CN111254159B - Soybean GmST1 gene mutant plant and preparation method thereof - Google Patents

Abstract

A soybean GmST1 gene mutant plant and a preparation method thereof, belonging to the technical field of plant biology. The invention provides a preparation method of a soybean GmST1 gene mutant plant for determining the disease resistance function of a soybean mosaic virus-resistant related gene, which comprises the following steps: designing a gRNA single-target-point primer according to a soybean GmST1 gene; preparing a gRNA single-target primer dimer; inserting the primer dimer into a Cas9/gRNA vector to construct a soybean GmST1 gene knockout vector; and (4) transforming the GmST1 gene knockout vector constructed in the step 3) into soybeans, and screening to obtain soybean GmST1 gene mutant plants. The invention determines that GmST1 can participate in the resistance reaction of resisting the soybean mosaic virus disease, and provides a theoretical basis for the research of the functions of the genes related to the soybean mosaic virus resistance.

Description

A kind of soybean GmST1 gene mutant plant and its preparation method

technical field

The invention relates to the field of plant biotechnology, in particular to a soybean GmST1 gene mutant plant and a preparation method thereof.

Background technique

Soybean Mosaic Virus, caused by Soybean Mosaic Virus (SMV), is one of the most serious viral diseases affecting soybean yield and quality. In the main soybean producing areas in Northeast my country, N1 and N3 strains are the dominant strains, and the abnormal growth of soybean plants caused by them can cause a large area of soybean production reduction of 25%-60%, or even extinction in severe cases. Due to the limitations of traditional breeding methods, it is particularly important to use molecular methods to screen and identify effective disease-resistant genes for the selection of disease-resistant varieties, which improves the efficiency and accuracy of breeding disease-resistant varieties and speeds up the selection process. The process of breeding disease-resistant varieties. Due to the large soybean genome and the polyploidization and low transformation rate of soybean, the functional analysis of disease resistance genes has become more difficult.

Contents of the invention

In order to clarify the disease resistance function of soybean anti-mosaic virus-related genes, the present invention provides a soybean GmST1 gene mutant plant, the mutant plant contains a mutated soybean GmST1 gene, and the mutation refers to the GmST1 gene coding region Base substitution or deletion occurs in the 5'AGAAAGGGGAGGAAATAA3' or 5'CTTCCTAGGGAGAGAGGT 3' nucleotide sequence, so that the encoded amino acid sequence changes. The nucleotide sequence of the soybean GmST1 gene is shown in SEQ ID NO:1.

The preparation method of the above-mentioned soybean GmST1 gene mutant plant comprises the following steps:

1) Design gRNA single-target primers according to the soybean GmST1 gene;

2) Preparation of gRNA single-target primer-dimer;

3) inserting the primer dimer into the Cas9/gRNA vector to construct a soybean GmST1 gene knockout vector;

4) Transforming soybean with the GmST1 gene knockout vector constructed in step 3), and obtaining soybean GmST1 gene mutant plants through screening.

Further defined, the forward primer of the soybean GmST1 gRNA single target primer in step 1) is GmST1-1-S, its nucleotide sequence is shown in SEQ ID NO: 2, and the reverse primer is GmST1-1-A, Its nucleotide sequence is shown in SEQ ID NO: 3; or the forward primer is GmST1-2-S, its nucleotide sequence is shown in SEQ ID NO: 4, and the reverse primer is GmST1-2-A, its nuclear The nucleotide sequence is shown in SEQ ID NO:5.

Further defined, the preparation of gRNA single-target primer-dimer in step 2) refers to 5 μL of forward primer, 5 μL of forward primer and 15 μL of H ₂ O in each 20 uL synthesis system, the reaction conditions are 95°C for 3 minutes, cooled to 25°C After ℃, stand at 16 ℃ for 5min.

To further define, the construction of the GmST1 gene knockout vector in step 3) means that in each 10uL reaction system, 1-7 μL of primer dimer, 1 μL of Cas9/gRNA vector, 1 μL of Solution1, 1 μL of Solution2, H ₂ O 0- 6 μL, react overnight at 16°C.

Further defined, in step 4), the GmST1 gene knockout vector is transformed into soybean through the Agrobacterium-mediated method; the Agrobacterium is Agrobacterium EHA105.

Further defined, the soybean described in step 4) is Dongnong 93-046.

To further define, the forward primer used for the screening in step 4) is CasJC-S, whose nucleotide sequence is shown in SEQ ID NO: 6, and the reverse primer is CasJC-A, whose nucleotide sequence is shown in SEQ ID NO: 7, used to screen positive mutant plants.

