CN115927393B - GsHSP gene and application thereof in improving salt tolerance of soybeans and increasing isoflavone content in bean seedling vegetables - Google Patents

Abstract

The invention discloses a GsHSP gene and application thereof in improving salt tolerance of soybeans and increasing isoflavone content in bean seedling vegetables, wherein the sequence of the GsHSP gene is shown as SEQ ID NO.1, and the GsHSP gene-containing recombinant vector, transgenic cells or recombinant bacteria and application thereof are also disclosed.

Description

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

Background

As an important agricultural crop in China, the soybean is rich in high-quality vegetable proteins, unsaturated fatty acids, calcium, B vitamins, isoflavone and other substances, and plays an important role in national nutrition. In recent years, with the improvement of the living standard of people and the improvement of the material requirements, the soybean yield in China cannot meet the increasing market demands of people. Therefore, improving the yield and quality of soybeans in our country is a serious civil problem that needs to be solved urgently at present.

Adverse factors such as salt stress are main factors influencing the yield and quality of soybeans, and cause great loss to soybean production, so that the improvement of soybean quality and stress resistance is a key for solving the problem. The traditional breeding method is adopted to improve the bad characters of the soybean, and is limited by the lack of soybean gene resources; resistance and environmental protection problems of plants caused by messy and excessive use of chemical agents also limit the application of chemical agents.

Soybean is an important crop and contains rich nutrients. The soybeans contain enough high-quality proteins, unsaturated fatty acids, calcium, isoflavone, various vitamins and the like, occupy important positions in the diet of residents in China and are an important source of high-quality plant proteins.

Soy isoflavones are a pale yellow powder material that has a slightly bitter taste and is associated with a slight astringent taste. Isoflavone is a compound that is structurally and biologically similar to human estrogen. After the soybean and its product enter human body, the soybean isoflavone contained in the soybean is metabolized and absorbed mainly in intestinal tract, and then converted into equol and other matters, which enter the blood circulation system of human body through capillary vessel. Soy isoflavones are recognized as natural active substances phytoestrogens, a widely accepted natural substitute for HRT (hormone replacement therapy) or HRT supplement.

The soybean isoflavone is used as a physiologically active substance, plays an important physiological function in the vital activity of human body, and plays an important role in the aspects of reducing female aging, improving climacteric symptoms, protecting cardiovascular, resisting tumor, preventing osteoporosis, protecting nervous system, reducing blood sugar, protecting liver, regulating immune system and the like. Isoflavone has been accepted by people more and more because of various nutrition and health care physiological functions. And the soyabean glycoside is used as an important component of the soyabean isoflavone and has pharmacological effects of preventing and treating cardiovascular diseases, resisting tumors and the like. While soybean glycoside is another important constituent of soybean isoflavone, and plays an important role in antiallergic and antithrombotic effects.

Improving the salt tolerance of soybeans is beneficial to expanding the planting area of the soybeans and improving the yield of soybean related products, and how to improve the content of isoflavone (such as daidzein and daidzein) in soybean foods such as bean sprouts and the like, and improving the nutrition and health-care quality of the soybean foods, which are the major concerns of researchers at present. Accordingly, there is a need for further improvements in the art.

Disclosure of Invention

Aiming at the problems, the invention provides a GsHSP gene and application thereof in improving the salt tolerance of soybeans and the content of soybean glycosides in bean seedling vegetables, and the GsHSP gene is found for the first time that the GsHSP gene can improve the salt tolerance of soybean root systems, over-expression of the GsHSP gene can promote the root growth of soybeans (bean seedling vegetables), and over-expression of the GsHSP gene can further improve the content of soybean glycosides and soybean glycosides in bean seedling vegetables under salt stress, and can be applied to cultivation of GsHSP recombinant soybean plants with salt tolerance, and the planting area, yield and quality of the GsHSP recombinant soybean plants are improved.

