CN114854775B - Application of BcSfp1 gene in plant gray mold control and disease resistance improvement - Google Patents
Application of BcSfp1 gene in plant gray mold control and disease resistance improvement Download PDFInfo
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- CN114854775B CN114854775B CN202210392053.7A CN202210392053A CN114854775B CN 114854775 B CN114854775 B CN 114854775B CN 202210392053 A CN202210392053 A CN 202210392053A CN 114854775 B CN114854775 B CN 114854775B
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Abstract
The invention providesBcSfp1The application of the gene in preventing and controlling plant gray mold and improving disease resistance,BcSfp1the gene number is BCIN01G 01160,BcSfp1the expression of the gene and the protein product coded by the gene are used as target factors for the design and screening of plant botrytis-resistant medicaments, and the screening of compounds capable of enhancing the expression of the gene and the expression, modification and positioning of the protein can effectively improve the disease resistance of plants and control the occurrence of botrytis, thereby being beneficial to developing novel bactericides and immunity inducers.
Description
Technical Field
The invention belongs to the technical field of microbial genetic engineering, in particular to botrytis cinereaBcSfp1The application of the gene in preventing and controlling plant gray mold and improving disease resistance.
Background
Botrytis cinereaBotrytis cinerea) The pathogenic bacteria of Botrytis cinerea, which are commonly called Botrytis cinerea, have the behavior of Botrytis cinerea @ FuBotryotinia fuckeliana(de Bary) Whetzal), a member of the genus Botrytis of the phylum ascomycota; the no-behavior is Botrytis cinereaBotrytis cinereaPers) are members of the genus Botrytis, the asexual fungus. The Botrytis cinerea host can infect more than 1400 plants including Solanaceae, cucurbitaceae, rosaceae, leguminosae, etc., and harm the flowers, fruits, leaves and stems of the host plants, belonging to typical dead body nutrition type pathogenAnd (3) fungi. Gray mold belongs to typical low temperature Gao Shibing, serious occurs when the temperature is 20-23 ℃ and the relative humidity is more than 90%, disease can occur from the seedling stage to the fruiting stage, and each part of the plant can be infected by gray mold, so that great loss is caused to agricultural production. The gray mold can still cause larger damage in the process of fruit transportation and storage, so that the storage time of the fruits is shortened, the quality of the fruits is reduced, the storage difficulty and the cost are increased, and the economic loss of 100-1000 hundred million dollars of gray mold can be caused in the global world according to incomplete statistics. Because of the wide host range and serious production hazard, and the mature technology of related molecular research, the gray mold is one of the most important mode plant pathogenic fungi, and is widely researched.
Botrytis cinerea is a typical dead-body vegetative pathogenic fungus that, when placed in relation to a host plant, kills the host plant's tissues and cells rapidly to obtain nutrients from them that are required for growth and development. Reactive oxygen species (Reactive oxygen species, ROS) are critical to plant defense and also to fungal attack during host interaction with pathogenic bacteria. When affected by pathogens, plants undergo oxidative burst (rapid) reactions, i.e., rapid and transient production of reactive oxygen species, mainly by the plant's Rboh's (respiratory burst oxidase homolog, a homolog of the mammalian NADPH oxidase gp91 phox). Thus, fungi require a powerful oxidative stress (Oxidative stress reaction, OSR) system to handle ROS; in order to balance the redox state within the cell, an effective antioxidant system, including the thioredoxin and glutathione systems, is essential.
Glutathione is a tripeptide consisting of glutamic acid, cysteine and glycine, is an important antioxidant in organisms and is an important component of host defense system. Glutathione may develop strategies to prevent oxidant damage by enhancing the transport system of glutathione precursor amino acids or by providing substrates that circumvent feedback inhibition of glutathione synthesis. Glutathione protects tissues from free radical damage through detoxification of active substances and repair of damage, and plays an important role in metabolism of drugs and endogenous substances.
Transglutaminases (TG) are very widely distributed in nature and are found in microorganisms, plants, fish, amphibians, invertebrates and birds. Glutamine transferase is a transferase that catalyzes an acyl transfer reaction. The transglutaminase uses gamma-carboxamido of glutamine residue in peptide chain as acyl donor, and acyl acceptor includes epsilon-amino, primary amino, water, etc. of lysine residue in polypeptide chain. In addition to the above, the enzyme may also directly decompose glutamine into glutamic acid and ammonia. Glutamine transferase has wide application in various fields such as food, industry, agriculture, biological medicine, textile, cosmetics and the like, however, the research on the glutamine transferase in Botrytis cinerea is not reported.
Members of the p-transglutaminase protein familyBcSfp1The research of the formula is not only helpful for revealing the molecular mechanism of the pathogenicity of dead nutritional pathogenic fungi such as botrytis cinerea, but also has important application value for researching and developing medicaments for preventing and treating plant pathogenic fungi including botrytis cinerea. The effect of the gene in the botrytis pathogenic and plant disease-resistant processes is evaluated, so that the potential functional genes can be conveniently excavated, and the gene can be used for screening novel fungicidal agents and the like.
