CN117247964B - Application of E3 ubiquitin ligase gene GmPUB20 capable of regulating and controlling soybean mosaic virus resistance - Google Patents
Application of E3 ubiquitin ligase gene GmPUB20 capable of regulating and controlling soybean mosaic virus resistance Download PDFInfo
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Abstract
The invention discloses an application of an E3 ubiquitin ligase gene GmPUB20 capable of regulating and controlling soybean mosaic virus resistance. According to the invention, the soybean genetic transformation is carried out after the gene editing is carried out on the GmPUB20 sequence by utilizing the Crispr/Cas9 technology, then a soybean mosaic virus inoculation experiment is carried out on a stable genetic transgenic soybean offspring plant from which the GmPUB20 is knocked out, and the transgenic soybean plant shows remarkable disease resistance, so that the GmPUB20 is a gene for negatively regulating and controlling the soybean mosaic virus. The gene and the protein sequence thereof obtained by the invention can improve the resistance of soybean varieties to soybean mosaic virus by using a biotechnology means.
Description
Technical Field
The invention belongs to the technical field of biological genetic engineering, and relates to application of an E3 ubiquitin ligase gene GmPUB20 capable of regulating and controlling soybean mosaic virus resistance.
Background
Soybean is a major source of edible oil and protein in our country and even worldwide. Its production is compromised by a variety of diseases. Wherein soybean mosaic virus is one of the main diseases of soybean. Soybean mosaic virus is found in all soybean production areas nationwide; the soybean yield loss caused by the soybean mosaic virus can reach 15% -70%; meanwhile, soybean mosaic virus can also cause mottle of the seed coats of soybean seeds, so that the quality is reduced. As no medicament for effectively killing the virus exists at present, the cultivation of disease-resistant varieties is the most economical and effective method for controlling the disease.
Plants evolved in the long-term co-existence and fight against pathogens to form a variety of mechanisms to fight pathogen invasion or reduce the rate of pathogen transmission within plants. The disease resistance gene (R gene) can identify the pathogen, so that the most effective resistance character is endowed to the plant. However, few genes have been shown to be able to combat soybean mosaic virus.
And is regulated by various signal paths in the disease-resistant reaction process of plants, including positive regulation and negative regulation. In recent years, along with the development and popularization of gene editing technology, the improvement of disease resistance of crop varieties by knocking out disease-sensitive genes becomes a new breeding method for cultivating disease-resistant varieties. Among them, the Crispr/Cas9 gene editing technique is considered to be the most effective method for improving crop resistance and quality.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing the application of the GmPUB20 gene.
The aim of the invention can be achieved by the following technical scheme:
The invention provides a gene GmPUB20 for resisting soybean mosaic virus, and the nucleotide sequence of the gene cDNA is shown as SEQ ID NO. 1.
The protein encoded by the soybean mosaic virus resistant gene GmPUB20 has an amino acid sequence shown in SEQ ID NO.2 in a sequence table.
As a preferred aspect of the present invention, the expression of GmPUB20 gene is knocked out or suppressed to increase the resistance of the susceptible variety to soybean mosaic virus.
The application of the gene editing system of the GmPUB20 gene in the cultivation of soybean mosaic virus resistant seeds.
A method for constructing soybean with soybean mosaic virus resistance comprises transferring a gene editing system of GmPUB20 gene into a variety to be improved, thereby obtaining the variety with soybean mosaic virus resistance.
As a preferred embodiment of the present invention, the gene editing system of the GmPUB20 gene is transferred into the variety to be improved by using the soybean cotyledonary node transformation method.
The invention has the beneficial effects that:
Soybean mosaic virus resistant breeding is one of the important targets of soybean variety breeding today. The isolated GmPUB20 gene is identified to be edited by using the Crispr/Cas9 gene, and then the gene can be introduced into a variety infected with soybean mosaic virus by a transgenic method, so that the acceptor variety is improved, the resistance of the soybean variety to the soybean mosaic virus is improved, the yield and quality of soybean are ensured, and the supply rate of domestic soybean is improved.