In order to solve the problems, the technical scheme provided by the application is as follows:

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

The applicant excavates a salt stress response gene GsHSP from a very salt-tolerant wild soybean variety for the first time, clones and bioinformatics analysis is carried out on the gene, tissue expression characteristics and salt stress expression modes of the gene are analyzed through real-time fluorescence quantitative PCR, and the GsHSP can improve the salt tolerance in soybean roots and improve the isoflavone metabolite content of soybean seedlings under salt stress by constructing a gene overexpression vector GsHSP and an overexpression soybean strain, so that the gene GsHSP can be used for cultivating high-quality soybean strains.

For example, gsHSP over-expression recombinant soybean can be constructed by adopting the gene, and experiments prove that the over-expression recombinant soybean of the gene has excellent milk salt capability, the root growth vigor under salt stress is better than that of a wild type, the yield increase of bean seedling vegetables is promoted, and the content of soyabean glycoside and soyabean glycoside in the body is obviously higher than that of the wild type, so that the soybean seedling soybean has higher nutritional value.

In a second aspect, the present invention provides a recombinant vector, a transgenic cell or a recombinant bacterium comprising the above-described GsHSP gene.

The recombinant vector is the existing cloning plasmid or expression plasmid and other vectors. Preferably, the recombinant bacterium is agrobacterium.

In a third aspect, the present application further provides an application of the above GsHSP gene, and a recombinant vector, transgenic cell or recombinant strain containing the GsHSP gene in increasing the isoflavone (especially soyabean glycoside and soyabean glycoside) content.

Experiments prove that the content of the soybean glycoside and the soybean xanthosine in the GsHSP over-expressed bean seedling vegetable is obviously improved, and especially the content of the soybean glycoside and the soybean xanthosine in the bean seedling vegetable under salt stress can be further improved by the GsHSP over-expression, and the gene can be utilized for over-expressing and cultivating high-quality bean seedling vegetable. Experiments prove that the overexpression of GsHSP can improve the content of soyabean glycosides and soybean xanthosine in the bean seedling under salt stress; and the content of genistin and glycitein in the soybean subjected to gene knockout of GsHSP is obviously improved.

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

In a fifth aspect, the application further provides an application of the GsHSP gene in improving the yield of bean seedling vegetables under a saline-alkali condition. As the over-expression of the GsHSP is found, the root growth of the bean seedling vegetable under the condition of salt stress is promoted, and the yield of the bean seedling vegetable under the condition of salt stress is further improved.

In a sixth aspect, the present application further provides a cultivation method of a GsHSP recombinant soybean plant, comprising the steps of:

(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, and connecting an amplified product of the enzyme cutting sites to a plant overexpression vector after enzyme cutting to obtain the GsHSP plant overexpression vector;

(2) Transforming agrobacterium rhizogenes K599 by using the constructed GsHSP plant overexpression vector, and carrying out soybean root system infection to obtain a GsHSP recombinant soybean plant (GsHSP overexpression strain).

Preferably, the expression vector is pSuper1300.

Preferably, the sequence of the upstream primer of the first primer pair is shown in 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 over-expression recombinant soybean plant, has excellent properties, has good salt tolerance, has high root growth vigor, side root length and quantity and wild type under the condition of salt stress, has much higher content of soyabean glycoside and soyabean glycoside than the wild type in vivo, has better quality and has better application prospect.

In a seventh aspect, the present application further provides a cultivation method of a GsHSP gene knockout soybean plant, comprising the steps of:

(1) Mixing and reacting the designed GsHSP gene knockout target site sequence primer pair to form an oligo dimer, inserting the oligo dimer into a Cas9/gRNA vector, transforming competent cells of escherichia coli, selecting positive single colonies for sequencing, and selecting a vector with correct sequencing to obtain a CRISPR/Cas9-GsHSP gene knockout vector;

(2) And transforming agrobacterium rhizogenes K599 by using the constructed CRISPR/Cas9-GsHSP gene knockout vector, and carrying out soybean root system infection to obtain a GsHSP gene knockout soybean plant.

