sequence of the GsHSP gene is shown in SEQ ID NO.1 specifically.

1.3.10GsHSP protein Structure analysis

Predicting the secondary structure of the wild soybean GsHSP by using (https:// npsa-prabi. Ibcp.fr/cgi-bin/secpred _ sopma.pl); the tertiary structure of the wild soybean GsHSP was predicted by SWISS-MODEL (https:// swissmodel. Expasy. Org /).

Through prediction of the secondary structure of the GsHSP protein, the protein is mainly composed of an alpha-helix, a beta-sheet, an extended chain and a random coil, wherein the alpha-helix accounts for 21.56%, the beta-sheet accounts for 5.99%, the extended chain accounts for 25.75% and the random coil accounts for 46.71% (FIG. 2A), and the tertiary structure of the GsHSP protein is shown in FIG. 2B.

1.3.11GsHSP Gene expression profiling

In order to research the expression characteristics of GsHSP in different tissues of the wild soybean, the relative expression quantity of the gene in different tissues is detected by adopting real-time fluorescent quantitative PCR. Secondly, wild soybean seedlings are treated by 100mM NaCl solution, and the gene expression level of GsHSP is detected under different stress time points.

(1) Experimental conditions of real-time fluorescent quantitative PCR:

the PCR reaction system is as follows: mu.L of 2 × SYBR Premix ExTaq, 0.4. Mu.L of 5'PCR primer, 0.4. Mu.L of 3' PCR primer, 2. Mu.L of cDNA template (diluted 5 times), 0.2. Mu.L of ROX dye, 2. Mu.L of ddH ₂ O (total volume 10. Mu.L).

The conditions of the PCR reaction were: 95 ℃ 10min → [95 ℃ 15s → 60 ℃ 1min ]. Times.40 → 95 ℃ 1min → 55 ℃ 1min → 95 ℃ 30s.

RT-PCR data processing: by comparison of C _T Method (2) ^-ΔΔCT ) Three independent biological replicates were performed with GsGAPDH as the reference gene.

(2) Results of the experiment

The result shows that GsHSP is expressed in the roots, stems, leaves and cotyledons of the wild soybean, and the expression level of the GsHSP is the highest in the cotyledons, about 4 times of the expression level in the stems and the second highest in the leaves and roots. This is probably because cotyledons provide certain energy for soybean seed germination and seedling growth, and GsHSP participates in plant growth and development, so that the expression level is relatively high in cotyledons. Under salt stress, gsHSP is obviously up-regulated in roots and reaches a peak value at 6h, which is about 8 times of 0h, but the expression level of the GsHSP is opposite in leaves, is obviously reduced, is the lowest at 1h and then slowly returns to a normal level. (see FIGS. 3 and 4, respectively)

Example 2 construction of GsHSP overexpressing Strain

2.1pSuper1300-GsHSP plant overexpression vector construction

(1) Designing a gene full-length specific amplification primer of a GsHSP with enzyme cutting sites (Xba I and Kpn I): the upstream primer P1 is 5.

Taking the plasmid carrying the GsHSP gene as a template, amplifying by using the primer, carrying out double enzyme digestion (Xba I and Kpn I) on an amplification product, connecting the enzyme digestion product with pSuper1300 subjected to the same enzyme digestion treatment, inserting the GsHSP gene into a plant over-expression vector pSuper1300, converting the connection product into DH5 alpha, and screening a positive bacterial colony to extract a plasmid pSuper1300-GsHSP;

then, the extracted plasmid is subjected to double enzyme digestion identification by using Xba I and Kpn I, and the electrophoresis result of the enzyme digestion product is shown in figure 5; as can be seen from FIG. 5, the restriction enzyme product has a target band of about 500bp, which indicates that the pSuper1300-GsHSP plant overexpression vector is successfully constructed.

(2) The constructed GsHSP plant overexpression vector is converted into agrobacterium rhizogenes K599, and colony PCR is carried out on a single colony which grows out, so that a band is shown to be about 500bp, and the target gene is transferred into the K599 and can be used for soybean root infection.

2.2 genetic transformation of Soybean root System

The constructed GsHSP overexpression vector pSuper1300-GsHSP, namely OX-GsHSP infects soybean root systems, and the specific operation method is as follows:

(1)H ₂ O ₂ ethanol sterilization method: 10mL of 30% (w/w) H was taken ₂ O ₂ 75mL 96% (v/v) ethanol, sterile water to 100mL.

