CN117327714A - Australian wild cotton splicing factor GauSR45a and application thereof - Google Patents
Australian wild cotton splicing factor GauSR45a and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Abstract
The invention discloses an Australian wild cotton splicing factor GauSR45a and application thereof, and an alternative spliceosome of the gene has a nucleotide sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6. The gene GauSR45a involved in the verticillium defense reaction is screened and analyzed from the third generation transcriptome library of the Australian cotton, and is separated, cloned, functionally verified and applied. The GauSR45a through genetic transformation can be applied to various new materials for plant cultivation of verticillium wilt resistance, and can be used as a marker gene for plant verticillium wilt resistance. The discovery and utilization of GauSR45a can help to cultivate plant varieties resistant to verticillium wilt, and solve the problem of verticillium wilt hazard in production.
Description
Technical Field
The invention belongs to the technical field of genetic engineering breeding, and particularly relates to a Australian wild cotton splicing factor GauSR45a and application thereof in improving broad-spectrum verticillium wilt resistance of plants.
Background
Verticillium wilt (Verticillium wilt, VW) is the most damaging plant microtubule bundle disease in the world, produced by verticillium dahliaeVerticillium dahlia) The caused soil-borne fungus disease, the microsclerotium of the verticillium dahliae survives in soil for a long time, the disease can be caused to occur in a large area under proper environmental conditions, and the disease can be continuously infected with aftercrop, especially the stable production of various high-added-value plants including cotton, tomatoes, eggplants, potatoes, melons and the like is seriously threatened, and great loss is caused to agricultural production (Atallah and Subbarao, 2012). More importantly, the mycelium exists in the xylem vascular tissue, so that the bactericide cannot reach thalli, the prevention and the control are particularly difficult, and the existing cultivar is generally not disease-resistant or weak in resistance, so that the improvement effect is not obvious. Therefore, the heterogeneous broad-spectrum disease-resistant gene is mined from wild plants, and the bottleneck of the lack of the broad-spectrum disease-resistant gene in the verticillium wilt-resistant breeding of plants in China can be broken.
As with all cultivated plants, targeted domestication of cotton varieties is accompanied by loss of genetic diversity along with improvement of traits (Dubcovsky and Dvorak, 2007). Cotton genusGossypiumspp.), of which 48 are wild species (Fryxell, 1992). Studies have shown that genes with high verticillium resistance are present in wild species (Gu Benkang et al, 1993). Cotton in AustraliaGossypium australe2n=gg=26) is a diploid wild cotton seed which is resistant to both fusarium and verticillium wilt and also has broad-spectrum resistance to multiple bacterial lines of verticillium wilt, and is an important gene resource for cotton disease resistance breeding. However, since australian cotton belongs to a three-level gene pool cotton species, it has long been unavailable for breeding due to the lack of necessary prior studies.
The invention takes the Australian cotton with high verticillium resistance as a research object and combines the second generation transcriptome sequencing and the third generation full-length transcriptome sequencing from verticillium dahliaeSix new species were obtained from the inoculated root transcriptome libraryGauSR45aIs a gene sequence of (a). Sequence analysis shows that the six sequences are different spliceosomes of the alternative splicing factor GauSR45a, the full length of the coding sequences is 1060 bp,1188 bp,396 bp,381 bp,399 bp,306 bp respectively, and the six sequences code for six proteins of 101-395 amino acid residues. Silencing the expression of SR45a using VIGS in australian cotton and gossypium barbadense resistant varieties Hai7124 appears to be more susceptible. Heterologous overexpression in Arabidopsis respectively by means of transgenesisGauSR45aIs a variant of the six alternative spliceosome. Overexpression ofGauSR45a-L1AndGauSR45a-L2shows a stronger resistance to verticillium wilt than wild type plants. Found in the inventionGauSR45aThe gene is from the third generation full length transcriptome sequencing of cotton in the wild cotton species Australia, and is different from the SR45a splicing factor gene which has been studied by all the former.
Disclosure of Invention
The invention aims to provide an Australian wild cotton splicing factorGauSR45aAnd its application in improving broad-spectrum verticillium resistance of plants.