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

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

On the basis of the above, the invention also provides the GsHSP recombinant soybean plant (over-expression plant) and the application of the 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 directly or as improved raw materials for cultivating high-quality soybeans, such as high-quality soybeans rich in isoflavone and salt-tolerant.

The invention has the following beneficial effects:

1. the invention provides a GsHSP gene and application thereof, and the applicant discovers for the first time that GsHSP can improve the salt tolerance of soybean root systems by reducing lipid film peroxidation, improving in vivo osmotic pressure, enhancing antioxidase activity and activating expression of stress-related Marker genes, so that the over-expression of GsHSP can promote the root system growth of soybean (bean seedling vegetable) under the saline-alkali condition, and can further improve the content of soybean glycoside and soybean xanthosine in bean seedling vegetable under the salt stress; the content of genistin and glycitein in the soybean subjected to gene knockout of the GsHSP is obviously improved, which shows that the GsHSP can be applied to cultivation of recombinant soybean plants with salt tolerance, the yield and quality of bean seedling vegetables are improved, and the GsHSP 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 reserves for improving the yield and quality of saline land crops.

Drawings

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

FIG. 2 shows predicted secondary and tertiary structures of GsHSP proteins;

FIG. 3 shows analysis of relative expression levels of GsHSP in soybean rootstocks, soybean cotyledons and soybean cotyledons;

FIG. 4 shows analysis of GsHSP expression characteristics under GsHSP salt stress;

expression level of a GsHSP in root of 0,1,3,6, 12h of 100mM NaCl stress treatment; expression level of B GsHSP in leaves subjected to 100mM NaCl stress treatment 0,1,3,6 for 12 hours;

FIG. 5 is an electrophoretogram of a double enzyme digestion identification pSuper1300-GsHSP vector;

FIG. 6 is a graph showing the phenotype of soybean root growth for each strain at 7d for normal treatment and 50mM NaCl treatment;

FIG. 7 is a graph showing the identification of transgenic plants;

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

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

FIG. 10 shows peak time of isoflavone metabolites; 1-daidzin; 2-soybean glycoside; 3-genistin; 4-daidzein; 5-genistein;

FIG. 11 shows the result of measuring the content of daidzin in the bean sprouts;

FIG. 12 shows the measurement result of the soybean glycoside content in the bean seedling vegetable;

FIG. 13 is a measurement of soybean meal content in a bean seedling vegetable;

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

FIG. 15 shows the measurement of genistin content in the bean sprouts.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example 1 acquisition of Soybean GsHSP protein and its coding Gene

1.1 Experimental materials

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

1.2 reagents

pMD19-T vectors

HS DNA Polymerase high-fidelity PCR enzyme was purchased from Takara Bio Inc.

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

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

Ampicillin stock: prepared into 50mg/mL mother liquor, and stored at-20 ℃ with the concentration of 50mg/L by filtration sterilization.

1.3 test methods

1.3.1 plant Material treatment

Immersing intact, disease-free, extremely salt-tolerant wild soybean 'G07256' seeds in concentrated H ₂ SO ₄ The mud film is removed by the medium treatment for 8 to 10 minutes, and the concentrated H is poured out ₂ SO ₄ Washing with sterile water for 3-4 times, inoculating on 1/2MS solid culture medium (pH 5.8), culturing at 25deg.C in dark, germinating, and culturing under light. Rapidly taking root, stem, leaf and cotyledon of wild soybean when seedling grows to 3 weeks old, and storing at-80deg.C.

Young leaves and roots of 3-week-old wild soybean seedlings were rapidly cut after 0,1,3,6 and 12 hours of treatment with 100mM NaCl and stored at-80 ℃.

