Application of a soybean mosaic virus resistance gene GmCSD

Abstract

This invention discloses the application of the soybean mosaic virus (SMV) resistance gene GmCSD. The candidate gene GmCSD was cloned from the soybean variety Qihuang 1, and its cDNA sequence is shown in SEQ ID NO.1. Using virus-induced gene silencing (VIGS) and overexpression technology based on Bean Pod Mottle Virus (BPMV), GmCSD was silenced in the resistant variety Qihuang 1 or overexpressed in the susceptible variety Nannong 1138-2. After inoculation with the SC3 strain, the virus content in the plants increased and decreased respectively compared to the control, indicating that this gene participates in the resistance response to SMV. The GmCSD gene obtained in this invention has important value for improving soybean resistance to SMV in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering and relates to a gene GmCSD resistant to soybean mosaic virus SC3 strain and application thereof. Background Art

[0002] When soybeans are infected with soybean mosaic virus, they will show two typical symptoms: mosaic and necrosis. It will also cause seed coat mottle and reduce seed vitality, which will affect the quality and yield of soybeans. Among them, the SC3 strain is a soybean mosaic virus strain prevalent in the Huanghuai and Yangtze River basins of China.

[0003] Reactive oxygen species (ROS) are byproducts of normal metabolism in living cells, including singlet oxygen ( 1 O2), superoxide anion (O 2·- ), hydrogen peroxide (H2O2) and hydroxyl radical (HO·), among which O 2·- It is the main ROS precursor and has poor stability. 2·- Increased levels usually lead to oxidative cell death, and superoxide dismutase (SOD) in cells is the first line of defense. 2·- The main scavenger can convert it into H2O2, which is then further decomposed by catalase (CAT) and ascorbate peroxidase (APX). In different parts of the cell, there are SODs that bind to different metal ions in their active sites, mainly including copper / zinc (Cu / ZnSOD), manganese (MnSOD) and iron (FeSOD). At present, there are few studies on the role of Cu / ZnSOD (CSD) in the plant response to viral infection, so it is necessary to explore its potential antiviral mechanism. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide an application of the gene GmCSD resistant to soybean mosaic virus SC3 strain.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The present invention provides a gene GmCSD for resisting soybean mosaic virus, and the cDNA nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0007] The amino acid sequence of the protein encoded by the soybean mosaic virus resistance gene GmCSD is shown in SEQ ID NO.2.

[0008] A recombinant expression vector for silencing the gene GmCSD comprises a specific silencing fragment for the gene GmCSD.

[0009] As a preferred embodiment of the present invention, the original vector is pBPMV-IA-V2.

[0010] A recombinant expression vector for overexpressing the gene GmCSD comprises the full-length CDS fragment of the gene GmCSD.

[0011] As a preferred embodiment of the present invention, the original vector is pBPMV-IA-V4.

[0012] The gene GmCSD is used to improve the resistance of soybean to soybean mosaic virus SC3 strain.

[0013] The gene GmCSD is used in breeding soybean varieties with resistance to soybean mosaic virus.

[0014] The recombinant expression vector is used to improve the resistance of soybean to soybean mosaic virus SC3 strain.

[0015] The recombinant expression vector is used in cultivating soybean varieties with soybean mosaic virus resistance.

[0016] Beneficial effects:

[0017] The GmCSD gene was silenced in the disease-resistant variety Qihuang No. 1, and the presence of the virus was detected in the upper leaves after inoculation with SC3, accompanied by a slight mosaic phenotype; while the virus content was effectively reduced after overexpression of the GmCSD gene in the susceptible variety Nannong 1138-2, indicating that the gene is involved in the resistance response to soybean mosaic virus. The target gene can be introduced into the susceptible variety by transgenic method for resistance improvement, so as to improve the resistance to soybean mosaic virus, which will help to increase the yield and quality of soybeans and ensure the soybean production in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Agarose gel electrophoresis of the PCR amplification product of GmCSD. The Marker is TAKARA's DNA marker 2kb ladder, GmCSD is the cDNA amplification result, and Control is the negative control.

