CN113493794B - Gene GmGRX4 with resistance to soybean mosaic virus and application thereof - Google Patents

Abstract

The invention discloses a gene GmGRX4 with resistance to soybean mosaic virus and application thereof. The cDNA nucleotide sequence of the gene GmGRX4 is shown as SEQ ID NO.1, and the encoded protein sequence is shown as SEQ ID NO. 2. The GmGRX4 gene in the disease-resistant variety Kefeng No.1 is silenced by utilizing VIGS, and then an SC3 strain is inoculated, so that the disease-resistant variety Kefeng No.1 shows the symptoms of flowers and leaves. And after the GmGRX4 gene is overexpressed in the susceptible variety Nannong 1138-2, the strain SC3 is inoculated, and the Nannong 1138-2 is resistant to the soybean mosaic virus. The expression level of the GmGRX4 gene is reduced, so that the disease resistance of the resistant variety can be changed, and the expression level of the GmGRX4 gene is improved, so that the resistant variety can obtain the resistance. The resistance of the recipient variety to soybean mosaic virus can be improved by introducing the GmGRX4 gene into the susceptible variety by using a transgenic technology.

Description

Gene GmGRX4 with resistance to soybean mosaic virus and application thereof

Technical Field

The invention belongs to the technical field of biological genetic engineering, and relates to a gene GmGRX4 with resistance to a soybean mosaic virus SC3 strain and application thereof.

Background

The soybean mosaic virus is a soybean disease commonly existing in the global soybean production area, has wide regional distribution, large hazard area and strong destructiveness. In general, SMV causes yield loss of 15-35%, and important occurrence years in some areas can cause yield loss of more than 70%, even absolute yield. The SC3 strain is a popular strain commonly existing in a plurality of soybean producing areas in China. In view of the fact that no medicament for effectively killing the virus exists at present, breeding and planting disease-resistant varieties are the most economical, effective and environment-friendly control methods for controlling the disease. The gene with resistance to soybean mosaic virus is the basis for breeding and planting disease-resistant varieties.

One important reason for the damage of soybean mosaic virus to a host is that it affects the photosynthetic system of the host leaf cells, resulting in a decrease in chlorophyll content in soybean leaves, affecting photosynthetic capacity, and that chloroplasts promote ROS production during infection, with excessive ROS inducing macromolecular damage and altering normal signaling. The GRXs protein family is a disulfide oxidoreductase that catalyzes the disulfide reversible reduction of substrate proteins by utilizing the reducing power of Glutathione (GSH) and plays a role in scavenging cellular ROS and regulating redox homeostasis within these cellular organelles. In recent years, GRXs proteins have been increasingly studied for their function in regulating responses of plants to biotic and abiotic stresses, and GRX has been involved in many cellular functions such as DNA synthesis, signaling and defense against oxidative stress by changing the redox state of target proteins, but reports of regulating resistance to pathogens in plants are rare. Therefore, the research of GRX genes has important significance for excavating novel disease resistance genes and further exploring the disease resistance mechanism thereof.

Disclosure of Invention

The invention provides a gene GmGRX4 (Glyma.18g240400) with resistance to soybean mosaic virus SC3 strain, and the nucleotide sequence of the gene cDNA is shown as SEQ ID NO. 1.

The protein coded by the gene GmGRX4 with resistance to the soybean mosaic virus SC3 strain has an amino acid sequence shown in SEQ ID NO. 2.

The invention aims to provide application of the gene in participating in soybean mosaic virus resistant SC3 strain, and specifically relates to the application of the gene in changing disease resistance of disease resistant variety Kefeng No.1 into infectious disease after silencing GmGRX4 gene by utilizing a VIGS technology. After the GmGRX4 gene is over-expressed, the resistance of the susceptible variety Nannong 1138-2 to the soybean mosaic virus SC3 strain can be obtained.

The invention has the beneficial effects that:

the virus-induced gene silencing technology using bean pod mottle virus as a vector is used for silencing GmGRX4 gene in disease-resistant soybean varieties, and the gene is overexpressed in the disease-resistant varieties, so that the comprehensive indication that the gene has resistance to soybean mosaic virus SC3 strain is provided. The transgenic technology is utilized to introduce GmGRX4 gene into the infected variety to improve the resistance of the acceptor variety, thereby ensuring the yield and quality of soybean and improving the national supply rate of soybean. And since the gene is derived from soybean, the safety of transgenic soybean is further improved.

