CN106632628A - Plant antiviral related protein GmSN1, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a plant anti-virus related protein GmSN1, its encoding gene and application. The protein provided by the present invention, named GmSN1 protein, is the following (a) or (b): (a) a protein composed of the amino acid sequence shown in sequence 1 in the sequence listing; (b) the amino acid sequence of sequence 1 is processed by a A protein derived from Sequence 1 which is related to the substitution and/or deletion and/or addition of several amino acid residues and is related to plant resistance to viral diseases. The gene encoding the GmSN1 protein (named as GmSN1 gene) also belongs to the protection scope of the present invention. The invention also protects a method for cultivating transgenic plants, which is to introduce the GmSN1 gene into the target plants to obtain the transgenic plants with higher resistance to plant virus diseases than the target plants. The invention has great application value for cultivating transgenic plants resistant to plant virus diseases.

Description

Plant anti-virus related protein GmSN1 and its coding gene and application

technical field

The invention belongs to the field of agricultural biotechnology, and relates to a plant anti-virus related protein GmSN1, its coding gene and application.

Background technique

Plant viruses are viruses that can infect higher plants. At present, more than 3,700 species are known, belonging to 7 orders and 111 families. Different kinds of plant viruses can infect different host plants and cause different symptoms in infected plants. Most plant viruses are single-stranded RNA (ssRNA) viruses, and some nucleic acid components are single-stranded DNA, double-stranded DNA and double-stranded RNA. Soybean mosaic virus disease is a common soybean disease, which is caused by soybean mosaic virus ( Potyviridae , Soybean Mosaic Virus, SMV), which is a species of Potyviridae Potyviridae. The common feature of soybean mosaic virus and other Potavirus members is that seeds are infected, and aphids are the main vectors. The in vitro preservation period of soybean mosaic virus is about 4-5 days, 15 days and 120 days at room temperature, 4°C and 0°C respectively, its lethal temperature is 58-66°C, and the dilution limit is 10 ^-5 .

Soybean mosaic virus can be transmitted between generations and different regions through seeds, and can be non-persistently transmitted in the field through sap rubbing inoculation and aphids. Soybean mosaic virus is a serious disease, widespread and difficult to control. The disease was first discovered in Northeast China in 1899, and was successively discovered in the United States, North Korea, Germany, the Soviet Union and other countries. It has become a worldwide disease and seriously affects the yield and quality of soybeans. In recent years, the disease has become more and more serious in Northeast China. In general epidemic years, it causes a 25%-60% reduction in production, and in a pandemic year it can result in extinction. For example, in 1978, soybean mosaic disease was rampant in Fuyang, northern Anhui, China, causing 1.5 million mu of soybeans to cease production.

The symptoms of soybean mosaic virus on plants are mainly divided into two categories: mosaic and necrosis. Mosaic symptoms are manifested as bright veins appearing on young leaves at the initial stage of infection. With the development of the disease, light mosaic leaves, mosaic leaves, macular mosaic leaves, and leaf curling downwards appear one after another, and some blister leaves, deformed leaves, and wrinkled leaves appear. Shrinkage, dwarfing, thickening and brittleness, the hairs on the stems and pods disappear, resulting in "light pods". The symptoms of necrosis are mainly manifested as small brown spots or necrosis of veins on the leaves at the initial stage of infection, and then the necrotic parts expand or even become one piece, and in severe cases, the leaves will fall off. After some varieties are infected with specific strains, the growth point of the main stem is necrotic, forming the so-called "top dead". Soybean mosaic virus disease not only affects the yield of soybean, but also affects the quality of soybean seeds. It can cause brown spots on the seeds of diseased plants. When the disease is severe, the rate of diseased seeds can reach more than 50%, which seriously reduces the commodity value of soybeans. The occurrence of streaks is affected by the soybean variety and the degree of disease. The stripes formed by soybean mosaic virus disease on the grains are the same as the navel color or slightly darker, and sometimes the stripes will spread all over the surface of the grains, most of which are radial or banded. Wu Zongpu divided the mottled spots into 7 types according to the color: light brown spot, dark brown spot, reddish brown spot, purple brown spot, black spot, gray blue spot and double color spot; There are 6 kinds of regular plaques, piebald spots and double-banded spots on one side of the navel.