1.3.2RNA extraction

The mortar and various glassware used in the test operation are all placed in a 180 ℃ oven for baking for 3 hours; the centrifuge tube, the pipette tip and the like are sterilization and enzyme deactivation products. Wild soybean RNA extraction was performed according to the method of TaKaRa MiniBEST Plant RNA Extraction Kit (Bao Ri doctor materials technology (Beijing) Co., ltd.) kit.

1.3.3RNA reverse transcription into cDNA

(1) RNA extension

After the reagents in the reaction system of Table 1 are uniformly mixed, the mixture is centrifuged for 5 seconds, reacted for 5 minutes at 70 ℃, and immediately placed on ice for at least 5 minutes.

TABLE 1 reaction system

(2) Reverse transcription reaction

The reagents of the reaction system in Table 2 were mixed uniformly, centrifuged for 5 seconds, reacted at 25℃for 5 minutes, reacted at 42℃for 1 hour, 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 homologous specific primer for amplifying full-length sequence of target gene by taking soybean genome sequence as reference, and using PrimeSTAR ^TM HS DNA Polymerase high-fidelity enzyme, the wild soybean total cDNA is used as a template and a gene specific primer for high-fidelity PCR amplification.

The PCR reaction system is as follows: 10 μL 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 LPrimeSTAR ^TM HS DNA Polymerase，ddH ₂ O was made up to 50. Mu.L.

The conditions for the PCR reaction were: 94℃for 5 min- [94℃for 30 s- & gt 60℃for 30 s- & gt 72℃for 30s ]. Times.35- & gt 72℃for 10 min- & gt 4 ℃.

(2) Experimental results:

as a result of electrophoresis, as can be seen from FIG. 1, a band of interest appears at 500bp, which corresponds in size to the expected band of interest.

1.3.5 recovery of the Gene of interest by gel

Specific test operation steps refer to the specification of the glue recovery kit

SV gel recovery and PCR product purification systems were purchased from Promega (Beijing) Biotechnology Co., ltd.). And (5) placing the target gene recovered by the gel in a refrigerator at the temperature of-20 ℃ for standby. And adding a tail after the sequence.

1.3.6 ligation of pMD19-T vector

After mixing the reaction systems according to Table 3, the mixture was allowed to stand at 16℃overnight.

TABLE 3 reaction system

1.3.7CaCl ₂ Preparation of competent cells of E.coli by methods

The specific preparation method of competent cells of Escherichia coli refers to the third edition of the molecular cloning experiment guide.

1.3.8 transformation of large intestine competence

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

1.3.9 processing and analysis of sequencing results

Based on the sequencing results, the sequencing low quality region and the vector sequence are removed, and the repeatability of a plurality of sequencing results is analyzed through DNAMAN multi-sequence alignment, so that the full-length gene sequence is determined. The CDS region of the GsHSP gene is 504bp, encodes 167 amino acids, has 3 conserved domains, namely an N-terminal conserved domain, a DnaJ_zf superfamity domain and a C-terminal conserved domain, and has the sequence shown in SEQ ID NO.1.

1.3.10 analysis of the Structure of GsHSP protein

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 using SWISS-MODEL (https:// swissmodel. Expasy. Org /).

The secondary structure of the GsHSP protein is predicted to find that the protein mainly consists of an alpha-helix, a beta-sheet, an extension chain and random coil, wherein the alpha-helix accounts for 21.56%, the beta-sheet accounts for 5.99%, the extension chain accounts for 25.75% and the random coil accounts for 46.71% (figure 2A), and the tertiary structure of the GsHSP protein is shown in figure 2B.

1.3.11GsHSP Gene expression profiling

In order to study the expression characteristics of GsHSP in different tissues of wild soybean, the relative expression amount of the gene in different tissues is detected by adopting real-time fluorescence quantitative PCR. Next, wild soybean seedlings were treated with 100mM NaCl solution, and the gene expression levels of GsHSP at different time points of stress were examined.

