CN116479020A - Peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance - Google Patents
Peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance Download PDFInfo
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- CN116479020A CN116479020A CN202310247962.6A CN202310247962A CN116479020A CN 116479020 A CN116479020 A CN 116479020A CN 202310247962 A CN202310247962 A CN 202310247962A CN 116479020 A CN116479020 A CN 116479020A
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Classifications
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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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)
- C12N15/8201—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)
- C12N15/8241—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
- C12N15/8281—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 bacterial resistance
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention discloses a peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance, relates to the technical field of plant genetic engineering, and discloses the peanut protease gene AhSBT1.7, wherein the nucleotide sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the gene is shown as SEQ ID No. 2. According to the scheme, a CaMV 35S promoter is constructed through a Gateway system to drive an excessive expression vector of AhSBT1.7 and transform a bacterial wilt-sensitive variety safflower Dajinyuan of tobacco, and molecular detection and bacterial wilt inoculation identification are carried out on a transgenic plant, so that the result shows that the excessive expression of a protease gene AhSBT1.7 in the tobacco can obviously improve the resistance of the transgenic tobacco to bacterial wilt, and the AhSBT1.7 possibly participates in the resistance reaction of the plant to bacterial wilt; the invention provides important gene resources for cultivating new varieties of peanut with bacterial wilt resistance by utilizing genetic engineering means, and has important application prospect.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance.
Background
Peanut is one of important oil crops in China, but bacterial wilt seriously threatens the development of peanut industry, and brings great influence to the production and economic benefit of peanut. At present, molecular breeding technology is widely applied to cultivation of new disease-resistant varieties on a plurality of crops, and disease-resistant gene cloning and mechanism research are key links of molecular breeding, so that the molecular mechanism of the novel disease-resistant varieties must be clarified on the basis of understanding the functions of plant disease-resistant genes.
Proteolysis is the basis of normal vital activities of plants, and plays an important role in the growth and development of plants and in the process of coping with vital activities such as stress. Proteolysis requires the catalysis of proteolytic enzymes, wherein serine proteolytic enzymes account for approximately 30% of the protease. The subtilisin type of serine proteolytic enzymes play a key role in plant and microbial interactions, regulating plant immune responses and enhancing host resistance to pathogens.
Based on the analysis result of a laboratory early-stage yeast two-hybrid screening library, the research obtains a peanut bacillus subtilis proteolytic enzyme gene AhSBT1.7, the construction of an AhSBT1.7 over-expression vector through agrobacterium-mediated transformation of tobacco can obviously enhance the resistance to bacterial wilt, and the assumption that the AhSBT1.7 gene may participate in the defense reaction of plants to the bacterial wilt can provide gene resources for genetic improvement of peanut bacterial wilt resistance.
Disclosure of Invention
Therefore, the invention aims to provide the peanut protease gene AhSBT1.7 and the application thereof in peanut bacterial wilt resistance, which provide gene resources for plant bacterial wilt resistance genetic engineering and have important application prospect and positive practical significance.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a peanut protease gene AhSBT1.7 is a peanut bacillus subtilis proteolytic enzyme gene, and the nucleotide sequence of the protease gene AhSBT1.7 is shown in SEQ ID No. 1; wherein the amino acid sequence of the protease gene AhSBT1.7 is shown as SEQ ID NO. 2.
In the scheme, the resistance of tobacco to bacterial wilt can be obviously improved by the heterologously expressed AhSBT1.7 gene, which provides a theoretical basis for the molecular mechanism of peanut bacterial wilt resistance.
According to the scheme, based on a laboratory early-stage yeast two-hybrid screening library analysis result, a peanut bacillus subtilis proteolytic enzyme gene AhSBT1.7 is obtained, a primer is designed according to a predicted CDS sequence of the peanut bacillus subtilis proteolytic enzyme gene, and the gene is amplified by polymerase chain reaction, and the result shows that the AhSBT1.7 sequence contains 2268 base pairs and encodes 755 amino acids, and the nucleotide sequence of the AhSBT1.7 is shown as SEQ ID NO. 1. The ahsbt1.7 encoded protein contained an I9 conserved domain 75 amino acids in length, a PA conserved domain 90 amino acids, and an S8 conserved domain 462 amino acids. Meets the structural characteristics of the bacillus subtilis proteolytic enzyme gene family. The coded amino acid sequence is shown as SEQ ID NO. 2.