Drawings
FIG. 1 agarose gel electrophoresis of PCR amplification products of GmPUB 20. Wherein, marker is TAKARA DNA MARKER 1kb ladder,GmPUB20 is cDNA amplification result.
FIG. 2 Wilimas82 is verified by sequencing after transgene knockout of GmPUB20 gene. GmPUB20 (Williams 82) shows the sequencing results of Williams 82, which are wild-type and not knocked out, gmPUB20#3-1 and GmPUB20#3-2 are the sequencing results of mutant lines after knocking out the target gene GmPUB 20. The results show that deletion of the fragment is caused by editing the Crispr/Cas9 gene, so that the target gene is mutated. The three peak diagrams are respectively the Williams 82 sequencing result of the wild type non-knocked-out GmPUB20 gene, the two later peak diagrams are peak diagrams formed after the GmPUB20 gene is knocked out, and the red frame is a knocked-out targeting sequence peak diagram.
FIG. 3 phenotype of transgenic recipient variety Williams 82 and transgenic lines after knockout of GmPUB20 Gene
FIG. 4 detection of the relative content of GmPUB20 gene in wild type Williams 82 leaves and progeny leaves of different transgenic lines after knocking out GmPUB20 gene.
FIG. 5 leaf phenotype after Williams82 inoculation with soybean mosaic virus SC73 weeks after knocking out GmPUB20 gene. Williams82 indicates wild type Williams82 leaves; gmPUB20#3-1 and GmPUB20#3-2 indicate progeny Williams82 leaves of different transgenic lines after knocking out the GmPUB20 gene.
FIG. 6 Western detection of soybean mosaic virus CP protein accumulation 3 weeks after Williams 82 inoculation of soybean mosaic virus with GmPUB20 gene knocked out. From the detection result, the accumulation amount of the CP protein of the leaf virus of the Williams 82 after the GmPUB20 gene knockout inoculated with the soybean mosaic virus is far lower than that of the leaf virus of the wild Williams 82.
FIG. 7 qRT-PCR detection of soybean mosaic virus RNA 2 weeks after Williams 82 inoculation of soybean mosaic virus after GmPUB20 gene knockout. From the detection results, the RNA content of the Williams 82 leaf viruses after the GmPUB20 gene knockout after the soybean mosaic virus inoculation is far lower than that of the wild type Williams 82.
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 GmPUB20 Gene
Extraction of RNA:
Sowing soybean variety Williams 82, and collecting tender leaves after emergence for total RNA extraction. Placing 0.1g of the above blade into a centrifuge tube, adding liquid nitrogen, rapidly grinding, adding 1ml of Trizol (Uyghur) and standing for 5min; adding 0.2ml of chloroform, shaking for 15sec, and standing for 2min; centrifuging at 12000rpm for 10min, sucking 400 μl of supernatant, adding into 400 μl of isopropanol, and mixing; centrifuging for 10min, discarding supernatant, adding 1ml 70% ethanol, and swirling for 1min; centrifuging for 5min, discarding supernatant, and air drying in a fume hood; mu.l of ddH 2 O was added for further use.
1. First strand cDNA Synthesis:
The first strand cDNA was synthesized using PRIMESCRIPT II RTASE (TAKARA Co.). Mu.l of the RNA was aspirated and added to a PCR tube, 1. Mu.l of Oligo dT Primer and dNTP mix were added, and ddH 2 O was added to 10. Mu.l. After the uniform mixing, the temperature is kept at 65 ℃ for 5min, and the mixture is rapidly cooled on ice. To the above mixture was added 5X PRIMESCRIPT II Buffer 4. Mu.l, RNase Inhibitor 0.5. Mu.l, PRIMESCRIPT II RTASE. Mu.l and ddH 2 O4.5. Mu.l; mixing, and maintaining at 42deg.C for 60 min.