Contents of the invention

The purpose of the present invention is to provide a plant anti-virus related protein GmSN1 and its coding gene and application.

The protein provided by the present invention is derived from soybean, named as GmSN1 protein, and is the following (1) or (2): (1) a protein composed of the amino acid sequence shown in sequence 1 in the sequence table; (2) the amino acid sequence of sequence 1 A protein derived from Sequence 1 whose sequence has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant resistance to viral diseases.

In order to make the protein in (1) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the sequence listing can be linked with the tags shown in Table 1.

Table 1 Sequence of tags Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in (2) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The protein-encoding gene in (2) above can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2 in the sequence listing, and/or making one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The gene encoding the GmSN1 protein (named as GmSN1 gene) also belongs to the protection scope of the present invention.

Specifically, the gene may be a DNA molecule of (1) or (2) or (3) or (4):

(1) A DNA molecule (open reading frame) whose coding region is shown in Sequence 2 of the Sequence Listing;

(2) The DNA molecule (genomic DNA) shown in sequence 3 of the sequence listing;

(3) A DNA molecule that hybridizes to the DNA sequence defined in (1) or (2) under stringent conditions and encodes a plant antiviral disease-related protein;

(4) A DNA molecule that has at least 90% identity with the DNA sequence defined in (1) or (2) and encodes a plant antiviral disease-related protein.

The above-mentioned stringent conditions may be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1 mM EDTA, washing at 65°C, 0.1×SSC, 0.1% SDS. The above stringent conditions can also be: hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

The expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the GmSN1 gene all belong to the protection scope of the present invention.

An existing expression vector can be used to construct a recombinant expression vector containing the gene. The expression vector may also include the 3′-untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When using the gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other promoters; in addition, When using the gene of the present invention to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate identification and screening, the recombinant expression vector can be processed, such as adding genes encoding color-changing enzymes or luminescent compounds, antibiotic-resistant markers or chemical-resistant reagent marker genes, etc.

The recombinant expression vector can specifically be the recombinant plasmid pB7FWG2-GmSN1 obtained by inserting the GmSN1 gene between the recombination sites of the pB7FWG2 vector.

The construction method of the recombinant plasmid pB7FWG2-GmSN1 is as follows: ① synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence table; PCR amplification to obtain the PCR amplification product; ③ BP recombination reaction between the PCR amplification product of step ② and the pDONOR221 vector to obtain the intermediate plasmid; ④ LR recombination reaction between the intermediate plasmid and the PB7FWG2 vector to obtain the recombinant plasmid pB7FWG2-GmSN1, the recombinant plasmid In pB7FWG2-GmSN1, between the attB1 site and the attB2 site is a double-stranded DNA molecule shown in sequence 2 of the sequence table; F1: 5´-GGGGACAAGTTTGTACAAAAAGCAGGCTTCACCATGAAGCTCGAGTTCGCAAA-3´; R1: 5´-GGGGACCACTTTGTACAAGAAAGCTGGGTGAGGGCATTTGTCCTTGCC-3´.

The invention also protects a method for cultivating transgenic plants, which is to introduce the GmSN1 gene into the target plants to obtain the transgenic plants with higher resistance to plant virus diseases than the target plants. Specifically, the GmSN1 gene can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and The transformed plant tissue is grown into plants. Specifically, the GmSN1 gene can be introduced into the target plant through the recombinant plasmid pB7FWG2-GmSN1. The target plant may be a monocotyledon or a dicotyledon. The dicot can specifically be soybean, such as soybean cultivar P03-8-23. The plant virus disease can specifically be a plant virus disease caused by the virulent No. 3 strain of soybean mosaic virus.

The invention also protects a method for cultivating transgenic plants, which is to introduce the GmSN1 gene into the target plant to obtain the transgenic plant with higher resistance to plant virus than the target plant. Specifically, the GmSN1 gene can be introduced into the target plant through the recombinant expression vector. In the method, the recombinant expression vector can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and The transformed plant tissue is grown into plants. Specifically, the GmSN1 gene can be introduced into the target plant through the recombinant plasmid pB7FWG2-GmSN1. The target plant may be a monocotyledon or a dicotyledon. The dicot can specifically be soybean, such as soybean cultivar P03-8-23. The plant virus can specifically be the virulent No. 3 strain of soybean mosaic virus.