[0019] Figure 2 Schematic diagram of the domain structure contained in the GmCSD gene, which was predicted and drawn using InterPro from EMBL-EBI. Figure 3Schematic diagram of cloning and corresponding vector construction of GmCSD gene silencing fragment (V2-Sequence) and overexpression fragment (V4-Sequence). The marker is TAKARA's DNA marker 2kb ladder, and pBPMV-IA-R1M, pBPMV-V2-GmCSD and pBPMV-V4-GmCSD all contain the BPMV genome. Figure 4 The propagation and detection of diseased leaves of the susceptible variety Nannong 1138-2 after inoculation with two vectors. The left picture shows the disease phenotype of BPMV after silencing or overexpressing the GmCSD gene, and the leaves have obvious mosaic symptoms, indicating that the two vectors have normal infection ability. The right picture shows the PCR detection of the two vector sequences in the diseased leaves, and the marker is the TAKARA DNA marker 2kb ladder.

[0020] Figure 5 qRT-PCR was used to detect the silencing efficiency and overexpression efficiency of the target gene GmCSD after Qihuang No. 1 was inoculated with the silencing vector and Nannong 1138-2 was inoculated with the overexpression vector. The error bars are the standard errors of three biological replicates. Compared with the control, the expression level of GmCSD in the plants inoculated with the silencing vector was significantly reduced, and the expression level of GmCSD in the plants inoculated with the overexpression vector was significantly increased.

[0021] Figure 6 Phenotypes of plants that have silenced or overexpressed the GmCSD gene (and their respective controls) after being inoculated with the soybean mosaic virus SC3 strain two weeks later. The lower row shows the partially enlarged leaf phenotypes. Compared with the control group, the Qihuang No. 1 plant with silenced GmCSD showed leaf curling and mild mosaic symptoms. The mosaic symptoms of the Nannong 1138-2 plant overexpressing GmCSD were milder.

[0022] Figure 7 RT-PCR was used to detect the accumulation of viral CP genes in plants that silenced or overexpressed the GmCSD gene and were inoculated with SC3 for two weeks. The reference gene was the soybean endogenous gene Tubulin. Among them, the viral CP gene could be detected in the upper leaves of the plants with silenced GmCSD in Qihuang No. 1 after inoculation with SC3, and the content was slightly higher than that of the control; while the viral content in the plants with overexpressed GmCSD in Nannong 1138-2 was lower than that of the control.

[0023] Figure 8DAS-ELISA was used to detect the accumulation of viral CP protein in plants with silenced or overexpressed GmCSD gene. The ordinate is the absorbance of the sample at a wavelength of 405nm. The error bar is the standard error of three biological replicates. From the average absorbance value, it can be seen that after inoculation with soybean mosaic virus, the accumulation of viral CP protein in Qihuang No. 1 GmCSD-silenced plants was higher than that in the control, while the accumulation of viral CP protein in Nannong 1138-2 GmCSD-overexpressing plants was lower than that in the control. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0025] Example 1: Acquisition of GmCSD gene

[0026] 1. RNA extraction:

[0027] Sow soybean variety Qihuang No. 1, collect tender leaves after emergence for total RNA extraction. First, put 0.1g of the above leaves into a centrifuge tube, add liquid nitrogen and grind quickly, then add 1ml Trizol (Invitrogen) and mix well, then stand at room temperature for 5min; add 200μl of chloroform and shake for 15sec, stand at room temperature for 2min; centrifuge at 4℃, 12000rpm for 10min, take 400μl of supernatant and add it to 400μl of isopropanol and mix well; centrifuge at 4℃, 12000rpm for 10min, discard the supernatant, add 1ml 75% ethanol and vortex for 1min; centrifuge at 4℃, 8000rpm for 5min, discard the supernatant, and place in a fume hood to dry; finally, add 50μl of ddH2O for standby.

[0028] 2. Synthesis of the first-strand cDNA:

[0029] The synthesis of the first-strand cDNA was performed using the PrimeScript II RTase (TAKARA) kit. First, 1 μl of the above RNA was added to a PCR tube, 1 μl of Oligo dT Primer and 1 μl of dNTP Mixture were added, and ddH2O was added to 10 μl. After mixing well, the mixture was incubated at 65°C for 5 min, and then quickly cooled on ice. 4 μl of 5× PrimeScript II Buffer, 0.5 μl of RNase Inhibitor, 1 μl of PrimeScript II RTase, and 4.5 μl of ddH2O were added to the above mixture, and the mixture was incubated at 42°C for 60 min.

