CN112813086A - Gene with verticillium wilt resistance function and application thereof - Google Patents
Gene with verticillium wilt resistance function and application thereof Download PDFInfo
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- CN112813086A CN112813086A CN201911122056.3A CN201911122056A CN112813086A CN 112813086 A CN112813086 A CN 112813086A CN 201911122056 A CN201911122056 A CN 201911122056A CN 112813086 A CN112813086 A CN 112813086A
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Abstract
The invention discloses a gene with a plant verticillium wilt resistance function and application thereof, wherein the gene is a BS2 gene with a plant verticillium wilt resistance function, which is modified by a codon, and the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown. The expression of the gene is helpful for improving the verticillium wilt resistance of plants. When the plant expresses the unmodified BS2 gene, the verticillium wilt resistance of the plant is favorably improved, and when the plant expresses the codon-modified BS2 gene, the verticillium wilt resistance of the plant can be obviously improved.
Description
Technical Field
The invention relates to the field of agriculture, in particular to a gene with anti-verticillium wilt function, and application and a method thereof in plant, especially cotton, anti-verticillium wilt.
Background
Cotton Verticillium dahliae Kleb is one of the most important diseases of cotton at present, and the occurrence and damage of cotton in China are gradually increased in recent years. However, cotton plays a very important strategic position in agricultural development in China, so that cultivation of a new variety of cotton with high verticillium wilt resistance has great practical significance for ensuring safe production of cotton in China. The method is a very effective method for improving the verticillium wilt resistance of cotton on land and is also an important way for ensuring the sustainable development of national cotton.
Chinese patent (ZL200510109118.9) discloses a BS2 protein separated and purified from bacillus subtilis B111 fermentation liquor, and a BS2 gene cloned. Antibacterial activity identification tests prove that the BS2 protein has the effect of inhibiting verticillium dahliae V991 of defoliating cotton. The disclosed BS2 gene sequence is cloned from Bacillus subtilis, and no research report of the BS2 gene for plant breeding exists at present. Therefore, the application of the BS2 gene in breeding of verticillium wilt-resistant cotton varieties is to be further researched.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, experiments prove that the BS2 gene has the function of improving the verticillium wilt resistance of plants.
It should be noted that the present invention is completed based on the following work of the inventors:
the inventor utilizes bioinformatics analysis to find that the BS2 protein contains a glutamic acid protease active center conserved sequence (glutamic active site 361-370VTAHEMTHGV) and is classified as neutral zinc metallopeptidase; the BS2 protein is presumed to be a secreted protein by positioning the BS2 protein on a cell membrane through subcellular localization and combining bioinformatics analysis and pathogenic mechanisms of verticillium dahliae.
Thus, according to a first aspect of the present invention, there is provided a gene which is highly expressed in plants and has the ability to improve plant resistance to verticillium wilt. According to an embodiment of the invention, the gene is:
the BS2 gene, wherein the BS2 gene has the nucleotide sequence shown in SEQ ID NO: 1.
That is, the BS2 gene can be used for breeding of verticillium wilt resistant plants.
SEQ ID NO: 1 is as follows:
the inventor researches and discovers that the anti-verticillium wilt capability of the plant can be favorably improved when the plant expresses the BS2 gene, and the anti-verticillium wilt capability of the plant can be obviously improved when the plant expresses the codon-modified BS2 gene.
Wherein, it is to be noted that the gene with plant verticillium wilt resistance function also comprises the nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and modification of one or more bases, and has a nucleotide sequence identical to that of SEQ ID NO: 1, the nucleotide sequence shown in the formula 1 has the same or similar anti-verticillium wilt function.
According to a second aspect of the invention, there is provided a vector. According to an embodiment of the invention, the vector contains the BS2 gene. The vector can be obtained, for example, by inserting the above-mentioned nucleotide sequence into a cloning vector or an expression vector, or can be obtained by artificial synthesis. For example, the vector may be a binary plant expression vector, such as the pBI121 plasmid.
According to a third aspect of the invention, the invention relates to a recombinant cell. According to an embodiment of the invention, the recombinant cell contains a plasmid carrying the BS2 gene.
According to some embodiments of the invention, the recombinant cell may be obtained by transforming a host cell with the aforementioned vector.
According to the present invention, the host cell can be Agrobacterium tumefaciens GV3101, which is capable of chemotactic infection of many dicotyledonous plants under natural conditions and inducing crown gall, and which has been used successfully in cotton genetic transformation, and is currently the mature method of transformation mediation.
According to a fourth aspect, the present invention relates to the use of the aforementioned gene or the aforementioned vector or the aforementioned recombinant cell for the resistance of plants against verticillium wilt.
The inventor finds that the crude protein extract of the transgenic tobacco generates an obvious bacteriostatic zone through a bacteriostatic activity detection test of the crude protein extract of the transgenic tobacco with the BS2 gene. Furthermore, a transgenic positive cotton plant is obtained by transforming cotton with the BS2 gene, and a verticillium wilt bacteria V991 infection test is carried out on the transgenic positive cotton plant, and the result shows that the BS2 gene plays an important role in improving the verticillium wilt resistance of cotton, and further the BS2 gene can be used for improving the verticillium wilt resistance of plants.
According to a fifth aspect of the invention, the invention relates to the use of the aforementioned gene or the aforementioned vector or the aforementioned recombinant cell for the preparation of a transgenic plant or for plant breeding.
As previously mentioned, according to an embodiment of the present invention, the plant may be a malvaceae plant, preferably, the plant may be cotton.
According to a sixth aspect, the present invention relates to the use of callus for the preparation of transgenic plants or for plant breeding. According to an embodiment of the invention, said callus is transformed with a gene as described above or a vector as described above or a recombinant cell as described above.
As previously mentioned, according to an embodiment of the present invention, the plant may be a malvaceae plant, preferably, the plant may be cotton.
According to a seventh aspect of the invention, the invention relates to a method of combating verticillium wilt in plants. According to an embodiment of the present invention, the method comprises transforming the aforementioned gene or the aforementioned vector into a plant, or infecting a plant with the aforementioned recombinant cell.
As previously mentioned, according to an embodiment of the present invention, the plant may be a malvaceae plant, preferably, the plant may be cotton.