The invention also protects the application of the GmSN1 protein, the GmSN1 gene or the recombinant vector in cultivating transgenic plants with increased resistance to plant virus diseases. The plant may be a monocot or a dicot. The dicot can specifically be soybean, such as soybean cultivar P03-8-23. The plant virus disease can specifically be a plant virus disease caused by the virulent No. 3 strain of soybean mosaic virus.

The invention also protects the application of the GmSN1 protein, the GmSN1 gene or the recombinant vector in cultivating transgenic plants with increased resistance to plant viruses. The target plant may be a monocotyledon or a dicotyledon. The dicot can specifically be soybean, such as soybean cultivar P03-8-23. The plant virus can specifically be soybean mosaic virus virulent No. 3 strain.

The invention has great application value for cultivating transgenic plants resistant to plant virus diseases.

Description of drawings

Fig. 1 Partial results of PCR identification of T2 generation plants of two transgenic lines (B6F7015 and B6F7016).

Figure 2 Detection of the expression level of GmSN1.

Figure 3 Southern blot identification of transgenic plants.

Fig. 4 The identification results of disease resistance of T1 transgenic soybeans, A is the seedling stage, B is the flowering stage.

Figure 5 shows the identification results of disease resistance (flowering stage) of T2 transgenic soybeans.

detailed description

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 test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

pDONOR221 vector: a component in Invitrogen’s “Multisite Gateway® Pro Plus Kit for 2-, 3- or 4-fragment recombination” kit, Cat.#12537-100, which is commercially available to the public.

pB7FWG2 vector: product of Plant system biology company, http://gateway.psb.ugent.be/vector/show/pB7FWG2/search/index/, the public can obtain it from commercial channels. Agrobacterium tumifaciens EHA101: Reference: Regeneration Study of Soybean Cultivars and Their Susceptibility to Agrobacterium tumifaciens EHA 101. Acta Agron Sin, 2003, 29(5): 664-669.

Soybean cultivars P03-8-23, Zhonghuang 35 and Jiunong 9: P03-8-23 is a soybean strain obtained by cross-breeding Jiunong 27 and Pingan 1008 by the Soybean Research Institute of Jilin Academy of Agricultural Sciences; the above varieties are public It can be obtained from Soybean Research Institute of Jilin Academy of Agricultural Sciences.

Soybean mosaic virus virulent No. 3 strain (SMV SC-3): References: Zheng Cuiming, Chang Ruzhen, Qiu Lijuan, Wu Zongpu, Gao Fenglan. Resistance identification of soybean germplasm resources to SMV3 strain. Soybean Science, 2000, 19(4): 299-306, publicly available from the Institute of Plant Protection, Jilin Academy of Agricultural Sciences.

See Table 2 for the formulations of each culture medium used in the examples. MS synthetic salt was purchased from Sigma Company, the product number is M5524. B5 synthetic salt was purchased from Sigma Company, the product number is G5768.

Table 2 Medium formula GM CCM SIM SEM RM MS synthetic salt MS salt mixture – – – 1× 1/2× B5 synthetic salt B5 salt mixture 1× 1/10× 1× – – 2-(4-morpholine)ethanesulfonic acid MES/g L ^–1 – 3.90 0.59 0.59 0.59 6-Benzyladenine 6-BAP/mg·L ^–1 – 1.67 1.67 – – Gibberellin GA ₃ /mg·L ^–1 – 0.25 – 0.50 – Acetosyringone AS/mM – 0.20 – – – L-cysteine L-Cys/mg·L ^–1 400.00 Dithiothreitol DTT/mg·L ^–1 154.20 Ticarcillin Tic/mg·L ^–1 250.00 250.00 250.00 Cephalosporin Cef/mg·L ^–1 – – 100.00 100.00 100.00 L-Asparagine L-Asp/mg·L ^–1 – – – 50.00 50.00 Glutamine Glu/mg·L ^–1 – – – 50.00 50.00 Glufosinate/mg·L ^–1 – – 5.00 5.00 – Indoleacetic acid IAA/mg·L ^–1 – – – 0.10 – Zeatin ZR/mg·L ^–1 – – – 1 – Indolebutyric acid IBA/mg·L ^–1 – – – – 1 Sucrose (w/v) 2.00 3.00 3.00 3.00 2.00 Agar Agar/g·L ^–1 4.50 8.00 8.00 Plant gel Phytagar /g·L ^–1 3.00 3.00 pH 5.80 5.40 5.70 5.70 5.60