[0030] 3. PCR amplification and gene cloning:

[0031] First, the candidate gene reference sequence was queried according to the phytozome database (http: / / phytozome.jgi.doe.gov / pz / portal.html) and the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and PCR primers were designed using SnapGene software for cloning of candidate genes. The primer sequences are as follows: upstream primer F1: 5'-ATGGTGAAGGCTGTGGCCGTTCTTG-3'; downstream primer R1: 5'-GTCAGTAA TAGTGAAGCTGAC-3'. The above cDNA was used as a template and PrimerSTAR-Max-DNA-Polymera se (TAKARA) was used for PCR reaction. The specific experimental steps are as follows: add 1 μl of upstream and downstream primers, 1 μl of template, 25 μl of PrimerSTAR-Max, and add ddH2O to 50 μl. The final PCR product was electrophoresed on a 1% agarose gel at 200 V for 10 min, then stained with EB dye and observed and photographed under ultraviolet light. The results are as follows: Figure 1 . The amplified product was purified and recovered using a gel recovery kit (Novozymes). The specific experimental steps were carried out according to the instructions. The purified fragment was A-added using an A-addition reaction kit (TAKARA) and then ligated to the T-Vector pMD19 (Simple) (TAKARA) vector. The ligation product was transformed into the Escherichia coli strain DH5α (Conway Century) by the heat shock method and cultured overnight on LB solid medium containing ampicillin. Single colonies were picked and detected using M13 primers, and single colonies containing the target insert were sent to the company (Universal Biotechnology) for sequencing. The sequenced sequence was compared with the reference gene sequence and found to be consistent with it. The result is shown in SEQ ID NO.1. The predicted amino acid sequence is shown in SEQID NO.2. The amino acid sequence was submitted to the InterPro tool of EMBL-EBI (www.ebi.ac.uk / interpro) for protein domain prediction. The results showed that the protein encoded by the GmCSD gene is a protein containing a copper / zinc binding domain domain, see Figure 2 .

[0032] Example 2: Demonstration of the disease resistance function of the GmCSD gene

[0033] 1. Construction of GmCSD silencing vector:

[0034] The specific silencing fragment of GmCSD was designed using the NCBI database combined with SnapGene software. The sequence is shown in SEQ ID NO.3. The vector construction schematic diagram is shown in Figure 3As shown. Cloning primers were designed to insert the fragment into the silencing vector pBPMV-IA-V2 in a reverse complementary manner. The primer sequences are as follows: upstream primer F2: 5'-GAATCCTCTGCATGAGGATCCGTCAGTAATAGTGAAGCTGAC-3'; downstream primer R2: 5'-CTCT CGAGGCCTGGAGTCGACATGGTGAAGGCTGTGGCC-3'. Using the vector pMD19-T-GmCSD constructed in Example 1 as a template, the silencing fragment was amplified, and the product size was 300bp. The amplified product was purified using a gel recovery kit (Novagen) for later use. The BPMV silencing empty vector pBPMV-IA-V2 was digested with restriction enzymes BamH I and Sal I at 37°C for 3h, and purified and recovered. The homologous recombination kit (Novozyme) was used to construct the vector. The system was as follows: 1 μl of the vector after restriction digestion, 2 μl of PCR purified product, 2 μl of 5×CE II buffer, 1 μl of Exnase II, and 10 μl of ddH2O were added to the PCR tube. The reaction conditions were 37°C, 60 min, and placed on ice for 5 min. After transformation of E. coli competent DH5α, a single clone was picked and sent to the company for sequencing after PCR detection with pBPMV-IA-V2 sequencing primer. The sequencing primer sequences are as follows: BPMV-R2-C2F: 5'-TGACATTCTCCTGGGAATTTCCC-3'; BPMV-R2-C2R: 5'-CACACTTCACAC ATCATTACGAC-3'. After sequence alignment, the positive monoclonal bacterial solution was selected for large shaking, and the extracted plasmid was the silencing vector pBPMV-V2-GmCSD.

[0035] 2. Construction of GmCSD overexpression vector:

[0036] SnapGene software was used to design primers for amplifying the CDS sequence of the GmCSD gene. The schematic diagram of vector construction is shown in the figure below. Figure 3As shown. The primer sequences are as follows: upstream primer F3: 5'-ACACCAAAGGGAAGCCTCGAGATGGT GAAGGCTGTGGC-3'; downstream primer R3: 5'-TTGCTTAGCTGGGGCCCCGGGTCCTTGCAGACCA ATGATACCAC-3'. The vector pMD19-T-GmCSD constructed in Example 1 was used as a template to amplify the GmCSD gene. The amplified product was purified using a gel recovery kit (Novagen) for later use. The BPMV overexpression vector pBPMV-IA-V4 (Zhang CQ, Bradshaw JD, Whitham SA, Hill JH. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol 2010, 153 (1): 52-65.) was digested with restriction enzymes Xho I and Sma I at 37°C for 3h and purified and recovered. The CDS sequence of the GmCSD gene was connected to the pBPMV-IA-V4 digested vector using a homologous recombination kit (Novozyme), and the reaction system and conditions were the same as above. After transforming the competent E. coli DH5α, a single clone was picked and sent to the company for sequencing after PCR detection with pBPMV-IA-V4 sequencing primers. The sequencing primer sequences are as follows: BPMV-R2-C1F: 5'-CTACTAA AGCACAATTTGTTAGTGG-3', BPMV-R2-C1R: 5'-GCAGAATGGTTCCTCCAACTG-3'. After sequence alignment, the positive monoclonal bacterial solution was selected for large shaking, and the extracted plasmid was the overexpression vector pBPMV-V4-GmCSD.