(2) Germination of soybean seeds

Sterilizing vermiculite in advance, sterilizing a tray and a bowl, filling the bottom of the vermiculite with BD solution, and soaking. Inoculating sterilized semen glycines into 0.5cm deep vermiculite, culturing in dark condition, spreading film for about four days, cutting cotyledon and partial hypocotyl of healthy seed seedling, shearing at a distance of 1cm below cotyledon node, and pricking 1-2 holes with fine needle at a position of 1mm below cotyledon node for infection.

(3) Preparation of Agrobacterium liquid and infection

Activating bacteria once a day before the infection of cotyledonary node, taking the frozen tube agrobacterium containing the target gene, and performing the steps of 1:1000 was inoculated into YEB liquid medium containing 50mg/L kanamycin and 50mg/L streptomycin, and shake-cultured at 28 ℃ for 24 hours for primary activation. Then, according to the following steps of 1: the culture was performed for 2 to 3 hours with shaking at a ratio of 10, and secondary activation was performed (OD 600=0.6 to 0.8). Before infection, the activated agrobacterium liquid is subpackaged into 50mL centrifuge tubes, each centrifuge tube is 40mL and 4000rpm, the supernatant is poured out after 5min of centrifugation, and the supernatant is resuspended by using a liquid resuspension culture medium with the same volume. The prepared cotyledonary node explants were placed in the invasion dye solution, and about 40 cotyledonary node explants were placed per 30mL of the invasion dye solution for 45-60 min with occasional shaking (about once every 10 min) to make full contact between the cotyledonary node and the bacterial solution.

The BD solution was: solution A, 2M CaCl ₂ (ii) a Solution B, 1M KH ₂ PO ₄ (ii) a Solution C, 20mM Fe-citrate; liquid D, 0.5M MgSO ₄ ，0.5M K ₂ SO ₄ ，2mM MnSO ₄ ，4mM H ₃ BO ₄ ，1mM ZnSO ₄ ，4mM CuSO ₄ ，0.2mM CoSO ₄ ，0.2mM Na ₂ MoO ₄ . 500. Mu.L of solutions A, B, C and D were added to 1L of BD solution.

The liquid resuspension medium was: 1/10B5 inorganic + B5 organic +3% sucrose +20mM MES +0.25mg/L GA3+1.67mg/L BAP + 200. Mu.m/L AS +1mM DTT, pH5.4.

(4) Cultivation of infected soybean seedlings

The infected soybean cotyledon node is horizontally placed on a filter paper paved with wet vermiculite in a square basin, sealed and moisturized, and cultured in the dark for 2 days, and then cultured in a greenhouse (14 h light/10 h dark, 28 ℃/25 ℃) for 2 weeks. During the culture, the culture medium is cultured with a medium containing 0-2mM KNO ₃ The BD solution was watered to ensure high humidity until hairy roots grew.

(5) Domestication of seedlings

When the domestication is just started, the membrane is punctured a few times every day, so that the membrane is slowly contacted with the outside air, the vermiculite in the basin is ensured to be wet, the membrane is transferred into water for domestication after a few days, and roots growing from the incision and the needle prick are basically transgenic. The small-scale seedling exercising can be performed by using a glass cup, the hairy root part is placed in water, and the overground part is in the air.

And then identifying and obtaining the GsHSP soybean over-expression strains OX-GsHSP-1 and OX-GsHSP-4 by a fluorescent quantitative PCR technology (figure 6).

Example 3 construction of GsHSP knockout Strain

1. CRISPR/Cas9-GsHSP gene knockout expression vector

(1) Designing a GsHSP gene knockout targeting site sequence and a primer according to a soybean CRISPR/Cas9 website:

CRISPR/Cas9-GsHSP-F：5’-ATTCTGTGGCCATGCAGGTTGCG-3’；

CRISPR/Cas9-GsHSP-R：5’-AACCGCAACCTGCATGGCCACAG-3’。

(2) After the primers are designed according to the instructions of a plant Cas9/gRNA plasmid construction kit (Beijing has a good friend biological science and technology, inc.), an oligo dimer is formed according to the operation steps, the oligo dimer is inserted into a Cas9/gRNA vector (figure 14), then DH5 alpha is transformed, a positive single bacterium is picked out and sent for sequencing, the sequencing result is consistent with an expected sequence, and the CRISPR/Cas9-GsHSP gene knockout vector is successfully constructed.

2. Genetic transformation of soybean root system

The constructed gene knockout vector CRISPR/Cas9-GsHSP, namely Cas9-GsHSP is infected in soybean root systems, and the specific operation method refers to the corresponding part of example 2.