To further define, the forward primer used for the screening in step 4) is GmST1-target1-S, its nucleotide sequence is shown in SEQ ID NO: 8, and the reverse primer sequence is GmST1-target1-A, its nucleotide sequence The sequence is shown in SEQ ID NO: 9, which is used to detect the soybean GmST1 gene mutant plant obtained by constructing primers GmST1-1-S and GmST1-1-A; or the forward primer is GmST1-target2-S, and its nucleoside The acid sequence is shown in SEQ ID NO: 10, the reverse primer is GmST1-target2-A, and its nucleotide sequence is shown in SEQ ID NO: 11, used for detection using primers GmST1-2-S and GmST1-2- A Construct the obtained soybean GmST1 gene mutant plants.

To further define, the present invention performs qRT-PCR quantitative detection of the mutated GmST1 gene expression in the obtained soybean GmST1 gene mutant plant, and the forward primer used for the detection is qGmST1-S, and its nucleotide sequence is as shown in SEQ ID NO : 12, the reverse primer is qGmST1-A, and its nucleotide sequence is shown in SEQ ID NO: 13; the gene used for the control is the viral coat protein gene, and the forward primer for detecting the expression of the gene is SMV- CP-S, its nucleotide sequence is shown in SEQ ID NO:14, and the reverse primer is SMV-CP-A, its nucleotide sequence is shown in SEQ ID NO:15.

Beneficial effect

The present invention uses CRISPR/Cas9 technology to carry out targeted mutation on the soybean GmST1 gene coding region, and obtains soybean mutant plants. It can be seen from the comparison with wild-type phenotype identification, GmST1 gene expression, and virus coat protein gene expression analysis that, After GmST1 gene mutation, its resistance to soybean mosaic virus is weakened, and it is determined that GmST1 can participate in the resistance response of soybean mosaic virus disease resistance. The present invention provides a theory for the research on the function of soybean mosaic virus-resistant genes in accordance with.

Description of drawings

Figure 1. Two single-target vectors of the GmST1 gene were used to construct bacterial liquid PCR, where M: DL2000, 1 and 2 are the PCR detection results of target 1, and 3 and 4 are the PCR detection results of target 2.

Fig. 2 PCR mutation detection of GmST1 gene knockout plants of T2 generation, where M: DL2000, 1-4: PCR products of plants transfected with GmST1-1 gene knockout vector, 5: negative control (Donnong 93-046).

Figure 3 The base deletion of GmST1-1 mutant, CK is Dongnong 93-046, WT is the sequence of GmST1 gene (Glyma.13G191400) on Phytozome, and 2-1 is the number of the mutant plant.

Fig. 4 Leaf changes of GmST1 knockout plants of T2 generation after inoculation with SMV N1.

Figure 5 qRT-PCR detection results of GmST1 mutants, where a is the qRT-PCR detection results of GmST1 gene in GmST1-1 mutants. CK is Dongnong 93-046, 1-4: gene knockout mutant, 1 is the mutant of GmST1-1 target, plant number 2-1, 2-4 is the mutant of GmST1-2 target, plant number 3-1, 3-2, 3-3 in sequence; 5-8: non-mutant; b is the qRT-PCR detection result of SMV CP gene in GmST1-1 mutant. CK is Dongnong 93-046, a is 1-4: gene knockout mutant, 1 is the mutant of GmST1-1 target, plant number 2-1, 2-4 is the mutant plant of GmST1-2 target , and the numbers are 3-1, 3-2, 3-3; 5-8: non-mutant.

Figure 6 PCR mutation detection of T2 generation GmST1 gene knockout plants, where M: DL2000, 1-4: PCR products of plants transfected with GmST1-2 gene knockout vector, 5: negative control (Donnong 93-046)

Figure 7 The base deletion of GmST1 gene knockout plants in T2 generation, CK is Dongnong 93-046, WT is the sequence of GmST1 gene (Glyma.13G191400) on Phytozome, 3-1, 3-2, 3-3 are mutations respectively Plant number.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In addition, the soybean variety Dongnong 93-046 (disease-resistant variety) is available to the public from Northeast Agricultural University; this variety is recorded in: Teng Weili. Soybean Mosaic Disease Resistance Inheritance, Cell Ultrastructure Analysis and Gene Mapping. Doctoral Dissertation. Northeast China Agricultural University. 2006. Both are available to the public from Northeast Agricultural University.

Wherein, the preparation method of the LB medium used is: prepare every liter of medium, add in 950ml deionized water: tryptone 10g, yeast extract 5g, NaCl 10g, shake the container until the solute is dissolved. Use 5mol/L NaOH to adjust the pH to 7.0. Dilute to 1L with deionized water. Steam sterilize under high pressure for 21min.