The invention provides a cotton plant which is derived from wild cotton seeds and can endow plants with broad-spectrum verticillium wilt resistanceGauSR45aA gene whose nucleotide sequence of an alternative splice is (1) or (2) as follows:
(1) Has the structure shown as SEQ ID NO. 1%GauSR45a-L1）、SEQ ID NO.2（GauSR45a-L2）、SEQ ID NO.3（GauSR45a-L3）、SEQ ID NO.4（GauSR45a-L4）、SEQ ID NO.5（GauSR45a-L5) Or SEQ ID NO. 6%GauSR45a-L6) The nucleotide sequence shown;
(2) A nucleotide sequence having at least more than 60% homology to SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6; preferably, a nucleotide sequence having at least more than 70% homology to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6; further preferably, a nucleotide sequence having at least 80% or more homology to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6; even more preferably, a nucleotide sequence having at least more than 90% homology to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6; most preferably, a nucleotide sequence having at least 95% or more homology to SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.
The saidGauSR45aThe nucleotide sequence of the alternative spliceosome of the gene is preferably SEQ ID NO.1 #GauSR45a- L1) Or SEQ ID NO.2 ]GauSR45a-L2) The nucleotide sequence shown. Studies have shown that by means of transgenesis, heterologous overexpression in the target plants, respectivelyGauSR45aIs a variant of the six alternative spliceosome. Overexpression ofGauSR45a-L1（SEQ ID NO.1）AndGauSR45a-L2（SEQ ID NO.1）shows a stronger resistance to verticillium wilt than wild type plants.
From the aboveGauSR45aA GauSR45a protein encoded by a gene, which is (i) or (ii) as follows:
a protein with an amino acid sequence shown as SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12;
(II) proteins with the same functions and obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequences shown as SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12.
Contains the aboveGauSR45aGenes or for silencing or editing the aboveGauSR45aThe biological material of the gene is recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.
Above mentionedGauSR45aThe gene, the GauSR45a protein or the biological material is applied to any one of the following (1) to (3),
(1) Improving verticillium wilt resistance of plants;
(2) Preventing and treating verticillium wilt of plants;
(3) Cultivating a new germplasm of a plant with verticillium resistance.
The application is as describedGauSR45aThe gene is a target gene, and the target plant is over-expressed by a genetic engineering methodGauSR45aAny variable cutter of the gene can improve the verticillium wilt resistance of plants or cultivate new plant germplasm with obviously improved verticillium wilt resistance and is applied in production.
The invention provides a novel composite material containingGauSR45aThe application of the recombinant expression vector of any one of the variable cutter of the gene in transforming plant hosts to cultivate new materials with verticillium wilt resistance. ComprisesGauSR45aThe recombinant expression vector of any one of six gene alternative spliceosomes can be used for transforming various plant hosts including cotton and cultivating new materials with verticillium wilt resistance.
A method for improving verticillium wilt resistance of crops, which comprises the following stepsGauSR45aThe gene is a target gene, and the target plant is over-expressed by a genetic engineering methodGauSR45aAny variable cutter of the gene can improve the verticillium wilt resistance of plants or cultivate new plant germplasm with obviously improved verticillium wilt resistance and is applied in production.
The plant is dicotyledon or monocotyledon; cotton, tobacco or arabidopsis are preferred, but not limited thereto.
Screening and analyzing genes involved in verticillium wilt-resistant responses from third generation transcriptome library of Australian cottonGauSR45aAnd isolating and cloning, functional verification and application thereof. By genetic transformationGauSR45aCan be applied to various plants to cultivate new materials with verticillium wilt resistance, and can be used as a marker gene with verticillium wilt resistance of plants.
The invention uses virus-induced gene silencing technique (VIGS) to silence the expression of SR45a in australian cotton and island cotton resistant varieties Hai7124, which are shown to be more susceptible. Heterologous overexpression in Arabidopsis respectively by means of transgenesisGauSR45aIs a variant of the six alternative spliceosome. Overexpression ofGauSR45a-L1AndGauSR45a-L2shows a stronger resistance to verticillium wilt than wild type plants. The discovery and utilization of GauSR45a can help to cultivate plant varieties resistant to verticillium wilt, and solve the problem of verticillium wilt hazard in production.
Drawings
FIG. 1 is a schematic view ofGauSR45aAlternative splicing analysis and induced expression profile of the gene after inoculation with verticillium.