Drawings

FIG. 1 agarose gel electrophoresis of PCR amplified products of GmGRX4. Wherein, marker is DNA Marker 5kb ladder,GmGRX4 of TAKARA, cDNA amplification result, control is blank Control and negative Control.

FIG. 2 GmGRX4 contains a specific domain. The results were predicted by the UniProt website and plotted as a graph.

FIG. 3 phenotype of Nannong 1138-2 vaccinated with BPMV silencing vector. The upper panel shows pBPMV-IA-V2, pBPMV-V-GmGRX4 from left to right after pot inoculation. The lower drawing is an enlarged view of the corresponding blade.

FIG. 4 phenotype of Nannong 1138-2 vaccinated with BPMV overexpression vector. The upper panel shows pBPMV-IA-V4, pBPMV-OE-GmGRX4 from left to right after pot inoculation. The lower drawing is an enlarged view of the corresponding blade.

FIG. 5 silencing efficiency and overexpression efficiency of the target gene GmGRX4 after 10 days of inoculation of silencing vector pBPMV-V-GmGRX4 and overexpression vector pBPMV-OE-GmGRX4 with RT-PCR detection. Wherein Marker is DNA Marker 5kb ladder of TAKARA, 1,6: inoculating a blank control of phosphate buffer; 2,7 empty pBPMV-IA-V control; 3-5,8-10:pBPMV-V-GmGRX4 plants; 1-5, detecting soybean internal reference gene Tubulin; 6-10: detecting the expression quantity of GmGRX 4; the upper row is the detection result of the over-expression vector, and the lower row is the detection result of the silencing vector.

FIG. 6 inoculation of silencing vector pBPMV-V-GmGRX4 and overexpression vector pBPMV-OE-GmGRX4 with qRT-PCR detection. After 10 days, the silencing efficiency and the overexpression efficiency of the target gene GmGRX4 are improved. Error bars are standard error of three biological replicates; the left graph shows that the expression level of GmGRX4 is remarkably reduced compared with pBPMV-IA-V2 after GmGRX4 silencing vector inoculation. The right graph shows that the expression level of GmGRX4 is obviously improved compared with pBPMV-IA-V4 after the GmGRX4 over-expression vector is inoculated.

FIG. 7 phenotype of the silenced plants after three weeks with the control plants after the kefeng No.1 silencing GmGRX4 by inoculating the soybean mosaic virus SC3 line. The upper row is a potted plant drawing, and the lower row is a corresponding blade enlarged drawing. The silenced plants had slight floral leaf symptoms compared to the control.

FIG. 8 phenotype of the overexpressed plants and control plants three weeks after the Nannong 1138-2 overexpressed GmGRX4 and inoculated with the soybean mosaic virus SC3 strain. The upper row is a potted plant drawing, and the lower row is a corresponding blade enlarged drawing. In contrast to the control, the overexpressed plants were normal, with no obvious floral leaf symptoms.

FIG. 9 DAS-ELISA detection of SMV-CP protein accumulation in silent plants and over-expressed plants. The ordinate is the absorbance of the ELISA plate at 405 nm; error bars are standard error of three biological replicates; from the average absorbance, it can be seen that after inoculation of the soybean mosaic virus SC3 strain, the accumulation of SMV viral CP protein on the Nannong 1138-2 overexpressed plants was much lower than that of the control. The accumulation of SMV viral CP protein on the kefeng No.1 silent plant was much higher than that of the control.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1: acquisition of GmGRX4 Gene

Extraction of RNA:

(1) Sowing and sprouting young leaves of DaLeguminosae Feng No.1, putting about 0.1g of the leaves into a centrifuge tube, adding liquid nitrogen, rapidly grinding, adding 1ml of Trizol (Yingwei Jieshiki company), uniformly mixing, and standing for 5min;

(2) Adding 0.2ml of chloroform, shaking for 15sec, and standing for 2min; centrifuge at 12000rpm for 10min.

(3) Mu.l of the supernatant was pipetted into 400. Mu.l of isopropanol and mixed well; centrifuging for 10min.