YEP liquid medium: prepare per liter of medium, add in 950ml deionized water: 10g tryptone, 5g yeast extract, 5g NaCl, shake the container until the solute is dissolved. Use 5mol/L NaOH to adjust the pH to 7.0. Dilute to 1L with deionized water. Steam sterilize at 15psi high pressure for 21min.

MS ₀ medium: Weigh 4.43g MS powder, 30g sucrose, add 600-800ml distilled water to dissolve, stir the glass rod to fully dissolve, adjust the pH value to 5.8, dilute to 1L, add 6g agar powder, steam under 15psi high pressure Sterilize for 21 minutes.

The CRISPR/Cas9 kit was purchased from Beijing Weishang Lide Biotechnology Co., Ltd., product number VK005-15.

Soybean mosaic virus N1 strain (referred to as SMV-N1 strain): The public can obtain it from Northeast Agricultural University; the strain is recorded in: Teng Weili. Soybean Mosaic Resistance Inheritance, Cell Ultrastructure Analysis and Gene Mapping. Ph.D. Thesis. Northeast Agricultural University. 2006. The phenotype of the affected leaves is: chlorosis between the veins, systematic mosaic.

Example 1. Preparation method of soybean GmST1 gene mutant plants.

This example takes the first target as an example (the target nucleotide sequence is 5'AGAAAGGGGAGGAAATAA3') located at the 47th to 64th bases of the GmST1 coding region, and describes the preparation method of soybean GmST1 gene mutant plants.

1) Design gRNA single-target primers based on soybean GmST1 gene.

First, the soybean GmST1 gene was amplified, and the soybean variety Dongnong 93-046 was used as the material. When the first group of three compound leaves grew, the material was collected, total RNA was extracted, and the first strand of cDNA was synthesized by reverse transcription. According to the sequence of the GmST1 gene (Glyma.13G191400) on Phytozome, use the Primer 5 software to design gene cloning primers (primer 1), and its nucleotide sequence is as follows:

Primer 1-S: 5'-GAAGATCTATGGCTCCAACAAATGTCAC-3' (shown in SEQ ID NO: 20);

Primer 1-A: 5'-CTTGGTTACCTTAAAATGACAAGCCTGAC-3' (shown in SEQ ID NO: 21).

The RT-PCR reaction was carried out using cDNA as a template. The reaction system was as follows. The reaction program: 94°C for 5 minutes; 38 cycles: 94°C for 30s, 58°C for 30s, 72°C for 1min; 72°C for 7min, and stored at 4°C. After the reaction, the PCR products were taken for 1% agarose gel electrophoresis detection and gel recovery and purification of the target fragment. The RT-PCR reaction system is as follows:

According to the steps of the PGM-T cloning kit of TIANGEN Company, the gel recovery product obtained above was connected with the cloning vector to obtain the cloning plasmid pGM-T-GmST1 with the target gene, which was transformed into Top10 large intestine competent cells, and single Cloned and verified by PCR and sequencing. Finally, the GmST1 gene with the target fragment size of 1035 bp was obtained, as shown in SEQ ID NO:1.

According to the obtained GmST1 gene sequence, gRNA single-target primers were designed. The forward primer is GmST1-1-S, 5'-ttgAGAAAGGGGAGGAAATAA-3' (shown in SEQ ID NO: 2), the reverse primer is GmST1-1-A, 5'-aacTTATTTCCTCCCCTTTCT-3' (SEQ ID NO: 3 shown), the above primers were synthesized by Primer Synthesis Company.

2) Preparation of gRNA single-target primer-dimer.

Mix the above forward and reverse primers according to the following ratio to form a dimer: GmST1-1-S/A (10 μM) 5 μL each, H ₂ O 15 μL after mixing, perform the following treatment: 95°C for 3 minutes; 95°C to 25°C for slow cooling ; 16°C for 5 min.

3) Insert the primer dimer into the Cas9/gRNA vector to construct the GmST1 gene knockout vector, which is denoted as the GmST1-1 gene knockout vector.

The reaction system is as follows (10 μl system): overnight reaction at 16°C.