According to an eighth aspect, the present invention relates to a method for producing a transgenic plant resistant to verticillium wilt. According to an embodiment of the invention, the method comprises: transforming the aforementioned gene or the aforementioned vector into plant callus, or infecting plant callus with the aforementioned recombinant cell; and regenerating a transgenic plant using the callus.
As previously mentioned, according to an embodiment of the present invention, the plant may be a malvaceae plant, preferably, the plant may be cotton.
Advantageous effects of the invention
The inventor researches and discovers that the BS2 gene is related to disease resistance of plants, particularly to cotton verticillium wilt resistance. Bioinformatics analysis shows that the BS2 protein contains a glutamic acid protease active center conserved sequence (glutamic acid active site 361-370VTAHEMTHGV), and is classified as neutral zinc metallopeptidase; the BS2 protein is presumed to be a secreted protein by positioning the BS2 protein on a cell membrane through subcellular localization and combining bioinformatics analysis and pathogenic mechanisms of verticillium dahliae.
When the plant expresses the unmodified BS2 gene, the verticillium wilt resistance of the plant is favorably improved, and when the plant expresses the codon-modified BS2 gene, the verticillium wilt resistance of the plant can be obviously improved.
The result of an antibacterial activity detection test of the crude protein extract of the transgenic tobacco by the BS2 gene shows that the crude protein extract of the transgenic tobacco generates an obvious antibacterial zone. Furthermore, a transgenic positive cotton plant is obtained by transforming cotton with the BS2 gene, and a verticillium wilt bacteria V991 infection test is carried out on the transgenic positive cotton plant, and the result shows that the BS2 gene plays an important role in improving the verticillium wilt resistance of cotton, and further the BS2 gene can be used for improving the verticillium wilt resistance of plants.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the construction of the plant expression vector pBI 121-BS2, A: pUC 57 vector BamHI/SacI digestion, B: pBI121 expression vector BamHI/SacI restriction enzyme, C: PCR identification of pBI 121-BS2, D: expression vector pBI 121-BS 2;
FIG. 2 shows the construction of the plant expression vector pBI GR79-35S-BS2, A: pUC 35S-BS2 vector EcoRI/ScaI cut, B: the pBI GR79 expression vector was digested with EcoRI, C: pBI GR79-35S-BS2 PCR identification, D: expression vector pBI GR79-35S-BS 2;
FIG. 3 shows the construction of the plant expression vector pBI GR79-vsp1-BS2, A: pUC vsp1-BS2 vector EcoRI cut, B: the pBI GR79 expression vector was digested with EcoRI, C: pBI GR79-vsp1-BS2 PCR identification, D: the expression vector pBI GR79-vsp1-BS 2;
FIG. 4 shows the acquisition of transgenic tobacco regeneration lines, A: infecting tobacco leaves with agrobacterium, B: callus, C: transgenic seedlings, D: transplanting;
FIG. 5 shows T0Inhibition of transgenic tobacco protein crude extract on V991, WT: a wild-type control; b1, B2, B3: transgenic tobacco with higher expression level;
FIG. 6 shows T1Verticillium wilt pathogen V991 resistance detection of generation transgenic tobacco, A: a tobacco phenotype; B-E: SOD, CAT enzyme activity and SA, ABA content determination ". about" and ". about" respectively show in P<0.05 and P<Significant differences at the 0.01 level;
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are not indicated by the manufacturer, and are conventional products available commercially, for example from Thermo Fisher corporation.
The experimental materials and reagents used in the examples of the present application:
1. plant material
NC89 is used as a transgenic receptor material of tobacco (Nicotiana tabacum L.) in the experiment; upland cotton Coker312 was used as transgenic acceptor material for cotton (Gossypium hirsutum L.).
2. Plasmids and strains
The plant expression vector pBI121 is provided by the kininoman researchers of the institute of biotechnology of Chinese academy of agricultural sciences, the cotton defoliating verticillium wilt strain V991 is provided by the doctor of the dawn of the institute of biotechnology of Chinese academy of agricultural sciences, the vsp1 promoter is provided by the doctor of the royal lyph of the institute of genetics and developmental biology of Chinese academy of agricultural sciences, the agrobacterium GV3101 competence, the intermediate vector pUC 35S-CSP2, the plant expression vector pBI GR79, the subcellular localization green fluorescence fusion vector 35S-eGFP and the bacillus subtilis B111 are provided by the three-pile researchers of the institute of biotechnology of Chinese academy of agricultural sciences, the TA/Blunt-Zero Cloning Kit is purchased from Nanjing Vazym company, and the escherichia coli DH10B competent cells are purchased from Zhongmeitai and biotechnology Limited.
3. Common reagent and kit
Yeast extract, Tris-base, sodium hypochlorite, agarose, ethylene diamine tetraacetic acid, beta-mercaptoethanol, kanamycin, ampicillin, 6-benzylaminopurine, indole acetic acid, rifampicin, cephalo, carboximidazole, M519 dry powder, calcium chloride, absolute ethanol, isopropanol, acetone, isoamyl alcohol, sucrose, spermidine, sodium chloride, glycerol, glacial acetic acid, vegetable gel, peptone and other common reagents commonly used in experiments are purchased from Beijing Bayer's Biotech Co.