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

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

The conditions for the PCR reaction were: 95℃for 10 min- & gt [95℃for 15 s- & gt 60℃for 1min ]. Times.40- & gt 95℃for 1 min- & gt 55℃for 1 min- & gt 95℃for 30s.

RT-PCR data processing: using comparison C _T Method (2) ^-ΔΔCT ) Three independent biological replicates were performed using GsGAPDH as an internal reference gene.

(2) Experimental results

The results showed that GsHSP was expressed in the roots, stems, leaves and cotyledons of wild soybean, and the expression level in the cotyledons was highest, about 4 times the expression level in the stems, and the expression level in the leaves and roots was second. This is probably because cotyledons provide a certain energy for soybean seed germination and seedling growth, while GsHSP participates in plant growth and development, so that its expression level is relatively high in cotyledons. Under salt stress, gsHSP obviously up-regulates expression in roots, and the expression quantity reaches a peak value at 6 hours and is about 8 times of 0 hours, but the opposite condition occurs in leaves, the expression quantity obviously decreases, the expression quantity at 1 hour is the lowest, and then the normal level is slowly restored. (see FIGS. 3 and 4, respectively)

EXAMPLE 2 construction of GsHSP overexpressing Strain

2.1 construction of pSuper1300-GsHSP plant overexpression vector

(1) Designing a gene full-length specific amplification primer of GsHSP with enzyme cutting sites (XbaI and KpnI): the upstream primer P1 was 5'-GGATCCTCATGGATATGGATTCAGTTTC-3' and the downstream primer P2 was 5'-TCTAGAGTCATTTTGGTCCCAGATG-3'.

Amplifying the plasmid carrying the GsHSP gene serving as a template by using the primer, carrying out double digestion (XbaI and KpnI) on an amplified product, connecting the digested product with pSuper1300 subjected to the same digestion treatment, inserting the GsHSP gene into a plant overexpression vector pSuper1300, converting the connection product into DH5 alpha, and screening positive bacterial colony to extract plasmids pSuper1300-GsHSP;

then, double digestion identification is carried out on the extracted plasmid by using XbaI and KpnI, and the electrophoresis result of the digestion products is shown in FIG. 5; as shown in FIG. 5, the target band of about 500bp exists in the enzyme digestion product, which indicates that the construction of the pSuper1300-GsHSP plant overexpression vector is successful.

(2) The constructed GsHSP plant over-expression vector is transformed into agrobacterium rhizogenes K599, colony PCR is carried out on single colonies which grow out, and the result shows that the single colonies have bands at about 500bp, which indicates that the target gene is transferred into K599 and can be used for soybean root system infection.

2.2 genetic transformation of soybean root lines

The constructed GsHSP overexpression vector pSuper1300-GsHSP, namely OX-GsHSP is used for infecting soybean root lines, and the specific operation method is as follows:

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

(2) Germination of soybean seeds

Sterilizing vermiculite in advance, sterilizing the tray and the bowl, filling the bottom of the vermiculite with BD solution, and soaking. The sterilized soybean seeds are planted into vermiculite with the depth of 0.5cm for about four days (dark culture and film laying), the cotyledons and part of hypocotyls of the healthy seed seedlings are cut off, the cotyledons are cut off at a position 1cm away from cotyledon nodes, and 1-2 holes are pricked at a position 1mm away from the cotyledon nodes by a fine needle head for standby infection.