(4) The supernatant was discarded, 1ml of 70% ethanol was added and vortexed for 1min; centrifuging for 5min.

(5) Discarding the supernatant, and airing in a fume hood; mu.l of ddH2O was added for later use.

2. First strand cDNA Synthesis:

first strand cDNA was synthesized using PrimeScript II RTase (TAKARA Co.).

(1) 1. Mu.l (2. Mu.g) of the RNA was aspirated and added to a PCR tube, and the following reaction system was prepared:

(2) After gentle mixing, reverse transcription reaction was carried out, and after 5min incubation at 65℃the mixture was cooled rapidly on ice.

(3) The following components were added to the above mixture:

(4) Mixing, and maintaining at 42deg.C for 60 min. Preserving at-20 ℃ for standby.

Amplification by PCR and Gene cloning

(1) Specific primers were designed using Primer5.0 software based on the soybean whole gene database (http:// phytozome. Jgi. Doe. Gov/pz/portal. Html):

upstream primer F1:5'-ATGTCGTGGTGTTACTGCGCTAA-3' the number of the individual pieces of the plastic,

downstream primer R1:5'-TCCATAGCTTCAGGACAACATTGCCT-3'.

PCR amplification is carried out by taking the cDNA as a template, and the amplification enzyme is PrimerSTAR-Max-DNA-Polymerase of TAKARA, and the PCR reaction system is as follows:

the PCR amplified products were electrophoresed using 1% agarose gel at 200V for 10min, then stained with EB dye and photographed under ultraviolet light observation, and the results are shown in FIG. 1. The amplified product was purified using an AxyPrep DNA gel recovery kit for use, and the purification process was all performed according to the instructions.

(2) The purified fragment was ligated to TAKARA T-Vector pMD19 (Simple) Vector after addition of A using TAKARA A-addition reaction kit. The ligation product was transformed into E.coli strain DH5a (century old) by the heat shock method and cultured overnight on LB solid medium containing ampicillin. Single colonies were picked and single colonies containing the insert of interest (common in Chuzhou) were detected using the M13 primer and sequenced. The sequencing results are compared in a soybean database, and the sequence is found to be consistent with the target sequence. The results show that the protein encoded by the GmGRX4 gene contains a Glutaredoxin protein, as shown in FIG. 2.

Example 2: proof of disease resistance function of GmGRX4 Gene

Construction of silencing vector for GmGRX4

Specific silent fragment (as shown in SEQ ID NO. 3) primers were designed using Primer5.0 software: upstream F2:5'-GCGGAATCCTCTGCATGAGGATCCACACTGTGGAAAGTCTTTAGT-3'. The downstream primer R2:5'-GATCTCTCGAGGCCTGGAGTCGACATGTCGTGGTGTTACTGCGC-3' was used to amplify the silencing fragment using the constructed vector pMD19-T-GmGRX4 (constructed in example 1) as a template. The amplified product was purified using an AxyPrep DNA gel recovery kit for use.

The BPMV silencing vector pBPMV-IA-V2 is digested with restriction enzymes BamH I and Sal I at 37℃for 3 hours, and purified. The silent fragment was ligated to the digested pBPMV-IA-V2 vector by homologous recombination as follows:

the reaction conditions were 37℃and 1hour, and the mixture was left on ice for 5 minutes. Coli competent DH 5. Alpha. Was transformed. The monoclonal was picked, PCR-detected with pBPMV-IA-V2 universal primer and sequenced by the company. The general primers were as follows:

BPMV-R2-C2F：5'-TGACATTCTCCTGGGAATTTCCC-3'

BPMV-R2-C2R：5'-CACACTTCACACATCATTACGAC-3'

after the sequencing result is correctly compared with the target sequence, the positive monoclonal bacterial liquid is further selected for culturing and extracting plasmids, and the extracted plasmids are recombinant silencing vectors and named as pBPMV-V-GmGRX4.

Construction of an overexpression vector of GmGRX4

Primers were designed to amplify the CDS sequence of the GmGRX4 gene using primer5.0 software: the GmGRX4 gene was amplified using the constructed vector pMD19-T-GmGRX4 (constructed in example 1) as a template, and the upstream primer F3:5'-TAGAGACACCAAAGGGAAGCCTCGAGATGTCGTGGTGTTACTGCG-3' and the downstream primer R3: 5'-AGCAATTGCTTAGCTGGGGCCCCGGGGGACAACATTGCCTTCTCCAA-3'. The amplified product was purified using an AxyPrep DNA gel recovery kit for use.