Amplification by PCR and Gene cloning
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'-GATACATAGAAACAATCGAAATTA-3', downstream primer R1:5'-CTGTTTCTACCAATATGACCT-3'. PCR amplification was performed using the above cDNA as a template, and the amplification enzyme used was PRIMERSTAR-Max-DNA-Polymerase of TAKARA, which was specifically used all according to the specification. 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 AxyPrep DNA gel recovery kit for use and the purification procedure was all according to the instructions. 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 harboring the insert of interest (Huada gene) were detected for sequencing using M13 primers. The sequencing sequence is put into biological software BIOXM, and the largest open reading frame is searched, and the result is shown as SEQ ID NO.1. Every 3 bases are translated into one amino acid, and the obtained amino acid sequence is shown as SEQ ID NO.2.
Example 2: demonstration of disease resistance of GmPUB20 Gene
1. Construction of soybean variety Williams 82GmPUB20 gene editing vector
Screening possible targets for a Crispr/Cas9 gene editing system according to the gene sequence obtained in example 1, comprehensively considering the transformation efficiency and transformation cycle of soybean, to ensure that GmPUB20 is successfully edited, two target sites were selected to be PAM1: GAAATATTCTGGTGGCGACATGG; PAM2: GTCGTCGAGGCTGGTCAGCTCGG. The two pairs of primers required for synthesizing and constructing the knockout vector in Huada gene company are respectively as follows: the primer whose target site is PAM1 is f2_1:5'-GGGTTGAAATATTCTGGTGGCGACA-3' and r2_1:5'-AAACTGTCGCCACCAGA'; the primer whose target site is PAM2 is f2_2:5'-GGGTTGTCGTCGAGGCTGGTCAGCT-3' and R2_2:5'-AAACAGCTGACCAGCCTCGACGACA-3'. The primers were diluted to 10. Mu.M with ddH 2 O, and 1. Mu.l to 18. Mu.l of TE buffer were mixed. The above mixed product was heated to 98℃and then slowly cooled to room temperature to form a double-stranded sequence. Mu.l of the above product was pipetted and mixed with 1. Mu.l of BsaI cleaved pCBSG vector, and 1. Mu. l T4 ligase (NEB Co.), 1. Mu.l of 10x T4 buffer and 6. Mu.l of ddH 2 O were added and mixed uniformly, and then left at room temperature for 3 hours for ligation. The ligated vector was transformed and plasmid sequencing of single colonies was extracted by the method described in example 1. The primers used for sequencing were U6-SG20-gRNA-CX: GCAAGTGCGGTGACAAGACAAG.
Genetic transformation of GmPUB20 Gene editing vector
The correctly sequenced plasmid was transformed into the Agrobacterium EHA105 strain by freeze thawing. The soybean variety Williams 82 was then transformed by agrobacterium infection to mature soybean cotyledonary node technology. And (5) transplanting the successfully transformed induced buds into nutrient soil and collecting seeds of the nutrient soil. Genomic DNA of T0 generation plants was extracted using primer F3:5'-ATTCTGGTGGCGACATGGAG-3' and R3:5'-TCACTTTCCTTCCCCCAAGC-3' amplified genomic knockout target site sequences were sequenced by sequencing company as shown in FIG. 2. The sequencing results were compared with the peak pattern of the non-knocked-out wild-type Williams 82 sequence to confirm the GmPUB20 gene knocked-out plants. The above test shows that the knockout homozygous different strains are used for subsequent experiments after seed collection, and wild type and offspring homozygous knockout plants are respectively planted in a phytotron as shown in fig. 3 for subsequent experimental study.