Embodiment 1, the discovery of GmSN1 protein and its coding gene

Sow Zhonghuang 35 (soybean mosaic virus-resistant variety) and Jiunong 9 (soybean mosaic virus-susceptible variety) in paper cups filled with sterilized soil, and put them into a light incubator for cultivation. 16 hours of light, 8 hours of darkness, and a temperature of 25°C. Each variety was divided into two groups—experimental group and control group. The experimental group was inoculated with soybean mosaic virus by friction on the first three compound leaves, and the control group was not inoculated with soybean mosaic virus. When the leaves of the susceptible variety experimental group showed slight mosaic leaves, the samples were taken from the newly infected leaves, and the leaves of the corresponding positions were taken from the control group. This results in a total of four samples.

RNA was extracted from each sample and hybridized with an Affymetrix chip (Affymetrix soybean chip #900525, available from Affymetrix, USA), and a batch of gene fragments with expression differences of more than 2 times in each combination were screened out. On this basis, the full-length gene was sequentially obtained and the antiviral function was verified.

After conducting a large number of verification tests, a protein was found, as shown in sequence 1 of the sequence listing, and it was named GmSN1 protein. The gene encoding the GmSN1 protein is named GmSN1 gene, its open reading frame is shown in sequence 2 of the sequence listing, and its genomic DNA is shown in sequence 3 of the sequence listing (in sequence 3 of the sequence listing, from the 265th to the 5' end Nucleotide 346 and nucleotides 484-668 are exons).

Embodiment 2, application of GmSN1 gene to breed disease-resistant soybean

1. Construction of recombinant plasmids

1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

2. Using the double-stranded DNA molecule synthesized in step 1 as a template, a primer pair composed of F1 and R1 is used for PCR amplification to obtain a PCR amplification product.

F1: 5´- GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACC ATGAAGCTCGAGTTCGCAAA-3´; (SEQ ID NO: 4)

R1: 5´- GGGGACCACTTTGTACAAGAAAGCTGGGTGAGGGCATTTGTCCTTGCC -3´. (Sequence Listing Sequence 5)

The underline in F1 marks the attB1 site, and the underline in R1 marks the attB2 site.

3. The PCR amplification product in step 2 undergoes a BP recombination reaction with the pDONOR221 vector to obtain an intermediate plasmid.

4. The LR recombination reaction between the intermediate plasmid and the pB7FWG2 vector was performed to obtain the recombinant plasmid pB7FWG2-GmSN1. In the recombinant plasmid pB7FWG2-GmSN1, between the attB1 site and the attB2 site is a double-stranded DNA molecule shown in sequence 2 of the sequence table.

The acquisition of transgenic plants

1. The recombinant plasmid pB7FWG2-GmSN1 is introduced into Agrobacterium EHA101 to obtain recombinant Agrobacterium.

2. Suspend the recombinant Agrobacterium in CCM liquid medium to obtain a bacterial suspension with OD ₆₀₀ nm = 0.5-0.8.

3. Take the seeds of soybean cultivar P03-8-23 and sterilize them in a closed container containing chlorine for 12-16 hours.

4. After completing step 3, take the seeds, place the hilum down on the GM medium plate, and culture in the dark at 23°C for 24 hours.

5. After completing step 4, take the seeds, cut them open along the hilum with a sterile scalpel, and remove the axillary buds, make 3 to 4 incisions in the groove above the axillary buds parallel to the central axis, and then place the bacteria obtained in step 2 Soak in the suspension for 30-40 minutes.

6. After completing step 5, transfer the seeds to a CCM medium plate covered with sterile filter paper, and culture in the dark at 23°C for 4-5 days.

7. After completing step 6, transfer the seeds to the SIM medium plate, culture at 25°C, 16h (light)/8h (dark), subculture once every 2-3 weeks, and subculture 3-5 times in total.