[0037] 2. Silencing of the target gene GmCSD in Qihuang No. 1 and overexpression of GmCSD in Nannong 1138-2:

[0038] After the concentration of the two plasmids pBPMV-V2-GmCSD and pBPMV-IA-R1M was concentrated to 1μg / ml, they were mixed at a ratio of 1:1; similarly, after the concentration of the two plasmids pBPMV-V4-GmCSD and pBPMV-IA-R1M was concentrated to 1μg / ml, they were mixed at a ratio of 1:1. Both were inoculated in the true leaf stage of Nannong 1138-2 by in vitro friction method. About 10 days later, when the disease occurred, the two diseased leaves were collected and then inoculated into Nannong 1138-2 for virus propagation. Figure 4It can be seen that mosaic symptoms appeared after inoculation, indicating that the constructed silencing vector and overexpression vector have normal infection ability. The RNA of the diseased leaves was extracted and tested by PCR, and sent to Universal Company for sequencing. The diseased leaves with correct sequencing alignment were stored at -80℃. The diseased leaves inoculated with the combination of pBPMV-IA-R1M+pBPMV-IA-V2 and pBPMV-IA-R1M+pBPMV-IA-V4 in the early stage were used as controls, and the diseased leaves of the silencing vector of the combination of pBPMV-IA-R1M+pBPMV-V2-GmCSD and the diseased leaves of the overexpression vector of pBPMV-IA-R1M+pBPMV-V4-GmCSD were frictionally inoculated in Qihuang No. 1 and Nannong 1138-2, respectively. After the disease, samples were first taken to detect the silencing efficiency and overexpression efficiency of the target gene, and the soybean mosaic virus SC3 strain was inoculated on the first pair of new compound leaves.

[0039] 3. qRT-PCR detection of silencing or overexpression efficiency of GmCSD gene in Qihuang No. 1 and Nannong 1138-2:

[0040] About 10 days after friction inoculation, RNA was extracted from the leaves of the above-mentioned diseased plants and reverse transcribed into cDNA (see Example 1 for steps). The soybean internal reference gene Tubulin was selected for qRT-PCR, and the primer sequences were as follows: qPCR-GmCSD-F: 5'-GCCCCTGAGGATGAGAATCG-3'; qPCR-GmCSD-R: 5'-CACAAGCTACT CTGCCACCA-3'; Tubulin-F: 5'-GGAGTTCACAGAGGCAGAG-3'; Tubulin-R: 5'-CACTTACGCATCACATAGCA-3'. The results are as follows Figure 5 As shown, 10 days after Qihuang No. 1 was inoculated with pBPMV-V2-GmCSD, the expression level of GmCSD gene was significantly lower than that of the plants inoculated with empty vector, while the gene expression level of Nannong 1138-2 after inoculation with pBPMV-V4-GmCSD was significantly higher than that of the control, indicating that the target gene GmCSD was effectively silenced or overexpressed in both.

[0041] 4. Phenotypic identification of Qihuang No. 1 and Nannong 1138-2 plants with GmCSD silenced and overexpressed respectively after inoculation with SC3 strain:

[0042] Two weeks after inoculation with soybean mosaic virus SC3 strain, the phenotypes of the two materials were identified. Figure 6As shown, the Qihuang 1 plant (pBPMV-V0) without silencing the GmCSD gene was resistant to soybean mosaic virus, with healthy new leaves and no obvious mosaic symptoms; while the Qihuang 1 plant with silencing the GmCSD gene had curled leaves and mild mosaic symptoms; in addition, overexpression of the GmCSD gene in Nannong 1138-2 could alleviate the mosaic symptoms compared with the control. The above results indicate that the GmCSD gene positively regulates the plant's resistance to the SC3 strain of soybean mosaic virus.