3. Relative fluorescent quantitative experiments (qRT-PCR) of the expression level of GmPUB20 gene in wild type and transgenic plants
The qRT-PCR experiment in this study was performed with ChamQ Universal SYBR QPCR MASTER Mix reagent (Q711-02) from Vazyme. Seeds of homozygous lines (GmPUB #3-1 and GmPUB # 3-2) after the Williams 82GmPUB20 gene knockout and wild type Williams 82 were sown in the greenhouse respectively, and leaves were harvested for the experiment after the 1 st pair of true leaves was fully developed. The gene Tubulin (NM_ 001252709.2) expressed by soybean is taken as an internal reference gene, and a primer F for qRT-PCR is designed: 5'-GCTTTGCGGTTTCGGTTCT-3' and R4:5'-ACACTCGGCTTTGCTCCTACA-3'. The operation is described according to SYBR QPCR MASTER Mix, and the specific reaction system is as follows:
fluorescent quantitative PCR procedure (three-step method):
the dissolution curve was read, and the experimental results were subjected to expression level calculation using 2 -ΔΔCt (CT value comparison method), and the difference in expression was detected by t-test.
Indeed, the mRNA levels of the GmPUB20 mutant were significantly reduced compared to the wild type, indicating that GmPUB20 was genetically inactivated in the knockout mutant, see fig. 4.
Phenotypic changes after soybean mosaic Virus lines (isolates) were inoculated into GmPUB20 Gene knockout plants
Different soybean mosaic virus strains (isolates) are inoculated on a susceptible variety Nannong 1138-2, and after 2 weeks, the upper leaves show flower leaf symptoms, and fresh leaves are collected for standby. Seeds of homozygous lines (GmPUB #3-1 and GmPUB # 3-2) obtained after the Williams 82GmPUB20 gene knockout and wild Williams 82 are respectively sown in a greenhouse, and the soybean mosaic virus inoculation experiment is carried out after the 1 st true leaf is completely unfolded. The traits exhibited by the upper leaves were investigated 3 weeks after inoculation and recorded by photographing. Williams 82 after inoculation with soybean mosaic virus showed a certain degree of floral leaf symptoms, whereas GmPUB20 gene knockout plants had no obvious floral She Zhousu phenomenon, as shown in FIG. 5. The GmPUB20 gene knockout can change Williams 82 from soybean mosaic virus-sensitive variety to disease-resistant variety.
Detection of CP protein accumulation after inoculation of GmPUB20 Gene knockout plants with different Soybean mosaic Virus strains
(Western Blot detection of SMV-CP content)
In order to further confirm that the resistance of the GmPUB20 gene knocked-out plant to the soybean mosaic virus is improved, the Western detection of soybean mosaic virus CP protein is carried out by selecting upper leaves of different plants inoculated with different soybean mosaic virus strains for 2 weeks or 3 weeks. 3 biological replicates were taken for each treatment. The detection steps are as follows:
(1) The soybean leaves are placed in a mortar which is pre-cooled in advance and ground into powder. Adding appropriate amount of protein lysate (10%glycerol,50mM Tris-HCl, pH 7.5,1mM EDTA,150mM NaCl,1%NP-40,1mM DTT and mixed protease inhibitor (CWBIO, china)) and mixing. After the protein is fully cracked, the mixture is centrifuged for 15min at 10000rpm at room temperature, 5X SDS loading buffer is added, and after the mixture is uniformly mixed, the water bath is carried out at 85 ℃ for 15min.
(2) Proteins were separated by electrophoresis in 10% SDS polyacrylamide gel, used during electrophoresis.
(3) The separated proteins were transferred onto PVDF membrane with 0.22 μm pore size by "sandwich" method at 90V for 1h.
(4) After the transfer, the PVDF membrane was taken out and placed in a sealing solution (5% skimmed milk was dissolved in 1×TTBS) and sealed at room temperature for 2h.
(5) The blocking solution was decanted and eluted with 1×ttbs solution 3-5 times for 5min each. Primary anti-hybridization solution (SMV-CP antibody) was added, and the primary antibody was diluted in proportion according to the instructions of the antibody, incubated at room temperature for 2h or overnight at 4 ℃.