Disclosure of Invention
The gene for regulating and controlling the pathogenicity of the botrytis cinerea provided by the invention is derived from the botrytis cinerea and is named asBcSfp1，BcSfp1The gene number is BCIN01G 01160,BcSfp1the gene open reading frame consists of 793 nucleotides, which contain 3 exons, and is located between nucleotides 1 to 84, between nucleotides 146 to 250, and between nucleotides 302 to 793 of the open reading frame, respectively, and the length of the coding region is 681 nucleotides. Due to the fact thatBcSfp1Deletion of the gene results in enhancement of the pathogenicity of the gray mold,BcSfp1the gene and the protein coded by the gene can be used as targets for developing anti-gray mold medicaments, and compounds capable of enhancing the expression of the gene and the expression, modification and positioning of the protein can be screened, so that the generation of gray mold can be effectively controlled and used as novel sterilizationAnd (3) an agent.
The regulation and control of the pathogenicity of the botrytis cinerea is realized by promoting the growth and development or inhibiting the growth and development of the botrytis cinerea.
The invention relates to gray mold fungusBcSfp1The activity and/or the expression quantity of the gene or/and the coded protein in the botrytis cinerea are increased to inhibit the growth and development of the botrytis cinerea.
The invention also provides a deviceBcSfp1The application of the gene or the coded protein thereof in preventing and controlling plant gray mold caused by gray mold bacteria.
The invention also provides a deviceBcSfp1Use of a gene or a protein encoded by the gene as a target for a drug for controlling plant diseases caused by botrytis cinerea.
The invention also provides a method for preventing and controlling plant gray mold, which comprises promoting the gray mold in the bacteriaBcSfp1Expression of genes, said promotion promoting in Botrytis cinereaBcSfp1The gene expression reagent isBcSfp1Gene promoter and composition containingBcSfp1An over-expression vector for the gene.
The invention also provides the followingBcSfp1Use of a gene and said protein for increasing disease resistance in plants, said use comprising overexpressing said geneBcSfp1The gray mold of the gene induces the plants to generate immunity, and the overexpressionBcSfp1The botrytis cinerea of the gene is used for preparing a plant botrytis cinerea inducer, and inducing the plant to generate immunity to the botrytis cinerea.
The invention also provides the followingBcSfp1The gene and the application of the protein in regulating and controlling the oxidation stress resistance of gray mold bacteria.
The invention relates to gray mold fungusBcSfp1The activity or/and the expression quantity of the gene or/and the coded protein in the botrytis cinerea is increased, so that the adaptability of the botrytis cinerea to oxidative stress is improved.
The invention also provides the followingBcSfp1The use of genes and said proteins for increasing the resistance of plants to oxidative stress. Will contain the saidBcSfp1Transformation of recombinant vectors of genes into plants, in particular by introducing the recombinant vectors into microorganisms of the genus Agrobacterium or other plantsTransformed into a microorganism of a plant body, and transformed into a plant.
The recombinant vector of the invention comprises an introducedBcSfp1Genes, also comprising and introduced intoBcSfp1A promoter to which the gene is operably linked. The said ANDBcSfp1The promoter of the gene operably linked enablesBcSfp1Gene expression, or enhancementBcSfp1Gene expression.
The invention also provides the followingBcSfp1The application of the gene in the breeding of gray mold resistant varieties and the identification of disease resistance of plants.
The object of the present invention is to provide the followingBcSfp1The application of the gene in obtaining a variety which is obviously resistant to gray mold diseases and oxidation stress after being introduced into plants.
The object of the present invention is to provide the followingBcSfp1The gene is applied to the soil adversity improvement restoration agent.
The plant is one of Solanaceae, cucurbitaceae, rosaceae and Leguminosae.
In addition, contains the aboveBcSfp1The over-expression vector, the knockout vector and the recombinant bacterium of the gene are also within the protection scope of the invention.
The beneficial effects are as follows:
the invention proves thatBcSfp1The deletion of the gene leads to the enhancement of the pathogenicity of the botrytis cinerea, and simultaneously, the adaptability of the botrytis cinerea to oxidative stress is reduced, which indicatesBcSfp1The gene participates in the pathogenic process of the botrytis cinerea on host plants, and plays an important role in the growth and development of the botrytis cinerea and the stress response of the botrytis cinerea. Thus (2)BcSfp1The gene and the protein encoded by the gene can be used as targets for designing and screening botrytis related medicaments,BcSfp1the gene overexpression body can be used as a microbial agent for biological control, plant disease resistance induction, plant stress resistance induction and the like to improve the disease resistance and stress resistance of plants.