4) Transforming the GmST1-1 gene knockout vector constructed in step 3) into Agrobacterium, infecting soybeans, and obtaining soybean GmST1 gene mutant plants after identification.

a Transformation: Take 10 μl of the above product and add it to 50 μl TOP10 competent medium that has just been thawed, flick and mix well, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, let stand on ice for 2 minutes, then add 500 μl of anti-antibody-free LB medium, place In a constant temperature shaker at 37°C, 170 rpm, after recovery for 1 hour, apply a Kana-resistant solid plate, culture overnight at 37°C, pick 3-5 single-spot shakers, and perform bacterial liquid PCR and sequencing, as shown in Figure 1 Show. The bacterial liquid PCR detection primers are:

CasJC-S: 5'-AAAAGTCCCACATCGCTTAG-3' (shown in SEQ ID NO: 6)

CasJC-A: 5'-TGTGCAAGGTAAGAAGATGG-3' (shown in SEQ ID NO: 7), the target fragment is 435bp, the sequencing result is compared with the vector sequence, and the positive bacterial liquid is stored at -80°C for later use.

b. Agrobacterium tumefaciens-mediated transformation of soybean shoot tips.

The GmST1-1 gene knockout vector was transformed into Agrobacterium EHA105, and the plasmid preserved in this experiment was used to transform Agrobacterium by freeze-thaw method. For this method, please refer to: Wang Tao. Soybean miRNA172 and target genes involved in flowering induction and stress response Functional Analysis. Ph.D. Dissertation. Northeast Agricultural University. 2016 Agrobacterium transformation method described.

The GmST1-1 gene knockout vector was transformed into the disease-resistant soybean variety Dongnong 93-046 by the Agrobacterium-mediated method, and the specific method was as follows:

(1) Bacterial solution preparation: Take the prepared bacterial solution and streak it on the YEP solid plate (50mg/mL Str, 50mg/mL Kan, 25mg/mL Rif) to culture at 28°C, pick a single colony and inoculate it in the YEP liquid culture In culture medium (50mg/mL Str, 50mg/mLKan, 25mg/mL Rif), shake culture at 28°C 200rpm for 1-2 days, inoculate 1-2ml bacterial liquid into 50ml fresh YEP liquid medium and shake culture until OD ₆₀₀ is 0.6-0.8.

(2) Seed sterilization: select plump, no-spot seeds in a petri dish, use chlorine gas sterilization, put the seeds in the desiccator of the fume hood, pour 96ml of sodium hypochlorite into the triangular flask of the desiccator, and quickly After adding 6ml of concentrated hydrochloric acid, quickly close the cap tightly. After sterilizing for 16 hours, place it in an ultra-clean workbench to blow off residual chlorine gas, and keep it sealed for about 30 minutes.

(3) Seed germination: the sterilized seeds were planted in MS0 medium with the hilum down, 10 seeds were planted in each bottle, and germinated for 5-6 days at 23°C, 16h light/8h dark conditions.

(4) Preparation of shoot tip explant: when the seed germinates until the cotyledon is about to break through the seed coat, remove the seed coat with tweezers and a scalpel, cut off half of the two halves of the cotyledon, scrape off the cotyledon and grow gently with a scalpel The axillary buds between the points, and 3-5 wounds were gently drawn at the cotyledon nodes, and the remaining parts were used as explants for infection.

(5) Infection and co-cultivation: put the prepared explants into the invasion solution for vacuum treatment, the vacuum condition is 0.6 Pa, 10 min. The explants after vacuum infection were washed three times with sterilized distilled water, and the infection solution on the surface of the explants was blotted dry with sterile paper, inserted into MS0 medium for co-cultivation for 3 days, and the resurrection was observed. The transplanting time is determined according to the situation. After transplanting into the soil, it is cultivated in an incubator at 25°C and under the conditions of 16h light/8h dark.

c. Identification of mutant plants: Get the leaves of the T1 generation and the control (Donnong 93-046) leaves of the soybean plants with the GmST1 gene knocked out, extract their DNA, and carry out PCR identification. The primers are:

CasJC-S: 5'-AAAAGTCCCACATCGCTTAG-3' (shown in SEQ ID NO: 6);

CasJC-A: 5'-TGTGCAAGGTAAGAAGATGG-3' (shown in SEQ ID NO: 7), the target fragment is 435bp, and a transgenic plant transfected with the GmST1-1 gene knockout vector was identified. The soybean seeds identified as transgenic plants in the T1 generation were harvested and planted, leaf DNA was extracted, and PCR detection was performed with single-target mutation detection primers. The forward primer was: GmST1-target1-S:5'-GATCGACCATTAGCACTCTA-3'(SEQ ID NO: 8); reverse primer: GmST1-target1-A:5'-TCCTTACATTCTTGACTTAGC-3' (SEQ ID NO: 9). The target fragment 121bp was amplified and the product was sequenced, as shown in Figure 2, the sequencing results found that one positive plant had a mutation at the target site, and a base deletion occurred compared with the wild type (as shown in Figure 3) , the mutated GmST1 gene sequence is shown in SEQ ID NO:16. The soybean seeds identified as transgenic plants in the T1 generation were collected and planted, and the SMV N1 virus inoculation experiment was carried out when the opposite true leaves of the T2 generation plants were fully expanded. The specific method was: prepare 0.01mol/L disodium hydrogen phosphate and diphosphate Phosphate buffer solution of sodium hydrogen (pH=7.0) (10mL/g virus leaves), put it into the mortar with virus leaves and a small amount of corundum, and grind it into a homogenous slurry. Sow the material in the sterilized soil. After the opposite true leaves are fully unfolded, use a brush to dip in the inoculum and rub along the veins of the true leaves for inoculation. After inoculation, rinse the surface of the leaves with clean water immediately to remove residues. A second inoculation was performed a week later.