8. After completing step 7, cut off the cotyledons on the explants that induced clustered buds, place them on the SEM medium plate, culture at 25°C for 16h (light)/8h (dark), and subculture 1 every 2 to 3 weeks. times, a total of 2 to 3 times of subculture, and the browned callus was removed during subculture.

9. After completing step 8, when the regenerated shoots grow to 4-8cm, cut off the regenerated shoots and transfer them to the RM medium plate, and culture them at 25°C for 16h (light)/8h (dark).

10. After completing step 9, when the regenerated plants grow more than 2 roots, move them to the seedling hardening room for 3-5 days for domestication, and then transfer them to the greenhouse for growth to obtain T0 generation plants.

11. Single plant selfing of T0 generation plants to obtain T1 generation plants.

12. Carry out PCR identification of T0 generation plants and T1 generation plants respectively, the method is as follows: extract genomic DNA, use genomic DNA as a template, and use the primer pair composed of F2 and R2 to perform PCR amplification, if the amplification of 264bp and 403bp is obtained at the same time product, the identification result is positive; if only the amplified product with a size of 403bp is obtained, the identification result is negative.

F2: 5'-ATGAAGCTCGAGTTCGCAAA-3'; (SEQ ID NO: 6)

R2: 5'-AGGGCATTTGTCCTTGCC-3'. (Sequence Listing Sequence 7)

13. The T1 generation plants were self-crossed to obtain T2 generation plants.

14. Take the T2 generation plants and carry out PCR identification according to the method in step 12.

Partial results of PCR identification of T2 generation plants of two transgenic lines (B6F7015 and B6F7016) are shown in FIG. 1 .

Figure 1. Results of PCR identification of some transgenic lines. (1, 2 are two individual plant amplification products of B6F7015 strain, 3, 4 are two individual plant amplification products of B6F7016 strain)

15. Detection of exogenous gene expression level in transgenic plants:

(1) Use Trizol reagent (Ambion, 15596-026) to extract total RNA from recipient plants, transgenic plants and negative plants respectively according to the instructions;

(2) Reverse transcription: SuperScript® (Invitrogen, 11754-050) reverse transcription kit was used to obtain cDNA according to the instructions.

(3) Detection of gene expression level: TOYOBO real-time quantitative PCR kit (Toyobo, QPK-212) was used, cDNA was used as template, F2 and R2 were used as primers, and the PCR instrument was Abi StepOnePlus. Expression levels in each strain. It can be seen from Fig. 2 that GmSN1 in the two transgenic lines was 672 and 100 times higher than that of the recipient plants and negative transgenic plants, respectively.

Figure 2. Expression level detection of GmSN1

16. Southern blot identification of transgenic plants.

The Southern hybridization method is as follows:

(1) Enzyme digestion of genomic DNA: extract genomic DNA from 3 individual plants of each of the two transgenic lines, recipient plants and transgenic negative plants, add 20 μg of genomic DNA to 20 μl Promega buffer H, 3 μl Eco RI (Promega), 2 μl 100X BSA, supplemented with ddH ₂ O to 200 μl, digested at 37°C for 24 hours. Then freeze-dry the digested sample with a lyophilizer, and add 50 μl ddH ₂ O to dissolve it.

(2) Labeled hybridization probe: Add the DNA to be labeled into a 0.2ml PCR tube (generally use 1ug labeling and use it several times), dilute the volume to 15μl with double distilled water, denature in boiling water for 10 minutes, and quickly put on ice. Add Hexanucleotide Mix (10×) 2μl, dNTP Labeling Mix 2μl, Klenow enzyme labelinggrade 1μl; mix and centrifuge briefly, react at 37°C for 24 hours, add 0.2M EDTA (pH8.0) 2μl to stop the reaction, or heat at 65°C for 10 minutes Stop the reaction.

(3) Electrophoresis: 1% agarose gel electrophoresis digested soybean genomic DNA, electrophoresis at 13V for 36 hours. Cut the separated agarose gel under the ultraviolet light, cut off the gel hole, and leave the bromophenol blue indicator. The length from the gel hole to the position where the bromophenol blue indicator is left should not exceed 10cm, otherwise it cannot be loaded into the hybridization tube. Cut off a corner in the upper right corner of the gel as a marker. Record the gel size. Use depurinating reagent (1.1% HCl) to treat the gel for 8-10 min, keep the gel shaking until the bromophenol blue turns from blue to yellow. Wash twice with deionized water and proceed to the next step. Use denaturing solution (NaCl 8.766%, NaOH 2%) to treat the depurinated gel for 30 minutes, keep the gel shaking, and you can find that bromophenol blue turns back to blue. Wash twice with deionized water, use a neutralizing solution (Trizma base 0.0605%, NaCl 0.08766%, adjust the pH to 7.5 with concentrated HCl), treat the denatured gel for 30 minutes, and keep the gel shaking. Wash twice with deionized water.