Primers used for experiments are synthesized by Shanghai Bioengineering, restriction enzymes and T4 ligase are purchased from New England Biolabs (Beijing) of America, DNA gel recovery kits are purchased from Omega, KOD DNA polymerase is purchased from TOYOBO biotech, DNA Marker is purchased from Baolin Biotech, In-Fusion HD Cloning Kit is purchased from TaKaRa, plant DNA extraction kits are purchased from Tiangen Biotech, plasmid extraction kits are purchased from GeneStar, RNA extraction kits are purchased from Beijing original Heizhen, DNA polymerase, reverse transcription kits and fluorescence quantitative PCR kits are purchased from Nanjing Vazyme, and physiological and biochemical index determination kits are purchased from Wuhan Libery Biotech. Specific models and uses are shown in table 1:
TABLE 1 reagents and uses
4. Preparation of experimental reagent
Culture medium for tobacco tissue culture:
1)1/2MS culture medium: weighing 2.217g of M519 dry powder, 30g of sucrose and 6g of agar powder into a cleaned conical flask, fixing the volume to 1L by using distilled water, and sterilizing for 15min by using high-pressure steam at 115 ℃;
2) MS0 medium: weighing 4.43g of M519 dry powder and 30g of sucrose into a clean conical flask, fixing the volume to 1L with distilled water, sterilizing with high-pressure steam at 115 ℃ for 15min, and storing at 4 ℃;
3) MS1 medium: weighing 4.43g of M519 dry powder, 30g of sucrose and 6g of agar powder into a cleaned conical flask, fixing the volume to 1L by using distilled water, sterilizing for 15min by using high-pressure steam at 115 ℃, cooling to 55-60 ℃, and adding 1mL of 2mg/mL of 6-BA and 0.5mg/mL of IAA respectively;
4) MS2 medium: weighing 4.43g of M519 dry powder, 30g of sucrose and 6g of agar powder into a cleaned conical flask, fixing the volume to 1L by using distilled water, sterilizing for 15min by using high-pressure steam at 115 ℃, cooling to 55-60 ℃, and adding 1mL of each of 2mg/mL of 6-BA, 0.5mg/mL of IAA, 100mg/mL of Car, 300mg/mL of Cef and 100mg/mL of Kan;
5) MS3 medium: weighing 2.217g of M519 dry powder, 15g of sucrose and 6g of agar powder into a cleaned conical flask, fixing the volume to 1L by using distilled water, sterilizing by using high-pressure steam at 115 ℃ for 15min, cooling the temperature to 55-60 ℃, and adding 1mL of each of 0.5mg/mL IAA, 100mg/mL Car, 300mg/mL Cef and 100mg/mL Kan.
The prepared culture medium is completely adjusted to pH 5.8 by 0.4M KOH, and then is sterilized.
Preparation of other media and TAE:
1) LB culture medium: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, putting into a washed conical flask, adding 800mL of distilled water, adjusting the pH to 7.0 with 0.4M KOH, then fixing the volume to 1L, and sterilizing for 20min by high-pressure steam at 121 ℃. 1.5% agar powder was added to the solid medium.
2) CM medium: weighing yeast extract 6g, sucrose 10g, and hydrolyzed retin 6g, placing into a cleaned conical flask, adding distilled water to constant volume of 1L, and sterilizing with high pressure steam at 121 deg.C for 20 min.
3) PDA culture medium: cleaning and peeling fresh potatoes, weighing 200g of the fresh potatoes, cutting the fresh potatoes into small pieces, adding 1000mL of distilled water, boiling for 25min, cooling, filtering residues with four layers of medical gauze into a cleaned conical flask, adding 20g of glucose and 20g of agar powder into filtrate obtained after filtration, keeping the volume of the distilled water to 1L, and sterilizing the mixture for 20min by high-pressure steam at 121 ℃.
4)50 × TAE electrophoresis buffer: 242g of Tris alkali and 242g of Na2EDTA & 2H2O 37.2.2 g are weighed into a cleaned conical flask, ddH2O 800mL is added, the mixture is fully stirred by a stirrer until the solid is completely dissolved, 57.1mL of glacial acetic acid is added, the pH is adjusted to 8.0, the volume of ddH2O is adjusted to 1L, and the mixture is stored at normal temperature.
Preparing other reagents:
the preparation conditions of other reagents of the experiment are shown in a table 2, wherein ampicillin, kanamycin, rifampicin, cefuroxime, carbenicillin, 6-benzylamino adenine and auxin reagents are required to be filtered and sterilized by a 0.22-micron filter, are subpackaged into 2.0mL sterilized centrifuge tubes and are stored at-20 ℃; potassium hydroxide and sodium hydroxide reagents were sterilized by filtration through a 0.22 μm filter and stored at 4 ℃.
TABLE 2 reagent preparation
Example 1
In this embodiment, codon plant preference modification is performed on the BS2 gene sequence, and the inventor finds an optimal modification method through multiple experiments to obtain a BS2 gene sequence that can be efficiently expressed in plants, and constructs a plant expression vector pBI 121-BS2 using a pBI121 plasmid as a vector, including the following specific steps:
1. artificially synthesized BS2 gene sequence
The base sequence of the BS2 gene is shown as SEQ ID NO: 1, and the following components:
2. construction of expression vector pBI 121-BS2
1) Taking an optimally synthesized target gene as a template, obtaining the full length of the gene with BamHI and SacI enzyme cutting sites at two ends by a PCR amplification method, and recovering a target fragment by agarose gel electrophoresis;
2) connecting the target fragment with a TA/Blunt-Zero vector by using T4 ligase, and carrying out double enzyme digestion on a coding region by using BamHI and SacI; the GusA fragment in the expression vector pBI121 was ligated and replaced, and the new vector was named pBI 121-BS2, as shown in FIG. 1.
3. Construction of expression vector pBI GR79-35S-BS2
1) Taking the optimally synthesized target gene as a template, amplifying to obtain the full length of the gene with PstI and XhoI enzyme cutting sites at two ends, and recovering a target fragment through agarose gel electrophoresis;
2) the target fragment was ligated with the TA/Blunt-Zero vector using T4 ligase, and the coding region sequences were double digested with PstI and XhoI, replacing CSP2 on the intermediate vector pUC 35S-CSP2 with BS 2.
3) Connecting an intermediate vector with a target gene BS2 with a linker to change a restriction enzyme cutting site, passivating a HindIII restriction enzyme cutting site, inserting an EcoRI restriction enzyme cutting site, and naming the new vector as pUC 35S-BS 2;
4) the expression cassette containing the target gene was obtained by digesting pUC 35S-BS2 with EcoRI and ScaI, and the expression cassette was ligated with plant expression vector pBI GR79 using T4 ligase to construct plant expression vector pBI GR79-35S-BS2 as shown in FIG. 2.
4. Construction of expression vector pBI GR79-vsp1-BS2
1) Obtaining a promoter fragment containing EcoRI/BamHI enzyme cutting sites at two ends by a PCR method, and recovering the promoter fragment by agarose gel electrophoresis;
2) cloning the recovered promoter fragment to a TA/Blunt-Zero vector, carrying out EcoRI/BamHI double enzyme digestion on the vector, connecting the vector to an intermediate vector pUC 35S-BS2, and naming a new vector as pUC vsp1-BS 2;
3) the vector pUC vsp1-BS2 was digested with EcoRI, ligated to the plant expression vector pBI GR79, to construct the plant expression vector pBI GR79-vsp1-BS2, as shown in FIG. 3.