As a result, it was found that the leaves of soybeans knocked out of the GmST1 gene shrank and appeared more mosaic after inoculation, and there was no significant change in the control soybean plants, as shown in Figure 4, which shows that the GmST1 gene is located in the target region (nucleotide The sequence is 5'AGAAAGGGGAGGAAATAA3'), the above-mentioned base deletion occurs, which makes the functional amino acid change, which leads to the weakening of the resistance of the disease-resistant variety Dongnong 93-046 to the mosaic virus.

Quantitative primers were designed respectively according to the sequences of the GmST1 gene and the viral coat protein gene:

GmST1 gene quantitative primers are: forward primer: qGmST1-S: 5'-TCCCCTTTCTCGGATTCATT-3' (SEQ ID NO: 12); reverse primer qGmST1-A: 5'-TGCCAATCAGTACTAGAGATCG-3' (SEQ ID NO: 13); Viral coat protein gene SMV-CP (GenBank accession number: U25673.1) quantitative primers are: forward primer: SMV-CP-S: 5'-AGGATCCAAAGAAGAGCACC-3' (SEQ ID NO: 14), reverse primer: SMV - CP-A: 5'-GCCTTTCAGTATTTTCGGAGT-3' (SEQ ID NO: 15).

The qRT-PCR method was used to detect the T2 generation plants inoculated with mosaic virus. As a result, it was detected that the relative expression level of GmST1 gene in soybean leaves with GmST1 gene knocked out was lower than that of control varieties. The results are shown in Figure 5. Based on the relative expression level 2.5 times higher than that of the control, a mutated soybean mutant plant with a GmST1 gene mutation was obtained, designated as 2-1. At the same time, it was detected that the relative expression level of the virus coat protein gene in the soybean leaves in which the GmST1 gene was knocked out was higher than that in the control variety. It shows that knocking out the GmST1 gene will make the soybean mosaic virus resistant variety Dongnong 93-046 produce certain disease susceptibility.

Example 2. The preparation method of soybean GmST1 gene mutant plants.

This example takes the second target as an example (the target nucleotide sequence is 5'CTTCCTAGGGAGAGAGGT 3') located at the 115th to 132nd bases of the GmST1 coding region, and describes the preparation method of soybean GmST1 gene mutant plants.

1) Design gRNA single-target primers based on soybean GmST1 gene.

According to the GmST1 gene sequence (shown in SEQ ID NO: 1), the method for obtaining the gene is the same as that described in Example 1. Design gRNA single-target primers. The forward primer is GmST1-2-S, 5'-ttgCTTCCTAGGGAGAGAGGT-3' (shown in SEQ ID NO: 4), the reverse primer is GmST1-2-A, 5'-aacACCTCTCTCCCTAGGAAG-3' (SEQ ID NO: 5 shown), the above primers were synthesized by Primer Synthesis Company.

2) Preparation of gRNA single-target primer-dimer.

The above-mentioned forward and reverse primers were mixed according to the following proportions to form dimers. After mixing 5 μL of GmST1-2-S/A (10 μM) and 15 μL of H ₂ O, the following treatment was performed: 95°C for 3 minutes; 95°C to 25°C for slow cooling; 16°C for 5 minutes.

3) Insert the primer dimer into the Cas9/gRNA vector to construct the GmST1 gene knockout vector, which is denoted as the GmST1-2 gene knockout vector.

The reaction system is as follows (10 μl system): overnight reaction at 16°C.