Wherein a is Australian cottonGauSR45aPattern diagram of alternative splicing factors. b:GauSR45aanalysis of protein domains encoded by different alternative spliceosomes. Sashimi plots map analysis of transcriptome sequencingGauSR45aAlternative splicing occurs. Semi-quantitative PCR (RT-PCR) analysisGauSR45aExpression levels of different alternative spliceosomes at 0, 3, 6, 12, 24, 48, 72 and 96 hours after inoculation with verticillium. e: RT-PCR analysisGbSR45aExpression levels of different alternative spliceosomes at 0, 3, 6, 12, 24, 48, 72 and 96 hours after inoculation with verticillium.
FIG. 2 is an analysis of serine arginine rich (SR) protein expression after verticillium inoculation of cotton.
Wherein, a, the expression quantity (TPM value) of the gene encoding the SR-like protein of the Australian cotton is analyzed by a transcriptome. b:GauSR45aexpression level (TPM) analysis of different transcripts. SR45a of island cotton Hai7124GbSR45a) Analysis of the expression pattern of the gene after inoculation of verticillium. SR45a of upland cotton TM-1GhSR45a) Analysis of the expression pattern of the gene after inoculation of verticillium. e:GbSR45aanalysis of the tissue expression pattern of the genes. f:GhSR45aanalysis of the tissue expression pattern of the genes.
FIG. 3 is a schematic view ofGauSR45aAndGbSR45apromoter activity is activated by verticillium.
Wherein, a GUS reporter gene is fused with GauSR45a and GbSR45a promoters to transiently express tissue staining after inoculation treatment of tobacco leaves. And b, quantifying the activity GUS enzyme activity of GauSR45 and GbSR45a promoters. Image J software analysis of average optical density of GauSR45 and GbSR45a promoter activity.
FIG. 4 shows subcellular localization of different alternative spliceosome proteins of GauSR45 a.
FIG. 5 shows sensitivity of VIGS silenced SR45a to verticillium in Australian cotton and island cotton.
Wherein a, VIGS silences SR45 in Australian cotton and island cotton Hai7124and c, verticillium pathogenicity test after the gene a. qRT-PCR analysis of the VIGS silencing efficiency of GauSR45a and GbSR45 a. And c, fungus resuscitation test. d: VIGS-GauSR45aAnd VIGS-GbSR45aAnd (5) observing the cross section of the cotton stalk. e VIGS-GauSR45aAnd VIGS-GbSR45aFungal biomass analysis of cotton. fVIGS-GauSR45aAnd VIGS-GbSR45aAnd (5) counting the disease index of cotton. Data are shown as mean ± standard error of three independent replicates.PThe values represent the results of the one-factor anova pairwise comparison. *P<0.05, **P<0.01, n.s., not significant.
FIG. 6 is a pathogenicity test of different spliceosomes overexpressing SR45a in Arabidopsis.
Wherein a: verticillium pathogenicity test after VIGS over-express different alternative spliceosomes of the GauSR45a gene in arabidopsis. And b, qRT-PCR analysis of the expression quantity of GauSR45a in transgenic Arabidopsis thaliana. And c, fungus biomass analysis of transgenic arabidopsis thaliana. And d, counting the disease index of the transgenic arabidopsis thaliana. Data are shown as mean ± standard error of three independent replicates.PThe values represent the results of the one-factor anova pairwise comparison. *P<0.05, **P<0.01, n.s., not significant.
Detailed Description
The following examples define the invention and describe the method of the invention for verifying the function of the GauSR45a gene by isolating and cloning DNA fragments comprising the complete coding segments of the different alternative spliceosomes of the GauSR45a gene. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Example 1: isolation cloning of different alternative spliceosomes of the GauSR45a gene
The applicant screens out a gene GauSR45a involved in resisting verticillium infection by integrating the third-generation full-length transcriptome of Australian cotton and the second-generation transcriptome treated by pathogen inoculation in the earlier stage, and utilizes RT-PCR to identify the alternative splicing and expression mode of the gene. Primers were designed at the 3 rd and 8 th exon regions of the GauSR45a gene, respectively, as shown in table 1:
TABLE 1 GauSR45a Gene 3 rd and 8 th exon region amplification primers
| RT-SR45a-F | GTGATCGATGTTCACCTTGT |
| RT-SR45a-R | CACGAATAGTTCTCAACCCC |
Alternative splicing and expression of the GauSR45a gene after inoculation with the pathogen were examined. As shown in FIG. 1, there are multiple alternative spliceosomes in the GauSR45a gene at different time points after inoculation, and their expression is induced by verticillium, further demonstrating its association with cotton verticillium resistance.