(4) Transfer film: Cut two pieces of filter paper and use a large rubber board to bridge, the size is 33cm×26cm. Then cut 3-4 pieces of filter paper slightly larger than the size of the gel (it is best to have horizontal and vertical stripes in the same direction as the filter paper when bridging), and 1 piece of nylon membrane. Mark the sample, enzyme digestion and time in the lower right corner of one side of the nylon membrane, and cut off a corner in the upper right corner for marking to match the gel. At this time, the marked side is the back side, and the other side has DNA. Cut a lot of absorbent paper that is the same size as the nylon membrane or slightly larger. Using the siphon effect, from bottom to top are: two filter papers for bridging, gel, nylon membrane, 3-4 pieces of filter paper, absorbent paper, glass plate, and heavy objects. There should be no air bubbles between each filter paper or gel or membrane. Use a glass rod to flatten, add transfer solution (sodium chloride 17.532%, sodium citrate 0.0882%, use solid NaOH to adjust the pH to 7.0), and then use plastic wrap to The gel is surrounded on all sides and covers the tray to reduce the evaporation of the transfer solution and ensure that the transfer solution can only be sucked upward through the gel, nylon membrane, filter paper, etc. Pay attention to replace the absorbent paper.

(5) Hybridization: hybridization: the DNA transferred to the nylon membrane was immobilized using an ultraviolet crosslinker, set at 70000uJ/cm ² . The DNA side of the membrane is facing up for UV crosslinking. Use 25ml blank hybridization solution (SDS 7%, formamide (deionize) 50%, 5×SSC, Blocking solution 2%, N-lauroylsarcosine 0.1%, Sodium Phosphate, pH7.0, 50mM) to pre-hybridize at 42°C for more than 2 hours. The hybridization solution containing the probe was taken out from the refrigerator, boiled in boiling water for 10 minutes, and immediately cooled on ice for denaturation. Add 1 μl of labeled Marker and mix it into the hybridization solution. Recover the blank hybridization solution, freeze it in the refrigerator, and replace it with the hybridization solution containing the probe for overnight hybridization. The next day, recover the probe-containing hybridization solution, freeze it in the refrigerator, add washing solution I (1×SSC, 0.1% SDS), wash the membrane once at 65°C for 15 minutes. Add wash solution II (0.5×SSC, 0.1% SDS), wash the membrane twice at 65°C for 20 minutes. The above two steps wash away excess probes. The following steps are carried out at room temperature, add an appropriate amount of Washing buffer (maleic acid buffer: maleic acid 0.1M, NaCl 0.15M, adjust the pH to 7.5 with NaOH; add 3μl/ml Tween-20), rinse gently for 1~5 minutes . Add Blocking solution (blocking reagent 1% dissolved in 100ml maleic acid buffer), and react in the hybridization tube, twice for 30 minutes each time, to block the protein. Centrifuge the Antibody (reagent No. 3 in the centrifugal detection kit) at 10,000rpm for 5 minutes. It needs to be centrifuged before each use. Take 1 μl of the supernatant and add it to the Blocking solution to prepare the Antibody solution. Pour out the Blocking solution in the previous step, add Antibody solution, and react in the hybridization tube for 60 minutes. Change the tube, take the membrane out of the Antibody solution, and replace it with Washing buffer (washing solution I: 1×SSC, 0.1% SDS; washing solution II: 0.5×SSC, 0.1% SDS) and rinse twice, each time for 15 minutes .