[0043] 5. RT-PCR detection of soybean mosaic virus accumulation in the upper leaves of plants with silenced and overexpressed GmCSD in Qihuang No. 1 and Nannong 1138-2, respectively:

[0044] The virus content in the upper leaves of the two plants inoculated with SC3 was detected. First, the RNA of the sample was extracted and cDNA was synthesized (see Example 1 for the steps). Then the initial concentration of the sample cDNA was adjusted to make it consistent. Each cDNA was used as a template and PCR amplification was performed with two pairs of primers, that is, the soybean mosaic virus CP gene and the soybean endogenous Tubulin gene were amplified respectively. The primer sequences are as follows: SMV-CP-F: 5'-AGCCCGCTATGCTTTTG ACT-3'; SMV-CP-R: 5'-CAAACAACTTGTTGTTAACTCCC-3'. Tubulin primers are as described above. PCR amplification uses Ex-taq enzyme (TAKARA), and the specific amplification method is carried out according to the instructions. The amplified product was electrophoresed in a 1.5% agarose gel, stained with EB and photographed under ultraviolet light. The results are as follows. Figure 7 As shown, after silencing the GmCSD gene and re-inoculating SC3 in Qihuang No. 1, the CP gene of the virus could be detected in its upper leaves, while the presence of the virus was basically undetectable in the control group; after overexpressing the GmCSD gene in Nannong 1138-2, the virus content in its upper leaves was significantly lower than that in the control group.

[0045] 6. DAS-ELISA detection of soybean mosaic virus accumulation in the upper leaves of plants with silenced and overexpressed GmCSD in Qihuang No. 1 and Nannong 1138-2, respectively:

[0046] In order to further confirm the changes in resistance of plants to the SC3 strain after silencing or overexpressing the GmCSD gene, the upper leaves of different plants inoculated with SC3 for two weeks were selected for ELISA detection of soybean mosaic virus CP protein. Three biological replicates were taken for each treatment. The detection steps are as follows: (1) The collected leaves were ground into liquid nitrogen and 10 times the weight of the leaves were added with extraction buffer to mix; (2) The coated antibody was diluted with the coating antibody buffer at a dilution ratio of 1:800, and 100 μl of the diluted antibody was added to each well of the ELISA plate. The plate was placed at room temperature (21-24°C) for 4 hours, and then the liquid in the plate was shaken off, and each well was filled with PBST buffer and quickly poured out. Repeat 6 times; (3) 100 μl of the sample to be tested was added to each well, and the plate was placed in a moisturizing box at room temperature for 2.5 hours, and then the liquid in the plate was shaken off, and each well was filled with PBST buffer and quickly poured out. Repeat 6 to 8 times; (4) Dilute the enzyme-labeled antibody with the enzyme-labeled antibody buffer at a dilution ratio of 1:800, add 100 μl of the diluted enzyme-labeled antibody to each well of the ELISA plate, leave it at room temperature for 2.5 hours, then shake off the liquid in the plate, fill each well with PBST buffer and quickly pour it out. Repeat 8 times; (5) Add 100 μl of 1 mg / ml ρNPP solution to each well of the ELISA plate, color develop for 30 minutes at room temperature in the dark, and finally add 50 μl 3M NaOH to terminate the reaction; (6) Measure the optical density (OD) at a wavelength of 405 nm on the ELISA instrument, and calculate the average value of different samples with the same treatment. The ELISA results of CP protein accumulation are shown in Figure 8 The accumulation of CP protein in Qihuang No. 1 plants after GmCSD gene silencing was significantly higher than that in the control, while the accumulation of CP protein in Nannong 1138-2 plants with GmCSD gene overexpression was slightly lower than that in the control, indicating that the soybean GmCSD gene is involved in the disease resistance response to the SC3 strain of soybean mosaic virus.

Claims

1. Application of overexpressing the gene GmCSD whose cDNA sequence is shown in SEQ ID NO.1 in improving the resistance of soybean to soybean mosaic virus SC3 strain.

2. Application of the gene GmCSD having a cDNA sequence as shown in SEQ ID NO.1 in breeding soybean varieties with resistance to soybean mosaic virus SC3 strain.

3. Use of a recombinant expression vector overexpressing the gene GmCSD of claim 1 in improving the resistance of soybean to soybean mosaic virus SC3 strain.

4. Use of a recombinant expression vector overexpressing the gene GmCSD of claim 1 in breeding soybean varieties resistant to soybean mosaic virus SC3 strain.

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