In addition to the above, the present invention provides an overexpression vector comprising the peanut subtilisin gene AhSBT1.7.
Meanwhile, the invention also provides a construction method of the over-expression vector, which comprises the following steps: it comprises the following steps:
constructing an entry vector pDONR207-AhSBT1.7 containing the protease gene AhSBT1.7 through BP reaction based on Gateway system, and constructing a plant expression vector pK7WG2.0-AhSBT1.7 driven by a CaMV 35S promoter through LR reaction.
Finally, the invention also provides application of the peanut bacillus subtilis proteolytic enzyme gene AhSBT1.7 or the over-expression vector in peanut bacterial wilt resistance genetic engineering. Transgenic tobacco plants which over-express AhSBT1.7 are obtained through agrobacterium-mediated genetic transformation, and the resistance identification is carried out on the transgenic tobacco plants, so that the result shows that AhSBT1.7 can positively regulate and control the resistance of tobacco to bacterial wilt.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that: according to the scheme, based on a peanut bacillus subtilis proteolytic enzyme gene AhSBT1.7 obtained by a laboratory early-stage yeast two-hybrid screening library analysis result, a cDNA sequence for encoding the protein is obtained through PCR cloning, a CaMV 35S promoter is constructed to drive an AhSBT1.7 gene plant expression vector pK7WG2.0-AhSBT1.7 through BP and LR reactions based on a Gateway system, GV3101 agrobacterium is transformed, the AhSBT1.7 is introduced onto a sensing variety safflower big golden member through a leaf disc method, molecular identification and bacterial wilt inoculation resistance identification are carried out on transgenic tobacco, and the result proves that AhSBT1.7 can positively regulate the resistance of tobacco to bacterial wilt. The invention provides gene resources for cultivating new peanut varieties with bacterial wilt resistance by utilizing genetic engineering means, and has important application value.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the construction of a peanut protease gene AhSBT1.7 overexpression vector based on Gateway system.
FIG. 2 is a peanut protease gene AhSBT1.7 clone, wherein M: DL15,000DNA Marker;1,2: experimental groups; CK: blank control.
FIG. 3 shows the results of partial identification of T0 generation of transgenic tobacco over-expressed with peanut protease gene AhSBT1.7, wherein (a) is identification on DNA level; (b) is an identification at the RNA level; m: DL15,000DNA Marker; +: a positive control; -: a negative control; CK: blank control.
FIG. 4 shows the phenotype of the transgenic tobacco inoculated with the bacterial wilt by the overexpression of the peanut protease gene AhSBT1.7, wherein (a) the resistance of the transgenic tobacco inoculated with the peanut protease gene AhSBT1.7 is enhanced compared with that of a control group after the bacterial wilt is inoculated with the transgenic tobacco, the disease index of the transgenic tobacco is reduced by the overexpression of the peanut protease gene AhSBT1.7, and (c) the propagation coefficient of the bacterial wilt in a transgenic plant is shown.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. It is specifically noted that the following examples are only for illustrating the present invention, but do not limit the scope of the present invention. Likewise, the following examples are only some, but not all, of the examples of the present invention, and all other examples, which a person of ordinary skill in the art would obtain without making any inventive effort, are within the scope of the present invention.
Example 1
Construction of AhSBT1.7 overexpression vector
According to the CDS sequence of the full-length gene AhSBT1.7 of the peanut bacillus subtilis proteolytic enzyme gene, a specific primer is designed:
AhSBT1.7-attb1-F(SEQ ID No.3):
GGGGACAAGTTTGTACAAAAAAGCAGGCTCATGGGCACGCCTCGTAAAGCGTCTCTTG
AhSBT1.7-attb2-R(SEQ ID No.4):
GGGGACCACTTTGTACA AGAAAGCTGGGTCTTAAAACCAACTAATTGAGATTGGGCTTC
PCR amplification is carried out by taking peanut variety Yueyou 92 leaf cDNA resisting bacterial wilt as template, and Takare company high-fidelity enzyme is adoptedMAX amplification, PCR reaction system: 1 μl of cDNA as a template, 5 μl +.>MAX mix, forward and reverse primers each 0.5 μl, and water was added to 10 μl. Reaction conditions: pre-denaturation at 94℃for 5min;94℃30s,60℃30s,72℃1min,25 cycles. And (3) performing gel cutting, purifying and recycling after agarose gel electrophoresis detection of the PCR product, and performing BP reaction on the target gene fragment and the pDONR207 in a no-load connection manner: 1 mu L (80-100 ng) of the purified AhSBT1.7 product, 1 mu L of the pDONR207 empty vector, 0.2 mu L of BP enzyme, and overnight connection at 25 ℃, the connection product is transformed into E.coli DH5 alpha competent cells, positive clones are screened for sequencing, the correct clone is sequenced to extract plasmids, and an entry vector pDONR207-AhSBT1.7 is constructed.