After the cotton of Australian cotton is inoculated with verticillium dahliae, total RNA is extracted from the root and cDNA is obtained by reverse transcription. The ORFs of the different alternative spliceosomes of the GauSR45a gene (shown as SEQ ID NOS.1, 2, 3, 4, 5, 6) were amplified using the primer pairs shown in Table 2 as templates.
TABLE 2 amplification primers for amplifying different alternative spliceosome ORFs of the GauSR45a gene
| SR45a-L1--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L1--R | cccttgctcaccatggatccAACCAGTACTTCGTTTAGGG |
| SR45a-L2--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L2--R | cccttgctcaccatggatccCGAGTAGCTCCTTGACCTCC |
| SR45a-L3--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L3--R | cccttgctcaccatggatccACACAAGAAAACTTTTACCT |
| SR45a-L4--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L4--R | cccttgctcaccatggatccAACCTTCTCAACTGTAATGT |
| SR45a-L5--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L5--R | cccttgctcaccatggatccAACCTGCTGCTGCCACAAAA |
| SR45a-L6--F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L6--R | cccttgctcaccatggatccACACAAGAAAACTTTTACCT |
By analyzing the transcription data, the expression level of SR genes of different transcriptome samples is quantified as TPM value. Further quantification of different transcripts of the same gene using Salmon software and DRIMseq+stager software is shown in FIG. 2.
Example 3: activity induction assay for GauSR45a and GbSR45a promoters Using GUS reporter System
The DNA sequence 2000 bp upstream of the start codon of SR45a gene was amplified in root cDNA of Australian cotton and sea island cotton, respectively, by primers shown in Table 3 and fused before GUS gene of pBI121 vector.
TABLE 3 investigation of GauSR45a and GbSR45a promoter Activity Induction test primers Using GUS report System
| GausSR45apro-GUS-F | accatgattacgccaagcttAGGCTAAACTGTCATTGAAG |
| GausSR45apro-GUS-R | gactgaccacccggggatccCTGGAAATATGAACAAAATA |
| GbSR45apro-GUS-F | accatgattacgccaagcttTCTTAAAAAACAATCATTGC |
| GbSR45apro-GUS-R | gactgaccacccggggatccCTGGAAATATGAACAAAATA |
And (3) using an agrobacterium infiltration mode to transiently express the constructed GUS report vector in tobacco leaves. After 48 hours of infiltration, the activity of the promoter was induced by She Bingjie bacteria verticillium using a syringe. Afterwards, GUS enzyme activity was quantified using the 4-MU method and the average optical density of GUS tissue staining was analyzed using imageJ software, as shown in FIG. 3.
Example 4: subcellular localization assay of different variable sheared bodies of GauSR45a
Amplification of the root cDNA of Australian cotton by primers shown in Table 4 the ORF sequences of the different variable cutter of the GauSR45a gene were fused before the Green Fluorescent Protein (GFP) gene of the pBInGFP4 vector.
TABLE 4 primer for testing subcellular localization of different alternative spliceosomes of GauSR45a gene
| SR45a-L1-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L1-GFP-R | cccttgctcaccatggatccAACCAGTACTTCGTTTAGGG |
| SR45a-L2-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L2-GFP-R | cccttgctcaccatggatccCGAGTAGCTCCTTGACCTCC |
| SR45a-L3-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L3-GFP-R | cccttgctcaccatggatccACACAAGAAAACTTTTACCT |
| SR45a-L4-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L4-GFP-R | cccttgctcaccatggatccAACCTTCTCAACTGTAATGT |
| SR45a-L5-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L5-GFP-R | cccttgctcaccatggatccAACCTGCTGCTGCCACAAAA |
| SR45a-L6-GFP-F | tttacgaacgatagggtaccATGTCGTACACTAAGAGGTC |
| SR45a-L6-GFP-R | cccttgctcaccatggatccACACAAGAAAACTTTTACCT |
And (3) transiently expressing the constructed GFP fusion vector in tobacco leaves by using an agrobacterium infiltration mode. After 48 hours of infiltration, subcellular localization of the different alternative spliceosomes of the GauSR45a gene was analyzed using confocal microscopy, as shown in figure 4.