4) Transforming the GmST1 gene knockout vector constructed in step 3) into Agrobacterium, infecting soybeans, and obtaining soybean GmST1 gene mutant plants after identification.

a Transformation: Take 10 μl of the above product and add it to 50 μl TOP10 competent just thawed, flick and mix well, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, let stand on ice for 2 minutes, then add 500 μl of no-antibody LB, and place at 37°C In a constant temperature shaker, 170 rpm, after recovery for 1 hour, apply Kana-resistant solid plate, culture overnight at 37°C, pick 3-5 single-spot shaking bacteria, and perform bacterial liquid PCR and sequencing, as shown in Figure 1. The primers used for PCR detection of the bacterial liquid are the same as CasJC-S and CasJC-A in Example 1. The sequencing results were compared with the carrier sequence, and the positive bacterial liquid was stored at -80°C for later use.

b. Agrobacterium tumefaciens-mediated transformation of soybean shoot tips, referring to the transformation method described in b in step 4) of Example 1, and obtaining plants after cultivation.

c. Identification of mutant plants: get the leaves of the T1 generation and the leaves of the control (Donnong 93-046) of soybean plants with the GmST1 gene knocked out, extract their DNA, and carry out PCR identification, (primers are the above-mentioned CasJC-S, CasJC-A) The target fragment was 435bp, and the transgenic plants transformed with the GmST1-2 gene knockout vector were finally identified. Soybean seeds identified as transgenic plants in the T1 generation were collected and planted, leaf DNA was extracted, and PCR detection was performed with single-target mutation detection primers.

Forward primer: GmST1-target2-S:5'-AGATCGACCATTAGCACTCTA-3', (SEQ ID NO: 10);

Reverse primer: GmST1-target2-A:5'-GACAATGGCAAAGGTGAGA-3', (SEQ ID NO: 11).

The target fragment 324bp was amplified and the product was sequenced, as shown in Figure 6, the sequencing results found that 3 transgenic positive plants had mutations at the target site, among which, the plants numbered 3-1 and 3-3 had more mutations than the wild type 2 bases were missing, and the plant numbered 3-2 had 1 base deletion compared with the wild type; as shown in Figure 7. The nucleotide sequence of the mutated GmST1 gene in the plant number 3-1 is shown in SEQ ID NO: 17, and the nucleotide sequence of the mutated GmST1 gene in the plant number 3-2 is shown in SEQ ID NO: 18, number 3 The nucleotide sequence of the mutated GmST1 gene in the -3 plant is shown in SEQ ID NO:19.

Collect the soybean seeds identified as transgenic plants in the T1 generation and plant them, and carry out the SMV N1 virus inoculation experiment when the opposite true leaves of the T2 generation plants are fully expanded (the inoculation method is described with reference to Example 1), and the results are found: knockout after inoculation The soybean leaf of GmST1 gene shrinks, and there are many mosaic phenomena, and contrast soybean plant has no obvious change, as shown in Figure 4, has illustrated that GmST1 gene is in the described target region (nucleotide sequence is 5' CTTCCTAGGGAGAGAGGT 3' ) The resistance of the disease-resistant variety Dongnong 93-046 to mosaic virus was weakened after the above-mentioned base deletion occurred.

Quantitative primers were designed respectively according to the sequences of the GmST1 gene and the viral coat protein gene:

GmST1 gene quantitative primers are: forward primer: qGmST1-S 5'-TCCCCTTTCTCGGATTCATT-3' (SEQ ID NO: 12); reverse primer: qGmST1-A 5'-TGCCAATCAGTACTAGAGATCG-3' (SEQ ID NO: 13).

The quantitative primers for virus coat protein gene SMV-CP (GenBank accession number: U25673.1) are:

Forward primer: SMV-CP-S:5'-AGGATCCAAAGAAGAGCACC-3' (SEQ ID NO: 14);

Reverse primer: SMV-CP-A: 5'-GCCTTTCAGTATTTTCGGAGT-3' (SEQ ID NO: 15).

The qRT-PCR method was used to detect the T2 generation plants inoculated with mosaic virus. As a result, it was detected that the relative expression level of GmST1 gene in soybean leaves with GmST1 gene knocked out was lower than that of control varieties. The results are shown in Figure 5. Based on the relative expression level of 2.5 times higher than that of the control, three mutated soybean mutant plants with GmST1 gene mutation were obtained, and the relative expression level of the virus coat protein gene present in the soybean leaves of the knockout GmST1 gene was detected to be higher than that of the control variety . It shows that knocking out the GmST1 gene will make the soybean mosaic virus resistant variety Dongnong 93-046 produce certain disease susceptibility.