The method used in the construction process of the vector comprises the following steps:
1. PCR amplification of Gene sequences
1) And (3) PCR reaction system:
PCR reaction procedure:
2) the enzyme system was placed in a 37 ℃ metal bath for 3 h.
A double enzyme digestion system:
single enzyme digestion system:
3) the ligation reaction system is as follows, the T4 ligase ligation reaction system is placed in a metal bath at 16 ℃ for 16 h; In-Fusion method the ligation was placed In a 50 ℃ metal bath for 30 min.
T4 ligase ligation reaction system:
In-Fusion ligation system:
4) linker Synthesis
linker-F and linker-R single chains were sterilized ddH2Dissolving and diluting O, mixing the linker-F and linker-R solutions with equal volumes in a centrifuge tube, sealing the tube opening of the centrifuge tube with a sealing film, putting the centrifuge tube into boiling water, timing for 5min, stopping heating, and naturally cooling to room temperature along with the boiling water to combine two single chains into a double chain.
2. Glue recovery
The DNA gel recovery kit is used for recovering the target fragment, and the specific steps are as follows with reference to the kit specification:
1) agarose gel electrophoresis: preparing 1% agarose gel with 1 × TAE, placing the agarose gel into an electrophoresis tank after the agarose gel is solidified, and adding samples for electrophoresis;
2) cutting the glue: irradiating the gel by using a gel imager, determining a target strip, cutting off a gel block with the target strip by using a blade under the irradiation of an ultraviolet lamp, and transferring the gel block with the target strip into a 2.0mL sterile centrifuge tube;
3) melting glue: adding 700 mu L of membrane binding solution (XP2) into a centrifuge tube, placing the centrifuge tube on a metal bath at 50 ℃, uniformly mixing the solution by reversing every two minutes until the gel block is completely dissolved, and uniformly mixing the solution by reversing the solution up and down again (the time is determined according to the volume of the gel block, and if the gel block cannot be completely dissolved, a proper amount of the membrane binding solution XP2 is added);
4) binding an adsorption column: and (3) putting an adsorption column into the collection pipe, transferring 700 mu L of the dissolved and uniformly mixed liquid into the adsorption column, centrifuging at 4000rpm at room temperature for 2min, taking out the adsorption column after centrifugation, pouring waste liquid in the collection pipe, and putting the adsorption column into the collection pipe. If the liquid after the gel is dissolved is more than 700 mu L, the gel passes through the column for multiple times;
5) cleaning: adding 300 μ L of membrane binding solution (XP2) into the adsorption column, centrifuging at 4000rpm for 1min, and discarding waste liquid after centrifuging;
6) rinsing: adding 700 μ L of rinsing solution containing anhydrous ethanol into adsorption column, centrifuging at 4000rpm for 1min, discarding waste liquid after centrifuging, and rinsing repeatedly;
7) removing ethanol residues: after the two times of rinsing, putting the adsorption column into a collecting pipe, opening a cover at room temperature for centrifugation, centrifuging at 12000rpm for 2min, after the centrifugation is finished, putting the adsorption column into a new 1.5mL centrifuge tube, and completely drying until no ethanol smell exists (generally standing for 10 min);
8) eluting the fragment: adding 30 μ L ddH to the center of the adsorption membrane2O (preheated to 65 ℃) and after standing for 2min, centrifuging at 12000rpm for 2min (eluting once more with eluent to improve the recovery concentration of DNA fragments);
9) and (3) storage: can be used or stored at-20 deg.C.
3. Heat shock transformation of E.coli competence
1) Thawing the escherichia coli competence stored at-80 ℃ on ice, adding the ligation product into the competent cells after the escherichia coli competence is completely thawed, gently mixing the ligation product and the competence, placing the centrifuge tube on ice for 30min, and placing the centrifuge tube in a metal bath at 42 ℃ for 45 s;
2) rapidly taking out competence after finishing, and immediately placing on ice for 2 min;
3) adding 500 μ L LB into the competence, putting the competence into a shaker at 37 deg.C and 200rpm, and culturing for 1-2h (time is adjusted according to the copy number of the carrier, the copy number is low, and the culture time is long);
4) coating the bacterial liquid on a culture medium containing corresponding carrier resistance, sealing the culture medium by using a sealing film, and culturing in an incubator at 37 ℃ (if more clones are needed, placing a bacterial liquid centrifuge tube in a centrifuge, centrifuging at 4000rpm for 1min, removing part of supernatant, and coating a plate after re-suspending bacterial cells); the culture time can be determined according to the characteristics of the carrier, after the plate is coated in the afternoon, part of the carrier can be taken away from the incubator at night and then placed in a room temperature environment, so that the better growth of the carrier is promoted, and the generation of star-shaped colonies and the difficulty in selecting monoclones are avoided.
4. Plasmid extraction
Extracting plasmids by using a plasmid miniextract kit, referring to a kit specification, and specifically comprising the following steps:
1) column balancing: placing the adsorption column into a collection tube, adding 500 μ L column equilibrium liquid (BL), standing for 1min, centrifuging at 12000rpm for 1min at room temperature, and pouring off waste liquid (using the adsorption column treated on the same day);
2) and (3) thallus enrichment: taking 4mL of cultured bacterial liquid (12-16h), centrifuging at 12000rpm for 10min at room temperature, pouring out supernatant after centrifugation is finished, and collecting bacterial precipitation (removing liquid as far as possible);
3) and (3) resuspending the thalli: adding 250 mu L of cell suspension S1 (containing RNaseA) into the collected thallus precipitate, performing vortex oscillation, and suspending and uniformly mixing thallus until the liquid state in the tube is turbid and has no precipitate;
4) and (3) cracking reaction: adding 250 μ L cell lysate S2 into the tube, slowly reversing the action, mixing the liquid, standing at room temperature for 5min until the liquid becomes viscous and translucent, and opening the cover to draw the fiber;
5) and (3) neutralization reaction: adding 350 μ L of neutralization buffer S3 into the liquid obtained by the above steps, slowly reversing the action for 5 times, mixing the liquid uniformly, changing the solution from viscous semitransparent state to clear state, and generating white precipitate, then placing at room temperature for 5min, and centrifuging at 12000rpm for 10 min;
6) DNA binding to adsorption column: after the centrifugation is finished, carefully taking out the centrifuge tube, transferring the supernatant into an adsorption column, centrifuging at 12000rpm for 1min at room temperature, and pouring out the waste liquid;
7) rinsing: adding 500 μ L of the rinse solution PW added with absolute ethanol into the adsorption column, centrifuging at 12000rpm for 30s, pouring off the waste liquid, and repeatedly rinsing once. Uncapping and centrifuging at 12000rpm for 2min, transferring the adsorption column into a 1.5mL sterile centrifuge tube, uncapping and completely drying (generally placing for 10 min);
8) and (3) elution: add 50. mu.L ddH to the center of the adsorption film2O (preheated to 65 ℃), standing for 2min, and centrifuging at 12000rpm for 2min (the concentration of the extracted particles can be improved by eluting with the eluent once again);
9) and (3) storage: can be used or stored at-20 deg.C.