Example 5: investigation of GauSR45a function Using Virus mediated Gene silencing technology (VIGS)
According to the primer design principle of VIGS, the primers shown in table 5 were used to amplify silencing fragments using the cdnas of roots of australian cotton and island cotton as templates, and constructed on pTRV2 vectors. And transforming into colibacillus DH5 alpha, and transferring positive plasmid into agrobacterium GV3101 after the sequence is correct. The pTRV2 empty vector is used as a negative control, the pTRV/CLA vector is used as a positive control, and the cotyledons of cotton are injected by using a sterile needle-removed injector after the auxiliary vector pTRV1 is mixed. When the positive control shows albino phenotype, qRT-PCR is used for detecting the silencing efficiency of SR45a gene in cotton.
TABLE 5 amplification primers for GauSR45a Gene silencing fragment in cotton
| VIGS-GausSR45a-F | taccgaattcTGTCGAGATCCTTGTCGAGC |
| VIGS-GausSR45a-R | taccggatccCAAGTAAGGAGGGAGCTCAT |
| VIGS-GbSR45a-F | taccgaattcTGTCAAGATCCTTGTCGAGC |
| VIGS-GbSR45a-R | taccggatccCGTCCCTCAAGAACAGAACCA |
When the albino phenotype appears, namely, conidium of verticillium dahliae V991 is used, the root dipping method is adopted to inoculate 20 ml with the concentration of 1 multiplied by 10 7 spores ml -1 Is a spore suspension of (a) a spore. As shown in fig. 5, the silenced plants exhibited more disease than the control plants.
Example 6: acquisition and disease resistance identification of GauSR45a gene-transferred arabidopsis thaliana
The overexpression vector selected was pBI121, and alternatively spliced L1-L6 of GauSR45a was cloned into the expression vector using the primers shown in Table 6, respectively. Transgenic lines of Arabidopsis were obtained by Agrobacterium-mediated genetic transformation using the floral dip method with Arabidopsis Col-0 as the receptor.
TABLE 6 cloning primers for alternative spliceosome of GauSR45a gene
| pBI121-SR45a-L1-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L1-R | tgtttgaacgatcggggaaattcAACCAGTACTTCGTTTAGGG |
| pBI121-SR45a-L2-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L2-R | tgtttgaacgatcggggaaattcCGAGTAGCTCCTTGACCTCC |
| pBI121-SR45a-L3-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L3-R | tgtttgaacgatcggggaaattcACACAAGAAAACTTTTACCT |
| pBI121-SR45a-L4-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L4-R | tgtttgaacgatcggggaaattcAACCTTCTCAACTGTAATGT |
| pBI121-SR45a-L5-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L5-R | tgtttgaacgatcggggaaattcAACCTGCTGCTGCCACAAAA |
| pBI121-SR45a-L6-F | gagaacacgggggactctagaATGTCGTACACTAAGAGGTC |
| pBI121-SR45a-L6-R | tgtttgaacgatcggggaaattcACACAAGAAAACTTTTACCT |
The stably inherited transgenic lines were screened by the following steps: seed of the current generation of the flower is spread on a seed containing 100ug mL - 1 On a resistance MS culture medium of kanamycin, the resistance seedlings growing for about 10 days are transplanted into soil, then young rosette leaves are taken through 20 d to extract DNA, and gene CDS conservation region primers are used for amplification detection to positive. The positive offspring single plants are harvested, spread on a kanamycin resistant medium, and offspring isolation ratio is selected to be 3:1, selfing the first generation, and harvesting the transgenic Arabidopsis overexpression line with stable inheritance.
The disease resistance of the transgenic strain is identified by dipping root method inoculation treatment by using verticillium wilt V991 spore suspension. The test results show that the GauSR45a-L1 and GauSR45a-L2 transgenic lines show higher disease resistance than wild arabidopsis thaliana, and the early disease occurrence rate is lower; whereas GauSR45a-L2 exhibited stronger disease resistance than GauSR45a-L1, as shown in FIG. 6.
Claims (10)
1. The method comprises the following steps ofGauSR45aThe gene, the alternative splice of the gene has a nucleotide sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
2. By claim 1GauSR45aThe GauSR45a protein coded by the gene has the amino acid sequence shown as SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.