(3) Preparation of Agrobacterium solution and infection

Activating bacteria once in the morning before cotyledonary node infection, taking frozen agrobacterium tumefaciens containing target genes according to the following weight ratio of 1:1000 was inoculated on YEB liquid medium containing 50mg/L kanamycin and 50mg/L streptomycin, and cultured at 28℃for 24 hours with shaking, and activated once. Then according to 1:10 for 2-3h, and performing secondary activation (OD 600 = 0.6-0.8). Before infection, the activated agrobacterium liquid is split into 50mL centrifuge tubes, each tube is about 40mL,4000rpm, after centrifugation for 5min, the supernatant is poured out and resuspended in an equal volume of liquid re-suspension medium. The prepared cotyledonary node explants were placed in an intrusion dye solution, and about 40 cotyledonary node explants were placed per 30mL of the intrusion dye solution, and the infection was carried out for 45 to 60 minutes with occasional shaking (about 10 minutes) to bring the cotyledonary node and the bacterial solution into sufficient contact.

The BD solution was: liquid A, 2M CaCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the Liquid B, 1M KH ₂ PO ₄ The method comprises the steps of carrying out a first treatment on the surface of the Solution C, 20mM Fe-citrate; solution 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 . Each 1L BD solution was added with 500. Mu.L of solutions A, B, C, and D.

The liquid re-suspension medium was: 1/10B5 mineral+B5 organic+3% sucrose+20 mM MES+0.25mg/L GA3+1.67mg/L BAP+200 μm/L AS+1mM DTT, pH5.4.

(4) Cultivation of infected soybean seedlings

Placing the infected soybean She Jieping on filter paper laid with wet vermiculite in square basin, sealing, keeping moisture, and culturing in dark for 2 daysThen cultured in a greenhouse (14 h light/10 h dark, 28 ℃ C./25 ℃ C.) for 2 weeks. During the culturing process, KNO with concentration of 0-2mM is used ₃ And (3) watering BD solution to ensure high humidity until hair roots grow out.

(5) Domestication of seedlings

When domestication is started, the membrane is poked, a plurality of holes are poked every other day, the membrane is gradually contacted with the outside air, the wetting of the vermiculite in the basin is ensured, the vermiculite is transferred into water for domestication after a plurality of days, and the roots growing from the incision and the needle-punched part are basically transgenic. The small-scale seedling hardening can be carried out by selecting a glass cup, placing the root part of the hair into water, and the overground part of the hair is in air.

Subsequently, the GsHSP soybean overexpression lines OX-GsHSP-1 and OX-GsHSP-4 were obtained by the identification of fluorescent quantitative PCR technology (FIG. 6).

EXAMPLE 3 construction of GsHSP knockout Strain

1. CRISPR/Cas9-GsHSP gene knockout expression vector

(1) Designing a GsHSP gene knockout target 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 were designed according to the instructions of the plant Cas9/gRNA plasmid construction kit (beijing fei Shang Lide biotechnology limited), an oligo dimer was formed according to the procedure, the oligo dimer was inserted into Cas9/gRNA vector (fig. 14), then dh5α was transformed, positive single colonies were picked up for sequencing, and the sequencing result was consistent with the expected sequence, indicating that CRISPR/Cas9-GsHSP gene knockout vector construction was successful.

2. Genetic transformation of soybean root lines

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

EXAMPLE 3 salt tolerance analysis of GsHSP soybean overexpression and knock-out strains

(1) The testing method comprises the following steps:

the obtained GsHSP soybean overexpression lines OX-GsHSP-1, OX-GsHSP-4 and GsHSP soybean gene knockout lines Cas9-GsHSP-2, cas9-GsHSP-3 and wild strain WT are respectively grown for 3 weeks under normal conditions, and after treatment for 1 week by 50mM NaCl salt stress, adverse physiological indexes such as MDA, SOD and the like are measured and the root length and the root number of soybean adventitious roots are counted.

Wherein, the measurement of stress physiological indexes such as MDA, SOD and the like refers to the study of the history welfare of the main editing of the botanic experimental guidance Liu Xin. Statistics on soybean adventitious root length and root number Image J software was used for statistics.