(6) Detection: wash off excess antibody, pour out the Washing buffer, add Detection buffer (Tris-HCl 0.1M, NaCl 0.1M, adjust pH to 9.5 with HCl), 2-5 minutes. Prepare CSPD solution in advance, take 10μl CSPD stock solution, dilute to 1.5ml with Detection buffer, spread plastic wrap, place the hybridization membrane with the DNA side facing up, add 1ml CSPD solution, adjust the plastic wrap left and right to quickly remove CSPD The solution is evenly applied to the entire film, and after fully reacting for 5 minutes, the excess CSPD solution is removed, and the CSPD solution that has been used once is recovered. Place the hybridized membrane at 37°C for 15 minutes. To enhance the luminescent response, place the membrane bare side up. Put the film face down on the plastic wrap, wrap the nylon film with the plastic wrap, wipe the front and back of the film flat with alcohol cotton to ensure that there are no air bubbles between the plastic wrap and the nylon film, and then place it on the X-ray film in the dark room for exposure 30 minutes or more. Put the pressed film into developer, stopper (low-concentration acetic acid solution, take about 2ml and dissolve in tap water), and fixer in sequence. Rinse with water afterwards and hang to dry. It can be seen that the plants of the two transgenic plant lines each have one copy of the exogenous gene (results of southern blot are shown in FIG. 3 ). Rinse the hybridized membrane in double-distilled water for 2-5 minutes, rinse twice with membrane washing solution (0.1% SDS, 0.2M NaOH) at 37°C for 15 minutes each time, and then rinse with 2×SSC for 5 minutes , wrapped in plastic wrap, and stored at -20°C for the next hybridization or direct pre-hybridization.

Figure 3. Southern blot identification of transgenic plants. Three individual plants were selected from each of the B6F7015 and B6F7016 strains, the plasmid vector was used as a positive control, P03-8-23 was used as a negative control for transformed receptors, and Marker was used as a molecular weight standard.

For a certain T1 generation plant, if both the plant and its T2 generation plant are identified as positive, the T1 generation plant is a homozygous transgenic plant, and the plant and its selfed offspring are transgenic lines. Follow-up studies were performed using homozygous transgenic lines.

3. Acquisition of empty vector plasmid:

There is a suicide gene ccdB in the pB7FWG2 vector, which expresses a lethal protein in common prokaryotic bacteria (including Escherichia coli and Agrobacterium) to kill the host, and can only survive normally in special strains, so it cannot be introduced into Agrobacterium for plant transformation. Instead, the ccdB gene must be removed before it can be used as an empty vector plasmid.

The pB7FWG2 vector was digested with restriction endonuclease Bsp 1407I (FastDigest®, Fermentas), the large fragment was recovered, and ligated with T4 DNA ligase to obtain an empty vector plasmid.

4. Acquisition of empty carrier plants:

The recombinant plasmid pB7FWG2-GmSN1 was replaced with the empty vector plasmid, and the other steps were the same as in step 2 to obtain empty vector-transferred plants.

5. Mosaic Virus Resistance Identification of Transgenic Plants:

The T1 generation plants and T2 generation plants of two transgenic lines (B6F7015 and B6F7016), the T1 generation plants and T2 generation plants of the empty vector control, and the soybean cultivar P03-8-23 were tested against soybean mosaic virus. For sexual identification, each strain identifies 20 T1 generation plants, and each strain identifies 60 T2 generation plants, the method is as follows:

Soybean mosaic virus virulent No. 3 strain (SMV SC-3) was inoculated by sap friction inoculation at the soybean seedling stage (the first compound leaf unfolded), and the resistance investigation was carried out before and after flowering. Soybean is resistant to mosaic virus disease See Table 3 for the division criteria of plant disease classification, Table 4 for the evaluation criteria of soybean mosaic virus resistance, and Table 5 for the results of the resistance investigation. The photos of B6F7015, B6F7016 of T1 generation and T2 generation and transformed recipient variety P03-8-23 after being inoculated with soybean mosaic virus virulent No. 3 strain are shown in Fig. 4 and Fig. 5 respectively.