3. Comprising the composition of claim 1GauSR45aThe biological material of the gene is characterized in that the biological material is a recombinant vector, an expression cassette or a recombinant bacterium.
4. The method of claim 1GauSR45aThe gene is applied to any one of the following (1) - (3),
(1) Improving verticillium wilt resistance of plants;
(2) Preventing and treating verticillium wilt of plants;
(3) Cultivating new germplasm of plant with improved verticillium resistance.
5. The use of the GauSR45a protein of claim 2 in any one of the following (1) - (3),
(1) Improving verticillium wilt resistance of plants;
(2) Preventing and treating verticillium wilt of plants;
(3) Cultivating new germplasm of plant with improved verticillium resistance.
6. The biomaterial according to claim 3, wherein the biomaterial is used in any one of the following (1) to (3),
(1) Improving verticillium wilt resistance of plants;
(2) Preventing and treating verticillium wilt of plants;
(3) Cultivating new germplasm of plant with improved verticillium resistance.
7. The use according to claim 4, 5 or 6, characterized in that in claim 1GauSR45aThe gene is a target gene, and the target plant is over-expressed by a genetic engineering methodGauSR45aAny one of variable cutter of gene to raise plantThe verticillium wilt resistance or the cultivation of new plant germplasm with significantly improved verticillium wilt resistance and the application in production.
8. The use according to claim 7, characterized in that: the plant is cotton, tobacco or Arabidopsis thaliana.
9. A method for increasing verticillium wilt resistance of plants, comprising the steps of claim 1GauSR45aThe gene is a target gene, and the target plant is over-expressed by a genetic engineering methodGauSR45aAny variable cutter of the gene can improve the verticillium wilt resistance of plants or cultivate new plant germplasm with obviously improved verticillium wilt resistance and is applied in production.
10. The method according to claim 7, wherein: the plant is cotton, tobacco or Arabidopsis thaliana.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187796A1 (en) * | 2017-04-07 | 2018-10-11 | Donald Danforth Plant Science Center | Methods for increasing resistance to cotton bacterial blight and plants produced thereby |
| CN108841837A (en) * | 2018-06-29 | 2018-11-20 | 山东农业大学 | Application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response |
| CN113234729A (en) * | 2021-04-30 | 2021-08-10 | 南京农业大学 | Gene GauRev2 capable of obviously improving verticillium wilt resistance of cotton and application thereof |
| WO2022073260A1 (en) * | 2020-10-09 | 2022-04-14 | 西南大学 | Application of tomato hydroxyproline-rich systemin precursor protein gene slhypsys in improving resistance of plants to verticillium wilt |
-
2023
- 2023-11-29 CN CN202311614361.0A patent/CN117327714B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187796A1 (en) * | 2017-04-07 | 2018-10-11 | Donald Danforth Plant Science Center | Methods for increasing resistance to cotton bacterial blight and plants produced thereby |
| CN108841837A (en) * | 2018-06-29 | 2018-11-20 | 山东农业大学 | Application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response |
| WO2022073260A1 (en) * | 2020-10-09 | 2022-04-14 | 西南大学 | Application of tomato hydroxyproline-rich systemin precursor protein gene slhypsys in improving resistance of plants to verticillium wilt |
| CN113234729A (en) * | 2021-04-30 | 2021-08-10 | 南京农业大学 | Gene GauRev2 capable of obviously improving verticillium wilt resistance of cotton and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| GAO X 等: "Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt", 《PLANT J》, vol. 66, no. 2, pages 293 - 305 * |
| LI Y 等: "Dual roles of the serine/arginine-rich splicing factor SR45a in promoting and interacting with nuclear cap-binding complex to modulate the salt-stress response in Arabidopsis", 《NEW PHYTOL》, vol. 230, no. 2, pages 641 - 655 * |
| 刘富杰: "利用RNA-seq挖掘澳洲棉7号染色体黄萎病抗性相关基因的研究", 《中国优秀硕士学位论文全文数据库农业科技辑》, no. 8, pages 046 - 126 * |
| 张源珊 等: "植物生长发育和逆境响应中SR-like蛋白的研究进展", 《北植物学报》, vol. 43, no. 1, pages 167 - 180 * |
| 无: "serine/arginine-rich splicing factor SR45a-like [Gossypium australe]", 《GENBANK》 * |
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