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<212>DNA

<213> GmST1-target2-A

<400> 11

gacaatggca aaggtgaga 19

<210> 12

<211> 20

<212>DNA

<213>qGmST1-S

<400> 12

tcccctttct cggattcatt 20

<210> 13

<211> 22

<212>DNA

<213>qGmST1-A

<400> 13

tgccaatcag tactagagat cg 22

<210> 14

<211> 20

<212>DNA

<213> SMV-CP-S

<400> 14

aggatccaaa gaagagcacc 20

<210> 15

<211> 21

<212>DNA

<213> SMV-CP-A

<400> 15

gcctttcagt attttcggagt 21

<210> 16

<211> 1034

<212>DNA

<213> 2-1 plant GmST1 gene mutation sequence

<400> 16

atggctccaa caaatgtcac atgcttcaga gaagaaaatg aatccgagaa aggggagaaa 60

taacaataga agaagacaag ctaagtcaag aatgtaagga gttgatactc tctcttccta 120

gggagagagg ttggagaaca cgttatatat atctatttca aggattttgg tgccagccat 180

tggaaatcca agcaataatc acttttcaga agcacttcca agctaaagac agtgatgtta 240

ttgtggccac aattccaaaa tcaggtacca cttggctgaa agctctcacc tttgccattg 300

tcaatcgcca tactcatagt atcactacat caatgtcatc acatcctttg cttacttcta 360

atcctcatga acttgtgcct ttcatagaat acaccgttta tggtaatgcc cctagccatg 420

ttccaaacct atccaacatg actgagccaa gactttttgg tacacatatt ccattccatg 480

cattggccaa gtcaatcaag gagttcaata gtagaataat ttatatatgt aggaacccac 540

ttgacacttt tgtgtctact tggattttcc tcaacaaaat taagccagaa catttacctg 600

aatttgaact agggggaagct tttgaaaagt attgcaaagg aataataggg tttggtccaa 660

cttgggacca aatgttgggt tattggaagg agagtatagc taggcctagt aaggttttgt 720

tcttgaagta cgaggatctt aaaaaagatg tcaattttca tgtgaaaaga atagcggagt 780

tcttaggatg gcctttcact tcggaggaag aaggtgatgg gactattgag agcataatca 840

agctatgcag cttcgagaag atgaaggaat tggaggcaaa taaatctgga aatttgcta 900

ggaactttga gagaaagtac ttgttccgaa aggctgaaat gggagattgg gtgaactacc 960

tttcccctga aatgggtgaa aagttatcgc aaattatgga agaaaagtta agtgggtcag 1020

gcttgtcatt ttaa 1034

<210> 17

<211> 1033

<212>DNA

<213> 3-1 Plant GmST1 gene mutation sequence

<400> 17

atggctccaa caaatgtcac atgcttcaga gaagaaaatg aatccgagaa aggggaggaa 60

ataacaatag aagaagacaa gctaagtcaa gaatgtaagg agttgatact ctctcttcct 120

ggagagaggt tggagaacac gttatatata tctatttcaa ggattttggt gccagccatt 180

ggaaatccaa gcaataatca cttttcagaa gcacttccaa gctaaagaca gtgatgttat 240

tgtggccaca attccaaaat caggtaccac ttggctgaaa gctctcacct ttgccattgt 300

caatcgccat actcatagta tcactacatc aatgtcatca catcctttgc ttacttctaa 360

tcctcatgaa cttgtgcctt tcatagaata caccgtttat ggtaatgccc ctagccatgt 420

tccaaaccta tccaacatga ctgagccaag actttttggt acacatattc cattccatgc 480

attggccaag tcaatcaagg agttcaatag tagaataatt tatatatgta ggaacccact 540

tgacactttt gtgtctactt ggattttcct caacaaaatt aagccagaac atttacctga 600

atttgaacta ggggaagctt ttgaaaagta ttgcaaagga aataagggt ttggtccaac 660

ttgggaccaa atgttgggtt attggaagga gagtatagct aggcctagta aggttttgtt 720

cttgaagtac gaggatctta aaaaagatgt caattttcat gtgaaaagaa tagcggagtt 780

cttaggatgg cctttcactt cggaggaaga aggtgatggg actattgaga gcataatcaa 840

gctatgcagc ttcgagaaga tgaaggaatt ggaggcaaat aaatctggaa catttgctag 900

gaactttgag agaaagtact tgttccgaaa ggctgaaatg ggagattggg tgaactacct 960

ttcccctgaa atgggtgaaa agttatcgca aattatggaa gaaaagttaa gtgggtcagg 1020

cttgtcattt taa 1033

<210> 18

<211> 1034

<212>DNA

<213> 3-2 plant GmST1 gene mutation sequence

<400> 18

atggctccaa caaatgtcac atgcttcaga gaagaaaatg aatccgagaa aggggaggaa 60

ataacaatag aagaagacaa gctaagtcaa gaatgtaagg agttgatact ctctcttcct 120