(6) The primary antibody hybridization solution was recovered and stored at-20 ℃. Eluting with 1×TTBS solution for 3-5 times each for 5min. Adding secondary antibody hybridization solution, diluting the secondary antibody according to the use instructions of the antibody in proportion, and incubating for 2 hours at room temperature.
(7) Recovering the secondary antibody hybridization solution and preserving at-20 ℃. Eluting with 1×TTBS solution for 3-5 times each for 5min.
(8) And (3) taking out the film, placing the film in a cut transparent self-sealing bag, and adding a luminescent liquid CHEMISTARTM HIGH-SIG ECL WESTERN Blotting Substrate kit (180-501, tanon, china) to perform a peroxidase-catalyzed chemiluminescent reaction.
(9) Developing to obtain the required picture. Finally, 3% ponceau S dye was used to stain the Rubisco large subunit as a load reference.
Western detection results of CP proteins show that the accumulation amount of the CP proteins of the plant subjected to GmPUB20 gene knockout is obviously lower than that of wild Williams 82, and the GmPUB20 gene knockout can enable disease resistance of soybean plants, as shown in figure 6.
6. Nucleic acid level detection of CP protein content in wild type and transgenic plants
To detect differences in soybean mosaic virus genome accumulation at the RNA level, leaves two weeks after SC3 and SC7 inoculation were selected for qRT-PCR experiments. Firstly, extracting RNA of a sample, synthesizing cDNA (same as in (3) of example 2), and then performing PCR (polymerase chain reaction) amplification by taking the cDNA as a template, wherein each cDNA is subjected to 2 pairs of PCR amplification by using a genomic sequence of soybean mosaic virus and soybean endogenous Tubulin, and the primers for amplifying the soybean mosaic virus sequence are SMV-F:5'-TTCTGAAAGTCCGTATATGCCTAG-3', SMV-R:5'-GCCTTTCAGTATTTTCGGAGTT-3' primer for amplifying soybean endogenous Tubulin is Tubulin-F:5'-GGAGTTCACAGAGGCAGAG-3' Tubulin-R:5'-CACTTACGCATCACATAGCA-3'. The RNA content of the Williams 82 leaf viruses after the GmPUB20 gene knockout is far lower than that of the wild Williams 82 leaf viruses after the soybean mosaic virus inoculation. The results are shown in FIG. 7.
The results are consistent with the phenotypic symptoms and the Western detection results, and further indicate that the deletion of the GmPUB20 gene can actually improve the disease resistance of soybean plants.
Claims (5)
- The application of the GmPUB20 gene in regulating and controlling the soybean mosaic virus resistance of soybean is disclosed, and the cDNA sequence of the GmPUB20 gene is shown as SEQ ID NO. 1.
- 2. Use according to claim 1, characterized in that the expression of the GmPUB20 gene according to claim 1 is knocked out or suppressed in order to increase the resistance of the susceptible variety to soybean mosaic virus.
- 3. The application of a gene editing system for knocking out or inhibiting the GmPUB20 gene in the cultivation of soybean mosaic virus resistant seeds is disclosed, and the cDNA sequence of the GmPUB20 gene is shown as SEQ ID NO. 1.
- 4. A method for constructing soybean with soybean mosaic virus resistance is characterized in that a gene editing system of a GmPUB20 gene is transferred into a variety to be improved, so that the variety with soybean mosaic virus resistance is obtained, and the cDNA sequence of the GmPUB20 gene is shown as SEQ ID NO. 1; the gene editing system of the GmPUB20 gene is a gene editing system for knocking out or inhibiting the GmPUB20 gene.
- 5. The method according to claim 4, wherein the gene editing system of the GmPUB20 gene is transferred into the variety to be improved by using the soybean cotyledonary node transformation method.
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