Example 3 analysis of salt tolerance of GsHSP Soybean overexpression and knockout strains

(1) The test method comprises the following steps:

after two obtained GsHSP soybean overexpression strains OX-GsHSP-1, OX-GsHSP-4 and GsHSP soybean gene knockout strains Cas9-GsHSP-2 and Cas9-GsHSP-3 and a wild type strain WT grow for 3 weeks under normal conditions, stress treatment is carried out for 1 week by using 50mM NaCl salt, and then determination of adversity physiological indexes such as MDA and SOD and statistics of adventitious root length and root number of soybean are carried out.

Wherein, the determination of adversity physiological indexes such as MDA, SOD and the like refers to the botanical experimental guidance and the history and welcome research compiled by Liuxin. The soybean is subjected to statistics on the length and the number of adventitious roots by using Image J software.

(2) GsHSP salt tolerant phenotype analysis

The experimental result shows that:

(1) under the normal growth condition, the growth states of WT and OX-GsHSP two groups of material roots are consistent and have no obvious change;

(2) after salt stress treatment, the growth and development of three groups of material roots are inhibited, but the soybean roots of the OX-GsHSP group with over-expressed genes have better growth potential than that of the WT group; the growth vigor of the soybean roots of the Cas9-GsHSP group is obviously weaker than that of the WT group (as shown in FIGS. 6 and 8);

the relative root length and the number of lateral roots measured and counted from tables 4 and 5 also demonstrate that the degree of salt stress injury of the root system of the GsHSP gene overexpression strain is obviously smaller than that of the wild-type plant. Specifically, under the salt stress condition, the root length of the gene over-expression OX-GsHSP-1 is prolonged by 49.8 percent and the number of lateral roots is increased by 40.5 percent compared with the wild type. The result shows that GsHSP is a salt stress positive regulation factor, can enhance the salt tolerance of soybeans and promote the growth of root systems of the soybeans.

TABLE 4 relative growth (mm)

TABLE 5 number of lateral roots

	Control	50mM NaCl
			WT	34	14
OX-GsHSP-1	34	20
			OX-GsHSP-4	33	20
Cas9-GsHSP-2	35	12
			Cas9-GsHSP-3	33	10

(3) GsHSP salt tolerance physiological index determination

The enzyme activities of the soybean root SOD and the podase of each strain were measured, and the results are shown in fig. 9, which shows that: the activities of antioxidase between WT and OX-GsHSP strains are in a normal state and have no great difference; under the condition of salt stress, the activity of antioxidant enzyme is obviously enhanced, and the SOD and POD enzyme activities in an OX-GsHSP strain are higher than those of a wild type and are respectively 1.25 times and 1.5 times of those of the wild type under the condition of salt stress. The result shows that the GsHSP gene can eliminate excessive active oxygen in plants by improving the activity of antioxidant enzyme under salt stress, thereby increasing the salt tolerance of the plants.

Example 4GsHSP increase the content of isoflavone components in Nostoc commune under salt stress

4.1 test materials

And culturing the GsHSP gene overexpression and wild soybean strains for one week under normal growth conditions and salt stress growth conditions to obtain soybean seedling vegetables of each group of strains. The overground part materials of each group are frozen by liquid nitrogen and then are frozen and stored at the temperature of minus 80 ℃. And then putting the mixture into a freeze drying instrument for freeze dehydration for about 48 hours, taking out the mixture, grinding the mixture, and sieving the ground mixture with a 80-mesh sieve for liquid chromatography sample extraction. The sample extraction method refers to national standard GB/T23788-2009 high performance liquid chromatography for determination method of soybean isoflavone in health food. Three biological replicates were performed for each set of materials.

4.2 test reagents

Chromatographic grade reagents such as methanol and dimethylsulfoxide were purchased from Solebao.

4.3 test methods

The method for measuring various metabolites of soybean isoflavone refers to the national standard GB/T23788-2009 high performance liquid chromatography for measuring method of soybean isoflavone in health food.

The peak time of each metabolite component of soybean isoflavone is shown in fig. 10, the peak time of daidzin is 13.104, the peak time of daidzin is 13.939, the peak time of genistin is 19.559, the peak time of daidzein is 33.395, and the peak time of genistein is 43.961.

TABLE 6 Soy isoflavone compositions and Standard working curves

4.4 results and analysis of the experiment

4.4.1 Soyabean glycoside content variation

The daidzin is used as important component of soybean isoflavone, and has pharmacological effects in preventing and treating cardiovascular diseases, resisting tumor, etc. The change of the content of the daidzein in the bean sprout vegetables of the WT, OX-GsHSP and Cas9-GsHSP three groups of materials under the normal growth condition and the salt stress growth condition is measured.