aggagagagg ttggagaaca cgttatatat atctatttca aggattttgg tgccagccat 180

tggaaatcca agcaataatc acttttcaga agcacttcca agctaaagac agtgatgtta 240

ttgtggccac aattccaaaa tcaggtacca cttggctgaa agctctcacc tttgccattg 300

tcaatcgcca tactcatagt atcactacat caatgtcatc acatcctttg cttacttcta 360

atcctcatga acttgtgcct ttcatagaat acaccgttta tggtaatgcc cctagccatg 420

ttccaaacct atccaacatg actgagccaa gactttttgg tacacatatt ccattccatg 480

cattggccaa gtcaatcaag gagttcaata gtagaataat ttatatatgt aggaacccac 540

ttgacacttt tgtgtctact tggattttcc tcaacaaaat taagccagaa catttacctg 600

aatttgaact agggggaagct tttgaaaagt attgcaaagg aataataggg tttggtccaa 660

cttgggacca aatgttgggt tattggaagg agagtatagc taggcctagt aaggttttgt 720

tcttgaagta cgaggatctt aaaaaagatg tcaattttca tgtgaaaaga atagcggagt 780

tcttaggatg gcctttcact tcggaggaag aaggtgatgg gactattgag agcataatca 840

agctatgcag cttcgagaag atgaaggaat tggaggcaaa taaatctgga aatttgcta 900

ggaactttga gagaaagtac ttgttccgaa aggctgaaat gggagattgg gtgaactacc 960

tttcccctga aatgggtgaa aagttatcgc aaattatgga agaaaagtta agtgggtcag 1020

gcttgtcatt ttaa 1034

<210> 19

<211> 1033

<212>DNA

<213> 3-3 plant GmST1 gene mutation sequence

<400> 19

atggctccaa caaatgtcac atgcttcaga gaagaaaatg aatccgagaa aggggaggaa 60

ataacaatag aagaagacaa gctaagtcaa gaatgtaagg agttgatact ctctcttcct 120

aggggagaggt tggagaacac gttatatata tctatttcaa ggattttggt gccagccatt 180

ggaaatccaa gcaataatca cttttcagaa gcacttccaa gctaaagaca gtgatgttat 240

tgtggccaca attccaaaat caggtaccac ttggctgaaa gctctcacct ttgccattgt 300

caatcgccat actcatagta tcactacatc aatgtcatca catcctttgc ttacttctaa 360

tcctcatgaa cttgtgcctt tcatagaata caccgtttat ggtaatgccc ctagccatgt 420

tccaaaccta tccaacatga ctgagccaag actttttggt acacatattc cattccatgc 480

attggccaag tcaatcaagg agttcaatag tagaataatt tatatatgta ggaacccact 540

tgacactttt gtgtctactt ggattttcct caacaaaatt aagccagaac atttacctga 600

atttgaacta ggggaagctt ttgaaaagta ttgcaaagga aataagggt ttggtccaac 660

ttgggaccaa atgttgggtt attggaagga gagtatagct aggcctagta aggttttgtt 720

cttgaagtac gaggatctta aaaaagatgt caattttcat gtgaaaagaa tagcggagtt 780

cttaggatgg cctttcactt cggaggaaga aggtgatggg actattgaga gcataatcaa 840

gctatgcagc ttcgagaaga tgaaggaatt ggaggcaaat aaatctggaa catttgctag 900

gaactttgag agaaagtact tgttccgaaa ggctgaaatg ggagattggg tgaactacct 960

ttcccctgaa atgggtgaaa agttatcgca aattatggaa gaaaagttaa gtgggtcagg 1020

cttgtcattt taa 1033

<210> 20

<211> 28

<212>DNA

<213> Primer 1-S

<400> 20

gaagatctat ggctccaaca aatgtcac 28

<210> 21

<211> 29

<212>DNA

<213> Primer 1-A

<400> 21

cttggttacc ttaaaatgac aagcctgac 29

Claims (1)

1. The application of soybean GmST1 gene mutant plants in improving soybean mosaic virus infection diseases is characterized in that the mutant plants contain mutant soybean GmST1 genes, the mutation is that nucleotide sequences of 5 'AGAAAGGGGAGGAAATAAA 3' or 5 'CTTCCTAGGGAGAGGGT 3' in coding regions of the GmST1 genes are subjected to base substitution or deletion, so that the coded amino acid sequences are changed, and the nucleotide sequences of the soybean GmST1 genes are shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the mutated soybean GmST1 gene is shown as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO:18 or SEQ ID NO: as shown at 19.

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