Table 3 Disease classification of soybean plants identified for resistance to mosaic virus disease disease level symptom description 0 Asymptomatic reaction. 1 Light mosaic type, the plant grows normally, the leaves are flat and not wrinkled, and there are light mosaic leaves in yellow-green and dark green. 3 Heavy flower leaf type, the plant growth is basically normal, the leaf veins are slightly wrinkled, and there are obvious yellow and green mottled. 5 Wrinkled leaf type, the plant growth is close to normal, not dwarfing, the leaves have wavy spots or curved leaves or vesicular protrusions along the leaf margins, and the yellowed leaves have yellow spots. 7 Shrunken type, the plant grows abnormally, slightly dwarfed and yellowed, the leaves are obviously shrunken, and the leaves are deformed and curled in a bubble shape. 9 Dwarf type: the plant is extremely dwarfed, the leaves are rigid and narrow, deformed and curled, and in severe cases, the top is necrotic, and the buds are withered or yellowish.

Disease index (DI) = ∑ (representative value of the disease level × number of diseased plants at this level) / (total number of plants under investigation × representative value of the highest level of disease) × 100

Table 4 Evaluation criteria of soybean resistance to mosaic virus disease Disease index Resistance evaluation 0 Immunity (IM) 0.1～20.0 High resistance (HR) 20.1～35.0 Moderate resistance (MR) 35.1～50.0 Middle sense (MS) 50.1～70.0 Sense (S) 70.1～100.0 High Sensitivity (HS)

Table 5 Results of the resistance survey strain T1 generation disease index T2 generation disease index Resistance evaluation B6F7015 4.99±0.78 0.79±0.12 HR/HR B6F7016 5.56±0.0 0.41±0.71 HR/HR Empty vector plants 20.99±6.98 8.81±5.26 R/HR Soybean cultivar P03-8-23 24.14±3.51 6.19±2.16 R/HR

The identification results of two consecutive generations of inoculation showed that among the two identified transgenic lines, the disease index of the transgenic soybean line B6F7015 was 0.79-4.99%, and the disease index of B6F7016 was 0.41-5.56%. Although there are significant differences in the identification results in different years, in the same year, its disease index is extremely significantly lower than that of the control variety P03-8-23 (the disease index is 6.19-24.14), showing high resistance (HR) or close to immune (IM). The results of this study showed that the overexpression of GmSN1 in soybean can significantly improve its antiviral level.

Figure 4. Identification of resistance to mosaic virus disease of T1 generation GmSN1 transgenic plants

Figure 5. Identification of resistance to mosaic virus disease in GmSN1 transgenic plants of T2 generation - 15 days after inoculation with soybean mosaic virus.

It can be seen that the plants transfected with GmSN1 gene do not appear any symptoms of soybean mosaic virus infection. However, both the recipient and the empty vector-transformed plants had symptoms of soybean mosaic virus infection with shrunken leaves.

Claims (10)

1. A protein that is (1) or (2): (1) A protein composed of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (2) The amino acid sequence of sequence 1 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues, and a protein derived from sequence 1 is related to plant resistance to viral diseases. 2. A gene encoding the protein of claim 1. 3. The gene according to claim 2, characterized in that: the gene is a DNA molecule as follows (1) or (2) or (3) or (4): (1) A DNA molecule whose coding region is shown in sequence 2 of the sequence listing; (2) The DNA molecule shown in sequence 3 of the sequence listing; (3) A DNA molecule that hybridizes to the DNA sequence defined in (1) or (2) under stringent conditions and encodes a plant antiviral disease-related protein; (4) A DNA molecule that has at least 90% identity with the DNA sequence defined in (1) or (2) and encodes a plant antiviral disease-related protein. 4. An expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the gene of claim 2 or 3. 5. A method for cultivating a transgenic plant, which is to introduce the gene according to claim 2 or 3 into the target plant to obtain a transgenic plant with higher resistance to plant virus diseases than the target plant. 6. A method for cultivating a transgenic plant, comprising introducing the gene of claim 2 or 3 into a target plant to obtain a transgenic plant with higher resistance to plant viruses than the target plant. 7. The method according to claim 5 or 6, characterized in that: the target plant is a monocot or a dicot. 8. The application of the protein according to claim 1, the gene according to claim 2, the gene according to claim 3 or the recombinant vector according to claim 4 in cultivating transgenic plants with increased resistance to plant virus diseases. 9. The application of the protein according to claim 1, the gene according to claim 2, the gene according to claim 3 or the recombinant vector according to claim 4 in the cultivation of transgenic plants with increased resistance to plant viruses. 10. The application according to claim 8 or 9, characterized in that: the starting plants of the transgenic plants are monocotyledonous plants or dicotyledonous plants.