Example 2
The plant expression vector pBI 121-BS2 constructed in the example 1 is used for transforming agrobacterium and a leaf disc method is used for transforming tobacco to obtain transgenic positive tobacco, and the specific steps are as follows:
1. preparation and transformation of agrobacterium tumefaciens electric stimulation competent cells
1) Selecting a single colony of the agrobacterium tumefaciens GV3101 to inoculate in 5mL LB containing Rif, putting the colony into an incubator at 28 ℃, and culturing overnight at 200 rpm;
2) transferring the cultured bacterial liquid into 50mL LB containing Rif according to the volume ratio of 1:100, putting into a 28 ℃ incubator, and carrying out shake culture at 200rpm for 5-6 h;
3) centrifuging at 8000rpm for 10min with 4 deg.C centrifuge, and adding pre-cooled sterile ddH2O, washing the collected thallus precipitate for 2 times;
4) washing thallus precipitate with pre-cooled sterile 10% glycerol, centrifuging again at 4 deg.C with a centrifuge at 8000rpm for 5min (repeating the step for 3 times);
5)1mL of sterile 10% glycerol is precooled and then used for resuspending the thalli, then the prepared competence is subpackaged into a sterile centrifuge tube and is put on ice for use or is preserved at minus 80 ℃;
6) adding 3 mu L of plasmid into the agrobacterium-infected cells which are just prepared or thawed on ice taken out at minus 80 ℃, gently mixing uniformly, and placing into a pre-cooled sterile electric cup;
7) placing the electric cup into an electrode of an electric conversion instrument, and selecting an agrobacterium electric excitation program for electric excitation;
8) taking out the electric cup, quickly adding 500 microlitre LB to mix evenly, transferring all liquid to a centrifuge tube of 1.5 mL;
9) placing in a shaking table at 28 ℃, and culturing for 6h at 200 rpm; spreading the cultured bacterial liquid on a culture medium containing Rif and Kan antibiotics, sealing, and inversely placing in an incubator at 28 ℃ for culturing for 2 d;
10) selecting single clone, shaking bacteria and PCR identification.
2. Tobacco transformation by leaf disc method
2.1 obtaining sterile seedlings
1) Taking an appropriate amount of NC89 tobacco seeds, putting the seeds into a 1.5mL centrifuge tube, adding 1mL 75% ethanol, inverting the centrifuge tube upside down for disinfection, and discarding liquid;
2) adding 1mL of sterile water into the centrifuge tube, turning the centrifuge tube upside down once, and discarding the liquid;
3) adding 1mL of 7.5% NaClO into a centrifuge tube, sterilizing for 30min, and discarding the liquid;
4) repeating the step 2 for 5 times, and finally reserving a little sterile water;
5) sucking out the liquid and seeds, placing on 1/2MS culture medium, shaking culture medium plate to disperse the seeds, placing in dark place at 28 deg.C for one day, and culturing under light.
2.2 Agrobacterium infection method for transformation of tobacco
1) Activating agrobacterium: taking GV3101 bacterial liquid containing an expression vector from-80 ℃, inoculating the bacterial liquid on a plate containing Rif and Kan, culturing for 2d at 28 ℃, selecting a single clone from the plate, inoculating the single clone on 4mL of liquid LB culture medium containing Rif and Kan, placing the liquid LB culture medium in an incubator at 180rpm and 28 ℃ for overnight culture, after PCR identification, inoculating the positive clone bacterial liquid in a volume ratio of 1:100 into 50mL of culture medium containing Rif and Kan, placing the culture medium in the incubator at 180rpm and 28 ℃ for about 10h, and detecting the OD value, wherein the OD value of the available bacterial liquid is about 0.6-0.8;
2) preparing a dip-dyeing bacterial solution: centrifuging the bacterial solution with a proper OD value at 4 ℃ and 6000rpm for 10min, removing a supernatant, adding 50mL of MS0 culture medium, and resuspending a thallus precipitate;
3) infecting the tobacco leaf disc: cutting tobacco leaves into small leaf discs in an aseptic culture dish by using an aseptic medical blade, removing veins, and putting the leaf discs into bacterial liquid for dip-dyeing for 10 min;
4) co-culturing: taking out the infected leaf disc by using a sterile forceps, placing the infected leaf disc on sterile filter paper, sucking the bacterial liquid on the leaf disc by using the sterile filter paper, paving the leaf disc with the front side upward on an MS1 culture medium with the sterile filter paper, placing a piece of sterile filter paper on the leaf disc, and culturing for 3d in a dark place;
5) selecting and culturing: transferring the leaf disc to MS2 culture medium, and performing subculture according to growth state;
6) rooting culture: when the sprouts grow to 2-3cm, selecting well-grown sprouts, completely cutting off callus at the lower parts of the sprouts, then transferring the sprouts to an MS3 culture medium, inducing the sprouts to take roots, and transferring the sprouts to an MS3 tissue culture bottle after the sprouts grow roots;
7) transplanting the regenerated seedlings: when the seedling leaves grow to be close to the mouth of the tissue culture bottle, opening the bottle cap, hardening seedlings for 2-3d, transplanting the seedlings into soil for culture, and showing in figure 4.