The BPMV overexpression vector pBPMV-IA-V4 is digested with restriction enzymes Xho I and Sma I at 37 ℃ for 3 hours, and purified and recovered. The CDS sequence of the GmGRX4 gene is connected to the vector after the pBPMV-IA-V4 is digested by utilizing a homologous recombination method, and the reaction system is the same as that described above.

The reaction conditions were 37℃and 1hour, and the mixture was left on ice for 5 minutes. Coli competent DH 5. Alpha. Was transformed. The monoclonal was picked, PCR-detected with pBPMV-IA-V4 universal primer and sequenced by the company. The general primers were as follows:

BPMV-R2-C1F：5'-CTACTAAAGCACAATTTGTTAGTGG-3'

BPMV-R2-C1R：5'-GCAGAATGGTTCCTCCAACTG-3'

and after the sequencing result is correctly compared with the target sequence, selecting positive monoclonal bacterial liquid to further culture and extract plasmids, wherein the extracted plasmids are recombinant over-expression vectors, and the plasmids are named as pBPMV-OE-GmGRX4.

3. Silencing target gene in Kefeng No.1 by using silencing vector and over-expressing target gene in Nannong 1138-2 by using over-expression vector

Two plasmids pBPMV-V-GmGRX4, pBPMV-IA-R1M (plasmid concentration up to 1 ug/ml) were combined at a ratio of 1:1, two plasmids pBPMV-OE-GmGRX4, pBPMV-IA-R1M (plasmid concentration up to 1 ug/ml) were mixed according to a ratio of 1:1, and respectively generating silent disease leaves and over-expression disease leaves of the gene by adopting in vitro friction inoculation of Nannong 1138-2 in a true leaf period. Approximately ten days after inoculation, diseased leaves were taken and seen in fig. 3, 4: flowers She Zhengzhuang appear 10 days after the Nannong 1138-2 is inoculated with the mixed vector, which indicates that the constructed silencing vector and the over-expression vector have normal infection capability. RNA is extracted and detected by PCR, and the PCR product is sent to Chuzhou general company for sequencing. The correctly diseased leaves were sequenced and compared and stored at-80 ℃.

The disease-causing leaf containing the recombinant silencing vector is inoculated with Kefeng No.1, the disease-causing leaf containing the recombinant overexpression vector is inoculated with Nannong 1138-2, and the leaf containing pBPMV-IA-R1M+pBPMV-IA-V2, pBPMV-IA-R1M+pBPMV-IA-V4 is respectively inoculated as corresponding control, and simultaneously, the phosphate buffer solution is inoculated as blank control. Samples were taken after onset for detection of silencing efficiency and overexpression efficiency of the target gene. And inoculating a virus sample of the soybean mosaic virus SC3 strain on the first pair of the grown multiple leaves.

RT-PCR and q RT-PCR for detecting expression level of GmGRX4

RNA was extracted from the obtained transgenic soybean leaves, and reverse transcribed into cDNA (see example 1 for steps). The following primers were used for RT-PCR and qPCR with the soybean housekeeping gene Tubulin as an internal reference. The results are shown in fig. 5 and 6, and 10 days after the kefeng No.1 is inoculated with the silencing vector pBPMV-V-GmGRX4, the expression level of GmGRX4 is obviously lower than that of the inoculated empty vector pBPMV-IA-V2, so that the target gene GmGRX4 is effectively silenced. 10 days after the Nannong 1138-2 is inoculated with the over-expression vector pBPMV-OE-GmGRX4, the expression level of GmGRX4 is obviously higher than that of the inoculated empty pBPMV-IA-V4, which indicates that the target gene GmGRX4 is effectively over-expressed.

q PCR-GmGRX4-F：5'-GTTGACCCCAACACGGATCT-3'

q PCR-GmGRX4-R：5'-AGAGCAGCATCATCGTTCCA-3'

Tubulin-F：5'-GGAGTTCACAGAGGCAGAG-3'

Tubulin-R：5'-CACTTACGCATCACATAGCA-3'