The entry vector plasmid and plant overexpression vector pk7wg2.0 were subjected to LR reaction: pDONR207-AhSBT1.7 plasmid 1. Mu.L (80-100 ng), pK7WG2.0 empty 1. Mu.L, LR enzyme 0.2. Mu.L, 25 ℃ overnight ligation, E.coli transformation, positive cloning verification, plant overexpression vector pK7WG2.0-AhSBT1.7 construction.
Wherein, the construction process of the pK7WG2.0-AhSBT1.7 overexpression vector is shown in figure 1, and the positive cloning verification diagram of the overexpression vector is shown in figure 2.
Example 2
Resistance analysis of heterologous expression AhSBT1.7 against ralstonia solanacearum
The overexpression vector pK7WG2.0-AhSBT1.7 obtained in the embodiment 1 is transformed into agrobacterium GV3101, and then is transformed into tobacco safflower Dajinyuan of bacterial wilt-susceptible variety by using agrobacterium tumefaciens-mediated leaf disc method, and positive transgenic strain is obtained by kanamycin screening and transgenic molecular identification.
To verify if the gene was recombined onto the tobacco genome, DNA (FIG. 3 a) and RNA (FIG. 3 b) level verification was performed on transgenic plants using 35s-F and AhSBT1.7-R primers, obtaining 11T strains 0 A generation positive plant, wherein the sequences of the 35s-F and ahsbt1.7-R primers are as follows:
35s-F(SEQ ID No.5)：TGATGTGATATCTCCACTGACGTAAG
AhSBT1.7-R(SEQ ID No.6)：TTAAAACCAACTAATTGAGATTGGGCTTC
subsequently, wild type and T are subjected to root irrigation 1 Where bacterial wilt inoculation is carried out on transgenic tobaccoAnd (3) growing under high-temperature and high-humidity conditions so as to cause bacterial wilt, and observing the disease after 14 days. And counting the morbidity conditions of the wild type and the transgenic tobacco according to the morbidity degree grade of the tobacco bacterial wilt, and calculating respective disease indexes according to the obtained data. Three days after inoculation, the stems of the tobacco were taken and subjected to a gradient dilution method (10 0 ，10 1 ，10 2 ，10 3 ) Calculating the content of the ralstonia solanacearum in the tobacco stems; the results shown in connection with fig. 4 demonstrate that overexpression of ahsbt1.7 can significantly enhance tobacco resistance to bacterial wilt.
The foregoing description is only a partial embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes using the descriptions and the drawings of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (6)
1. A peanut protease gene ahsbt1.7, characterized in that: the nucleotide sequence of the protease gene AhSBT1.7 is shown in SEQ ID No. 1.
2. The peanut protease gene ahsbt1.7 according to claim 1, characterized in that: the amino acid sequence of the protease gene AhSBT1.7 is shown as SEQ ID NO. 2.
3. An overexpression vector, characterized in that: comprising the protease gene ahsbt1.7 according to claim 1 or 2.
4. The method for constructing an overexpression vector according to claim 3, wherein: it comprises the following steps:
constructing an entry vector pDONR207-AhSBT1.7 containing the protease gene AhSBT1.7 through BP reaction based on Gateway system, and constructing a plant expression vector pK7WG2.0-AhSBT1.7 driven by a CaMV 35S promoter through LR reaction.
5. Use of the peanut protease gene ahsbt1.7 according to claim 1 or 2 in peanut bacterial wilt resistance genetic engineering.
6. Use of the over-expression vector of claim 3 in peanut bacterial wilt-resistant genetic engineering.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310247962.6A CN116479020A (en) | 2023-03-15 | 2023-03-15 | Peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance |
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| CN202310247962.6A CN116479020A (en) | 2023-03-15 | 2023-03-15 | Peanut protease gene AhSBT1.7 and application thereof in peanut bacterial wilt resistance |
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