Example 3
Further, the transgenic tobacco obtained in the embodiment 2 is subjected to positive screening, and a bacteriostatic activity experiment is performed on positive plants, and the specific steps comprise:
1. activation and culture of verticillium dahliae
Taking a proper volume of verticillium dahliae V991 spore liquid stored at-70 ℃, uniformly coating on a potato glucose agar (PDF) culture medium, placing in an incubator at 25 ℃ for dark culture for 4 days, carrying out primary activation on the pathogenic bacteria, taking a verticillium dahliae mycelium block which is activated on a PDA culture medium by using a sterile gun head, placing in a sterilized 1000mL triangular flask containing 200mL of CM culture solution, culturing in the dark for 5 days on a shaking table at 25 ℃ and 150r/min, filtering by using sterile double-layer medical gauze, and removing mycelium clusters. In thatUnder microscope, spore concentration was calculated using a hemocytometer, and the spore suspension concentration was diluted to 5X 10 with sterile water7one/mL.
2. Detection of bacteriostatic activity of crude tobacco protein extract
Taking 1.0g of the 3 rd to 5 th leaf of the seedling from the top to the bottom, grinding the seedling into powder by using liquid nitrogen, and transferring the powder into a 50mL centrifuge tube; adding 10mL protein extract (PBS), vortex shaking for 1min, and standing for 10 min; centrifuging at 8000r/min for 15min, and collecting supernatant in ultrafiltration centrifuge tube (cut-off molecular weight of 30.0 kDa); centrifuging at 8000r/min until the residual volume of the liquid is about 2 mL; determining the antibacterial activity of the protein concentrated solution by adopting an oxford cup method; dark culture is carried out for 4d at 25 ℃, and the result of observing the inhibition zone shows that: the crude protein extract of wild tobacco did not generate inhibition zone, and the crude protein extracts of transgenic tobacco B1, B2 and B3 all generated obvious inhibition zone, as shown in FIG. 5.
3. Verticillium wilt infection of tobacco
Will T0Seeds for generating transgenic tobacco and wild tobacco are spread on the surface of soil (nutrient soil: vermiculite is 1: 1), a culture pot is placed in a greenhouse for culture, and when 4-6 true leaves grow on tobacco seedlings, the tobacco seedlings are inoculated by adopting a root dipping method, namely: gently removing the tobacco seedlings from the soil, and reducing the root injury degree as much as possible; washing root soil with sterilized water, and then washing the root of tobacco seedling at 5 × 107Soaking in conidium suspension per mL for 2 min; and replanting the soaked and inoculated tobacco seedlings in a new culture pot. And 7d, inoculating the strain, and then measuring physiological and biochemical indexes.
4. Measurement of physiological and biochemical indexes
(1) Principle of
The enzyme immunoassay kit can detect various biochemical index levels in some samples including plant tissues, and the method is a double-antibody sandwich method, utilizes the combination of antigen and antibody, and has the characteristics of strong specificity and high sensitivity. Synthesizing an antibody marker by a chemical method, purifying the antibody marker, coating the antibody marker by a microporous plate, fixing the antibody marker on a solid phase carrier, sequentially adding a detected target substance into micropores coated with corresponding monoclonal antibodies, combining the detected target substance with a detection target substance antibody marked by HRP to form an antibody-antigen-enzyme-labeled antibody compound, thoroughly washing the microporous plate, and then adding a substrate TMB. The HRP enzyme catalyzes TMB to display blue, and finally displays yellow after adding acid, wherein the shade of the yellow is positively correlated with the concentration of the detected target.
(2) Dilution of standard
Catalase (CAT):
superoxide dismutase (SOD):
salicylic Acid (SA):
abscisic acid (ABA):
(3) procedure for the preparation of the
1) Diluting a standard product: diluting according to the table;
2) sample adding: the ELISA plate is provided with three blank holes (same operation except that no sample or enzyme is added), a standard hole and a sample hole to be detected. Add 50. mu.L of standard sample to the standard well, and add 50. mu.L of standard and 10. mu.L of sample to be tested to the sample well in sequence (sample is finally diluted 5 times). When a sample is added into the hole of the ELISA plate, the sample is added to the bottom of the plate hole as much as possible without touching the hole wall of the plate hole, and the mixed solution slowly shakes and is mixed uniformly;
3) and (3) incubation: sealing the enzyme label plate with a sealing plate membrane, and incubating at 37 deg.C for 30 min;
4) preparing liquid: diluting the concentrated washing solution by 30 times with distilled water, and diluting to 30 times;
5) washing: removing a sealing plate film on the ELISA plate, discarding liquid, spin-drying, filling the plate hole with a cleaning solution, standing for 30s, discarding the liquid, repeating the step for 5 times, and finally performing patting for 1 time;
6) adding an enzyme: adding 50 mu L of enzyme labeling reagent into each plate hole except the blank hole;
7) and (3) incubation: sealing the enzyme label plate with a plate sealing film, and incubating at 37 deg.C for 30 min;
8) washing: removing a sealing plate film on the ELISA plate, discarding liquid, spin-drying, filling washing liquid in each plate hole, standing at room temperature for 30s, discarding liquid again, repeating the step for 5 times, and finally 1 time drying;
9) color development: adding 50 mu L of color developing agent A into a plate hole, then adding 50 mu L of color developing agent B, shaking and uniformly mixing the mixed solution in the enzyme-labeled hole slowly, and standing for 10min in a dark place at 37 ℃;
10) and (4) terminating: adding 50 mu L of stop solution into each plate hole of the enzyme label plate to stop the reaction (the color changes from blue to yellow);
11) and (3) determination: OD value was measured at a wavelength of 450nm, and after blank wells were set to zero, OD value was measured sequentially for each well on the microplate (measurement was performed within 15min after addition of the stop solution).
(4) Computing
And drawing a standard curve by taking the standard substance and the OD value as an abscissa and an ordinate respectively. And detecting the OD value of the sample, finding out the corresponding concentration on the standard curve, and multiplying the concentration by the dilution factor of the detection sample. Or calculating a corresponding regression equation according to the data of the standard substance, substituting the OD value of the detected sample into the regression equation, calculating the concentration of the sample, and multiplying the calculated value by the dilution multiple of the sample to obtain the actual concentration of the sample.