(2) GsHSP salt tolerance phenotype analysis

The experimental results show that:

(1) under normal growth conditions, the growth states of the WT and OX-GsHSP material roots are consistent, and no obvious change exists;

(2) after salt stress treatment, the growth and development of three groups of material roots are inhibited, but the growth vigor of the soybean roots of the gene overexpression OX-GsHSP group is better than that of the soybean roots of the WT group; whereas the soybean roots from the gene knockout Cas9-GsHSP group are significantly weaker in growth than WT groups (see fig. 6 and 8);

the determination and statistics of the relative root length and lateral root number from tables 4 and 5 also demonstrate that the root system of the GsHSP gene over-expressed strain is significantly less vulnerable to salt stress than the wild type plant. Specifically, under salt stress conditions, the root length of the gene over-expressed OX-GsHSP-1 is prolonged by 49.8% and the lateral root number is increased by 40.5% relative to the wild type. The result shows that GsHSP is a positive regulation factor for salt stress, and can strengthen the salt tolerance of soybeans and promote the growth of root systems of the soybeans.

TABLE 4 relative growth (mm)

TABLE 5 lateral root number

	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 results of measuring the enzyme activities of the soybean root SOD and POD enzymes of each strain are shown in fig. 9, and it is clear that: the activities of the antioxidant enzymes between the WT and the OX-GsHSP strains are in a normal state, and the difference is not great; under the condition of salt stress, the activity of antioxidant enzyme is obviously enhanced, and the activity of SOD and POD enzyme in the OX-GsHSP strain is higher than that of wild type, which are respectively 1.25 times and 1.5 times that of the wild type under the condition of salt stress. The result shows that the GsHSP gene can remove excessive active oxygen in plants by improving the antioxidant enzyme activity under salt stress, so that the salt tolerance of the plants is improved.

Example 4GsHSP increases the content of Isoflavonoids in Isodon japonicus under salt stress

4.1 test materials

And culturing the GsHSP gene overexpression and wild soybean lines for one week under normal growth conditions and salt stress growth conditions to obtain soybean seedling dishes of each group of lines. The above-ground materials are frozen in liquid nitrogen and then frozen and stored at-80 ℃. And then putting the mixture into a freeze dryer for freezing and dehydrating for about 48 hours, taking out the mixture, crushing and grinding the mixture, and sieving the mixture with a 80-mesh sieve for extracting liquid chromatographic samples. 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 dimethyl sulfoxide are available from Soy Corp.

4.3 test methods

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

The peak time of each metabolite component of soybean isoflavone is shown in figure 10, the peak time of soybean glycoside is 13.104, the peak time of soybean glycoside is 13.939, the peak time of genistin is 19.559, the peak time of soybean Huang Suchu is 33.395, and the peak time of genistein is 43.961.

TABLE 6 soy isoflavone formulations and standard working curves

4.4 experimental results and analysis

4.4.1 changes in Soy glycoside content

The soybean glycoside is used as an important component of soybean isoflavone and has pharmacological effects of preventing and treating cardiovascular diseases, resisting tumor, etc. And determining the change of the soybean glycoside content in the soybean seedling vegetable of the three groups of materials of WT, OX-GsHSP and Cas9-GsHSP under normal growth conditions and salt stress growth conditions.

The results are shown in FIG. 11 and Table 7, and under normal growth conditions, the soybean glycoside contents in the two groups of bean seedling vegetable materials are not greatly different and are about 260 mg/kg; under the condition of salt stress growth, the soybean glycoside content in the two groups of bean seedling vegetable materials is increased, the soybean glycoside content in the WT is about 427mg/kg, and the soybean glycoside content in the OX-GsHSP group is increased to the maximum, namely 519mg/kg, which is improved by 21.5 percent compared with the wild type. The result shows that the salt stress can induce the synthesis of the soyabean glycosides in the bean seedling vegetable to a certain extent, and the content of the soyabean glycosides in the bean seedling vegetable under the salt stress can be further increased after the GsHSP gene is over-expressed.