5. Phenotype identification after inoculation of SC3 strain to silencing plant Kefeng No.1 and overexpressing plant Nannong 1132

Three weeks after inoculation of the soybean mosaic virus SC3 strain, phenotype identification was performed on silencing plant kefeng No.1 and overexpressing plant nannong 1138-2 and the light, respectively, and the results are shown in fig. 7: kefeng No.1 without silencing Gm GRX is resistant to soybean mosaic virus, and the new-born leaves are healthy and asymptomatic; kefeng No.1, silencing Gm GRX, presents symptoms of mild flowers and leaves. The results are shown in FIG. 8: the Nannong 1138-2 plant over-expressing Gm GRX has resistance to mosaic virus, and the new-born leaf is healthy and asymptomatic; the Nannong 1138-2 plants without over-expression of Gm GRX showed typical flower and leaf symptoms.

DAS-ELISA detection of SMV-CP protein accumulation in silenced plants and overexpressed plants

In order to further confirm the weakening or strengthening of the resistance of the GmGRX4 gene to the soybean mosaic virus SC3 strain in silent plants and over-expression plants, the ELISA detection of soybean mosaic virus CP protein is carried out by experimentally selecting upper leaves of different plants after 3 weeks of inoculation of the SC3 virus sample. 3 biological replicates were taken for each treatment. The detection steps are as follows:

(1) Adding liquid nitrogen into the collected blades for grinding, and adding extraction buffer solution with the weight 10 times of the weight of the blades for uniformly mixing;

(2) According to 1: the coated antibody is diluted by the coated antibody buffer solution according to the dilution ratio of 800, 100 mu l of the diluted antibody is added into each hole of the ELISA plate, the plate is placed for 4 hours at room temperature (21-24 ℃), then the liquid in the plate is thrown off, the PBST buffer solution is filled in each hole, and the plate is rapidly poured off. Repeating for 6 times;

(3) 100 μl of sample to be measured is added into each well, the mixture is placed in a humidity-preserving box at room temperature (21-24 ℃) for 2.5 hours, then the liquid in the plate is thrown away, the PBST buffer solution is filled into each well, and the mixture is rapidly poured out. Repeating for 6-8 times;

(4) According to 1: the enzyme-labeled antibody is diluted by enzyme-labeled antibody buffer solution according to the dilution ratio of 800, 100 mu l of the diluted enzyme-labeled antibody is added into each hole of the enzyme-labeled plate, the plate is placed for 2.5 hours at room temperature (21-24 ℃), then the liquid in the plate is thrown away, the PBST buffer solution is filled in each hole, and the plate is rapidly poured out. Repeating for 8 times;

(5) To the wells of each ELISA plate, 100. Mu.l of 1mg/ml ρNPP solution was added, and the reaction was stopped by adding 50. Mu.l of 3M NaOH, and developed for 30min at room temperature (21-24 ℃) in the dark.

(6) The Optical Density (OD) was measured at a wavelength of 405nm with a microplate reader and the average of the different samples from the same treatment was calculated. ELISA detection results are shown in FIG. 9. ELISA results of CP protein accumulation show that in Kefeng No.1 plants after GmGRX4 gene silencing, the CP protein accumulation is obviously higher than that of control, and in Nannong 1138-2 plants after GmGRX4 gene overexpression, the CP protein accumulation is obviously lower than that of control, so that the soybean GmGRX4 gene participates in resisting the disease of soybean mosaic virus, and the infection of the soybean mosaic virus can be effectively reduced after the excessive expression.

Sequence listing

<110> Nanjing agricultural university

<120> a Gene GmGRX4 having resistance to Soybean mosaic Virus and use thereof

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cgaccctctt ctcctcttgt tctgacccac aacctccact tccaacccaa actcaaacgc 180

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acctttcctc aagtctacat agaaggagag ttttttggtg gctgtgatat cactgttgat 480

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Claims (2)

1. Application of gene GmGRX4 shown in SEQ ID No.1 in improving resistance of soybean nan nong 1138-2 to soybean mosaic virus SC3 strain.
2. 2. Application of recombinant expression vector containing gene GmGRX4 shown in SEQ ID NO.1 in improving resistance of soybean nan nong 1138-2 to soybean mosaic virus SC3 strain.

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