The wild tobacco and the transgenic line have basically consistent growth vigor and no obvious difference in phenotype in the control group which is not infected with verticillium wilt bacteria V991; in the verticillium wilt V991 infection group, 1 st to 3 rd true leaves of wild tobacco turn yellow, the true leaves of transgenic tobacco are all green, and the phenotypic character of the transgenic tobacco is obviously superior to that of the wild tobacco (figure 6-A).
Physiological and biochemical index detection results (FIGS. 6-B, C, D):
effect of BS2 on the Activity of plant enzymes
After plants are infected by verticillium wilt, active oxygen substances can be synthesized through various ways, so that active oxygen in vivo is rapidly accumulated, and a certain damage is caused to organisms; meanwhile, the plant body can also be combined into various antioxidant enzymes and non-enzyme antioxidant substances to prevent active oxygen from damaging the plant body. The enzymes related to plant oxidation resistance include SOD, CAT and the like, and the SOD can remove oxygen free radicals in organisms and protect cells from being damaged by the oxygen free radicals. CAT has the function of eliminating hydrogen peroxide in organisms and protecting plants from the toxicity of hydrogen peroxide, and both SOD and CAT are related to the verticillium wilt resistance of cotton. According to the invention, through comparison, the SOD activity and verticillium wilt pathogen infection groups of the transgenic line and the wild type tobacco are lower than those of a control group which is not infected with verticillium wilt pathogens, and the SOD and CAT activities of the transgenic line and the control group which is not infected with verticillium wilt pathogens and the verticillium wilt pathogen infection groups are obviously higher than those of the wild type tobacco, so that the high-efficiency expression of a corresponding active oxygen scavenging mechanism is caused by the large accumulation of active oxygen substances in the transgenic tobacco body in order to inhibit the infection of pathogenic bacteria, and the reason for enhancing the verticillium wilt resistance of the transgenic tobacco is explained from the side.
Effect of BS2 on the content of endogenous hormones in plants
The plant hormone is a small molecular substance, has important regulation effect on the growth and development of plants and adversity stress response, and has SA and ABA and the like related to disease resistance. SA plays a crucial role in plant defense against pathogenic bacteria invasion, in Arabidopsis thaliana, verticillium wilt virus causes exogenous cAMP to enter cells to increase cytoplasmic cAMP, which can regulate calcium-permeable ion channels and increase cytoplasmic Ca2+Concentration, cytosolic Ca2+Increased concentration promotes SA synthesis[148]. With cell lysisIncreased concentrations of plasma SA trigger the development of an oxygen burst response leading to cell death, induce the expression of disease-course associated Protein (PR) genes, and ultimately produce systemic acquired resistance, and studies have provided strong evidence of the positive defense function of SA in cotton and other plants. ABA can reduce the incidence and degree of incidence of cotton to a certain extent. The research finds that the SA and ABA contents of the transgenic lines of the control group and the verticillium wilt pathogen infection group which are not infected with verticillium wilt pathogens are obviously higher than those of wild types, which shows that the expression of the BS2 gene can improve the expression level of the transgenic plant hormone, so that the resistance of the transgenic plant hormone to the verticillium wilt pathogens is better improved.
Example 4
The expression vectors pBI GR79-35S-BS2 and pBI GR79-vsp1-BS2 constructed in example 1 were transformed into cotton by Agrobacterium mediated transformation to obtain transgenic cotton, respectively, according to the transformation method described in example 2.
The identified transgenic positive cotton is further subjected to an antibacterial activity experiment, and the specific method refers to example 3, wherein the crude protein extract of wild cotton does not generate an antibacterial zone, and the crude protein extract of transgenic cotton generates an obvious antibacterial zone.
The physiological and biochemical indexes of the transgenic positive cotton are determined, the specific method refers to example 3, and the results are shown in the following table:
comparative example
Constructing a plant expression vector by using the BS2 gene which is not modified by the codon, wherein the sequence of the BS2 gene which is not modified is shown as SEQ NO: 2, transforming the expression vector into tobacco, screening to obtain transgenic positive tobacco, and further performing a bacteriostatic activity test on the transgenic positive tobacco, wherein the specific method refers to examples 1, 2 and 3.
The results prove that: the crude protein extract of the transgenic tobacco can generate a bacteriostasis zone by transforming the BS2 gene which is not modified by the codon, but the bacteriostasis zone is obviously smaller than that of the crude protein extract of the transgenic tobacco obtained in the embodiment 2, and under the same experimental conditions, the radius of the bacteriostasis zone is half of that of the crude protein body fluid of the positive plant obtained by transforming the BS2 gene into the tobacco after the codon modification. Description of the drawings: the expression level of the BS2 gene provided by the invention in transgenic plants is obviously improved, a technical guarantee is provided for breeding transgenic cotton resistant to verticillium wilt, and the application prospect is wide.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Sequence listing
<110> gene having verticillium wilt resistance and use thereof