As shown in FIG. 11 and Table 7, the difference between the daidzein content of the two groups of bean sprout vegetable material under normal growth conditions was about 260 mg/kg; under the condition of salt stress growth, the content of daidzin in the materials of the two groups of bean sprouts is increased, the content of the daidzin in WT is about 427mg/kg, while the content of the daidzin in OX-GsHSP group is increased to the maximum, namely 519mg/kg, and is increased by 21.5 percent compared with the wild type. The result shows that salt stress can induce the synthesis of the daidzin in the bean sprout vegetables to a certain extent, and the content of the daidzin in the bean sprout vegetables under the salt stress can be further increased after the GsHSP gene is over-expressed.

TABLE 7 Glycine content (mg/kg)

	Control	50mM NaCl
			WT	271	427
OX-GsHSP	254	519
			Cas9-GsHSP	253	423

4.4.2 changes in daidzein content

Daidzin is a component of soybean isoflavone, and its metabolite, daidzein, plays an important role in anti-allergy and anti-thrombosis. The changes in daidzin content of each group under normal and salt stress growth conditions were determined.

As shown in fig. 12 and table 8, under normal growth conditions, the content of daidzin in the OX-GsHSP group was slightly higher than that of the WT group, and both groups had daidzin at 700 mg/kg more; under the condition of salt stress, the content of daidzin in the two groups of bean sprout vegetables is increased, the content of daidzin in the WT group is 971mg/kg, and the content of daidzin in the OX-GsHSP group is increased most obviously, reaches 1093mg/kg, and is increased by 12.6 percent compared with the WT group. This shows that the content of daidzin in bean sprout can be increased by the overexpression of the GsHSP gene, and the salt stress can further induce the metabolism and synthesis of the daidzin by the GsHSP.

TABLE 8 daidzin content (mg/kg)

	Control	50mM NaCl
			WT	736	971
OX-GsHSP	798	1093
			Cas9-GsHSP	682	979

4.4.3 Genistein content variations

Genistin as one of the soybean isoflavone effective components has important effects in resisting tumor, improving metabolism and female climacteric syndrome.

The content of genistin in each group of materials was determined by high performance liquid chromatography, and as a result, as shown in fig. 15, in the OX-GsHSP group, the content of genistin in the group was not much different from that in the WT group (i.e., the K599 group in the figure) under both normal and salt stress growth conditions; the content of genistin in the Cas9-GsHSP group is far higher than that of the WT group under two growth conditions, and the content of the genistin is improved by not less than 60 percent compared with that of the WT group. The above results indicate that GsHSP may be a negative regulator of genistin anabolism. The GsHSP gene-knocked-out soybean can be applied as high-quality soybean with high genistin content or used as high-quality resource for further cultivation of high-quality soybean rich in genistin.

TABLE 9 genistin content (mg/kg)

	Control	50mM NaCl
			K599 (i.e., WT)	502	547
OX-GsHSP	570	565
			Cas9-GsHSP	814	877

4.4.4 changes in Soyaflavin content

The change of the content of daidzein in each group and under different growth conditions is shown in FIG. 13, salt stress treatment increased the content of daidzein in the seedlings of WT and OX-GsHSP groups, which were increased to a lesser extent than in the wild type; the content of daidzein in the Cas9-GsHSP group (GsHSP gene knockout strain) under normal growth conditions is obviously higher than that in the wild type group (K599), and the content of daidzein in the group is reduced under salt stress conditions.

Therefore, the GsHSP can inhibit the synthesis of the daidzein under the condition of salt stress to a certain extent and is a negative regulation factor for the salt stress to induce the synthesis of the daidzein. When the content of daidzein in the bean sprout vegetables needs to be increased, a gene knockout strain of GsHSP can be constructed, so that the negative regulation of GsHSP on daidzein is relieved, the content of daidzein in the bean sprout vegetables is increased, and the gene knockout bean sprout vegetables of GsHSP are suitable for old people suffering from cardiovascular and cerebrovascular diseases and osteoporosis.

TABLE 9 Soyaflavin content (mg/kg)

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Claims (5)

1. GsHSP gene is characterized in that the sequence is shown in SEQ ID NO.1.
2. 2. A recombinant vector, transgenic cell or recombinant bacterium containing the GsHSP gene according to claim 1.
3. 3. The GsHSP gene of claim 1 and application of a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene in increasing the isoflavone content of the bean sprout vegetables.
4. 4. The GsHSP gene of claim 1 and the application of a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene in improving the salt tolerance of soybeans.
5. 5. The application of the GsHSP gene of claim 1 and a recombinant vector, a transgenic cell or a recombinant bacterium containing the GsHSP gene in improving the yield of the bean sprout vegetables under the saline-alkali condition.

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