TABLE 7 Soy glycoside content (mg/kg)

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

4.4.2 changes in Soy daidzin content

The soybean flavonoid is used as a component of soybean isoflavone, and the metabolite of the soybean flavonoid plays an important role in antiallergic and antithrombotic effects. The variation of the soybean glycoside content of each group under normal and salt stress growth conditions was measured.

As shown in FIG. 12 and Table 8, under normal growth conditions, the content of soybean glycoside in the OX-GsHSP group was slightly higher than that in the WT group, and the content of soybean glycoside in both groups was 700 mg/kg; under the condition of salt stress, the soybean glycoside content in the two groups of bean seedling vegetables is increased, the soybean glycoside content in the WT group is 971mg/kg, the increase of the soybean glycoside content in the OX-GsHSP group is most obvious, and the increase is 1093mg/kg, and is improved by 12.6 percent compared with the WT group. This shows that the overexpression of GsHSP gene can increase the soybean glycoside content of the bean seedling vegetable, and the salt stress can further induce the metabolic synthesis of GsHSP to soybean glycoside.

Table 8 Soy daidzin content (mg/kg)

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

4.4.3 variation of genistin content

Genistin is used as one of the active ingredients of soybean isoflavone, and has important effects in resisting tumor, improving metabolism, female climacteric syndrome, etc.

In this example, the content of genistin in each group of materials was measured by high performance liquid chromatography, and as shown in fig. 15, in the OX-GsHSP group, the content of genistin in the group was not very different from that in the WT group (i.e., K599 group in the figure) under either normal or salt stress growth conditions; and the content of genistin in the Cas9-GsHSP group is far higher than that in the WT group under two growth conditions, and the content of genistin is improved by not less than 60 percent relative to the WT group. The above results indicate that GsHSP may be a negative regulator of genistin anabolism. The gene knockout soybean of GsHSP can be applied as high-quality soybean with high genistin content or used as a 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 daidzein content

The change of the daidzein content under various groups and different growth conditions is shown in fig. 13, the salt stress treatment improves the daidzein content of the WT and OX-GsHSP groups, and the degree of improvement in the OX-GsHSP group is lower than that of the wild type; the content of the glycitein in the Cas9-GsHSP group (GsHSP gene knockout strain) is obviously higher than that in the wild type group (K599) under the normal growth condition, and the content of the glycitein in the group under the salt stress condition is reduced.

Therefore, gsHSP can inhibit the synthesis of glycitein under salt stress to a certain extent, and is a negative regulator for salt stress induced glycitein synthesis. When the content of the glycitein in the bean seedling vegetable is required to be improved, the negative regulation and control of the GsHSP on the glycitein can be relieved by constructing a gene knockout strain of the GsHSP, the content of the glycitein in the bean seedling vegetable is improved, and the gene knockout bean seedling vegetable of the GsHSP is suitable for the old people suffering from cardiovascular and cerebrovascular diseases and osteoporosis.

TABLE 9 daidzein content (mg/kg)

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims (4)

1.GsHSPThe application of the gene overexpression vector and the recombinant bacteria thereof in improving the content of the daidzin and the daidzin in the bean seedling under the stress of salt is characterized in thatGsHSPThe sequence of the gene is shown as SEQ ID NO.1.

2.GsHSPThe gene knockout carrier and the recombinant strain thereof are characterized in that the content of xyloside and daidzein in the bean seedling vegetable under the condition of improving salt stress is improvedGsHSPThe sequence of the gene is shown as SEQ ID NO.1.

3.GsHSPThe application of the gene over-expression vector and the recombinant bacteria thereof in improving the salt tolerance of soybean is characterized in thatGsHSPThe sequence of the gene is shown as SEQ ID NO.1.

4.GsHSPThe application of the gene overexpression vector and the recombinant strain thereof in improving the yield of the bean seedling vegetable under the saline-alkali condition is characterized in thatGsHSPThe sequence of the gene is shown as SEQ ID NO.1.

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