<120> institute of biotechnology of Chinese academy of agricultural sciences
<130> PIDC4190169
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1566
<212> DNA
<213> Artificial Synthesis
<400> 1
gttggtcttg gaaagaaatt gtcagttact gttgctgctt cattcatgtc tcttacaatt 60
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gttcctaagc attcacttgt tcaatctgaa ttgccatcag tttctgataa ggctattaaa 180
caatacctta agcaaaacgg aaaggttttc aaaggtaacc cttcagagag acttaagttg 240
atcgatcata ctacagatga tttgggatac aagcatttta ggtatgttcc tgttgttaat 300
ggtgttccag ttaaggattc acaagttatt acacatgttg ataagtctaa taacgtttat 360
gctatcaacg gtgaacttaa taacgatgct tctgctaaga ccgctaattc aaagaaactt 420
tctgctaacc aggctttgga tcatgctttc aaggctatcg gaaaatcacc agaagctgtt 480
tctaatggta acgttgctaa taagaacaaa gctgagctta aggctgctgc tacaaaggat 540
ggtaaataca gattggctta tgatgttacc attaggtaca tcgaacctga gccagctaat 600
tgggaagtta ccgttgatgc tgagactgga aaggttctta agaaacaaaa caaagttgaa 660
catgctgctg ctaccggtac tggaaccact cttaagggaa aaactgtttc attgaacatt 720
tcttcagaga acggaaagta tgttatgaga gatctttcta aacctacagg aacccaaatt 780
atcacttacg atttgcaaaa caggcaatat aatcttccag gtacattggt ttcttcaaca 840
accaatcaat tcactacatc ttcacaaaga gctgctgttg atgctcatta caaccttgga 900
aaggtttacg attacttcta ccaaactttc aagagaaact catacgataa caggggtgga 960
aagatcgttt cttcagttca ttacggatct aggtataata acgctgcttg gattggagat 1020
caaatgatct acggagatgg agatggttca ttcttttctc ctttgtcagg ttctatggat 1080
gttacagctc atgaaatgac tcatggtgtt acacaagaaa ccgctaatct taactacgag 1140
aatcaaccag gagctttgaa cgagtcattc tctgatgttt tcggttactt cactgataca 1200
gaagattggg atattggaga gggtatcacc gtttcacaac ctgctcttag atcattgtct 1260
aatcctacta agtacggaca acctgatcat tacaaaaact accaaaacct tcctaacacc 1320
gatgctggag attacggtgg agttcatact aactctggta ttccaaataa ggctgcttac 1380
aacaccatca ctaagatcgg agttaagaaa gctgaacaaa tctattacag ggctcttact 1440
gtttacttga caccatcttc atcttttaag gatgctaaag ctgctcttat tcaatctgct 1500
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gtgccgaagc attctttggt gcaatctgaa ttgccttcag tcagtgacaa agcaatcaag 180
caatacttga aacaaaacgg caaagtcttc aaaggcaacc cttctgagag actgaagctg 240
attgaccaca cgaccgatga tctcggctac aagcacttcc gttatgtgcc tgtcgttaac 300
ggtgtgcctg tgaaagactc gcaagtcatt actcacgtcg ataaatccaa caatgtctat 360
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tgggaagtaa ccgttgatgc ggaaacaggg aaagtcctga aaaagcaaaa caaagtggag 660
catgccgctg caaccggaac aggtacgact cttaaaggaa aaacggtctc attaaatatt 720
tcttctgaaa acggcaaata tgtaatgcgt gatctttcga agcctaccgg aacacaaatt 780
attacgtacg atctgcaaaa ccgacaatat aacctgccgg gcacgctcgt atcaagcact 840
acaaaccagt tcacaacttc ttctcagcgc gctgcggttg atgcgcatta caatctcggc 900
aaagtgtatg attatttcta tcagacgttt aaacgcaaca gctacgacaa tagaggcggc 960
aaaatcgtat cttccgttca ttacggcagc agatacaata acgcggcctg gatcggcgac 1020
caaatgattt acggtgacgg tgacggctca ttcttctcgc ctctttccgg ttcaatggac 1080
gtaacggccc atgaaatgac acacggcgtt acacaggaaa cagccaacct gaactatgaa 1140
aatcagccgg gcgctttaaa cgaatccttc tccgatgtat tcgggtactt caccgatact 1200
gaggactggg atatcggtga gggtattacg gtcagccagc cggctctccg cagcttatcc 1260
aatccgacaa aatacggaca gcccgaccat tacaaaaatt atcaaaacct tccgaacact 1320
gatgccggcg actacggcgg cgtgcataca aacagcggaa ttccgaacaa agccgcttac 1380
aacacgatta caaaaatcgg cgtgaaaaaa gcggagcaga tttactatcg cgcactgacg 1440
gtatatctca ctccgtcatc aagctttaaa gatgcaaaag cagctttgat tcaatcagcg 1500
cgggaccttt acggctctca agacgctgca agcgtagaag cggcctggaa tgcggtcggc 1560
ttgtaa 1566
Claims (10)
1. A codon-engineered BS2 gene having plant verticillium wilt resistance, wherein the BS2 gene has the amino acid sequence of SEQ ID NO: 1.
2. A vector comprising the BS2 gene.
3. The vector of claim 2, wherein the vector is a plant binary expression vector.
4. A recombinant cell comprising the vector of claim 2 or 3.
5. Use of the gene of claim 1 or the vector of claim 2 or 3 or the recombinant cell of claim 4 for plant resistance to verticillium wilt,
optionally, the plant is a malvaceae plant, preferably cotton.
6. Use of the gene of claim 1 or the vector of claim 2 or 3 or the recombinant cell of claim 4 for the preparation of a transgenic plant or for plant breeding,
optionally, the plant is a malvaceae plant, preferably cotton.
7. Use of callus transformed with a gene according to claim 1 or a vector according to claim 2 or 3 or a recombinant cell according to claim 4 for the preparation of a transgenic plant or for plant breeding,
optionally, the plant is a malvaceae plant, preferably cotton.
8. A method for combating verticillium wilt in plants, comprising transforming a plant with the gene of claim 1 or the vector of claim 2 or 3, or infecting a plant with the recombinant cell of claim 4,
optionally, the plant is a malvaceae plant, preferably cotton.
9. A method of producing a transgenic plant resistant to verticillium wilt comprising:
transforming the gene of claim 1 or the vector of claim 2 or 3 into plant callus, or infecting plant callus with the recombinant cell of claim 4; and
regenerating a transgenic plant using the callus.
10. A method according to claim 9, wherein the plant is a malvaceae plant, preferably cotton.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888052A (en) * | 2005-10-18 | 2007-01-03 | 中国农业科学院生物技术研究所 | Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene |
| CN101323842A (en) * | 2008-05-23 | 2008-12-17 | 中国农业科学院生物技术研究所 | Breeding method of disease-resistant and high-yield cotton and use |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888052A (en) * | 2005-10-18 | 2007-01-03 | 中国农业科学院生物技术研究所 | Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene |
| CN101323842A (en) * | 2008-05-23 | 2008-12-17 | 中国农业科学院生物技术研究所 | Breeding method of disease-resistant and high-yield cotton and use |
Non-Patent Citations (2)
| Title |
|---|
| 张胜昔等: "棉花抗黄萎病转基因育种研究进展", 《湖北农业科学》 * |
| 白玮等: "枯草芽孢杆菌B111中性蛋白酶基因BS2在毕赤酵母中的表达", 《中国农业科学》 * |
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Application publication date: 20210518 |