Drawings
FIG. 1 is a schematic view ofBcSfp1A functional prediction map of the encoded protein;
wherein the method comprises the steps ofBcSfp1The gene number is BCIN01G01260, NCBIThe results of the protein blast show that the amino acid sequence comprises 5 functional domains, namely GATase1_PfpI_2 (6-197), glxA (2-196), DJ-1_PfpI (64-183), ftrA (122-183) and non_thiJ (64-178);
FIG. 2 shows Botrytis cinereaBcSfp1Schematic representation of the knockout strategy of the gene (gene replacement by homologous recombination);
wherein B05.10 is wild-type strain, pXEH is knockout vector, deltaBcSfp1Is thatBcSfp1A gene deletion mutant;
FIG. 3 is a schematic view ofBcSfp1PCR verification electrophoresis pattern of gene deletion mutant;
m: marker, wherein hygromycin primer is used, deletion mutant has band and is consistent with the size of plasmid template band as positive control (shown as channels 2, 4, 6, 8, 10); usingBcSfp1Gene primers, positive control wild DNA template with bands and deletion mutants without bands (as shown in channels 1, 3, 5, 7, 9);
FIG. 4 is a knockout mutant deltaBcSfp1With wild strain B05.10 and complement deltaBcSfp1-C culture profile comparison photograph;
wherein delta isBcSfp1For knockout mutants, B05.10 is wild type, deltaBcSfp1C is complement, the culture medium is PDA, and the culture time is 48 hours; a is a morphology of colonies grown on PDA plates for 48h; b is a histogram of colony diameters grown for 24h and 48h on PDA plates;
FIG. 5 is H 2 O 2 Knock-out mutant deltaBcSfp1With wild strain B05.10 and complement deltaBcSfp1-oxidative stress analysis of C;
wherein delta isBcSfp1For knockout mutants, B05.10 is wild type, deltaBcSfp1C is complement, the culture medium is CM, and the culture time is 48h. a is H at different concentrations 2 O 2 A 48h colony morphology map was grown on CM plates; b and c are respectively 24H and 48 different concentrations H 2 O 2 Knock-out mutant deltaBcSfp1With wild strain B05.10 and complement deltaBcSfp1-percentage of bacteriostatic efficiency of C;
FIG. 6 is a knockout mutant deltaBcSfp1With wild-type strain B05.10Pathogenicity contrast analysis;
wherein delta isBcSfp1B05.10 is wild type, the host is Arabidopsis leaves, and living body inoculation is adopted for knocking out the mutant;
FIG. 7 shows the knockout mutant deltaBcSfp1ROS detection of arabidopsis leaves inoculated as host plant with wild strain B05.10;
wherein delta isBcSfp1For knockout mutants, B05.10 was wild type, col-1 was wild type Arabidopsis thaliana, left side was DAB stained leaves, right side was microscopic observation of stained leaves.
Detailed Description
In order to better describe the present invention, the following description will be given by way of specific examples, which are conventional methods unless otherwise indicated.
The examples of the present invention demonstrate that Botrytis cinereaBcSfp1The gene is a pathogenicity related gene of gray mold and provides gray mold pathogenicity related geneBcSfp1The new functions of the gene and the protein encoded by the gene are realized by constructing a gene knockout carrier, and the gene is knocked out from botrytis cinerea to obtain a knocked-out mutant; the gene is complemented into the knockout mutant by constructing a gene complement vector and utilizing a random insertion method to obtain a complement mutant or an over-expression body. After the gene is knocked out, the growth speed of the knocked-out mutant in hypha is increased, the pathogenicity is obviously increased, and the pathogenicity of the anaplerotic mutant is restored to the wild pathogenicity level.
The higher the activity or the expression level of the botrytis cinerea BcSfp1 gene or the coded protein thereof in the botrytis cinerea, the weaker the pathogenicity of the botrytis cinerea, the slower the growth and development of the botrytis cinerea and the higher the adaptability to oxidative stress; the lower the activity or expression level of the botrytis BcSfp1 gene or the coded protein thereof in the botrytis, the stronger the pathogenicity of the botrytis, the faster the growth and development of the botrytis and the lower the adaptability to oxidative stress.
The invention provides a gray mold fungusBcSfp1The gene is used for the gene expression,BcSfp1the gene number is BCIN01G01260, and the control pathogenicity gene is derived from botrytis cinerea and is named asBcSfp1，BcSfp1The gene open reading frame consists of 793 nucleotides, which contain 3 exons, and is located between nucleotides 1 to 84, between nucleotides 146 to 250, and between nucleotides 302 to 793 of the open reading frame, respectively, and the length of the coding region is 681 nucleotides. The Botrytis cinereaBcSfp1The amino acid sequence encoded by the gene consists of 226 amino acids.
In the embodiment of the inventionBcSfp1The method for breeding the knockout mutant comprises the following steps:
1. by using the gray mold fungusBcSfp1Constructing a knockout vector by genes;
2. transforming the knockout vector into agrobacterium to obtain a transformant;
3. transforming the transformant into a receptor bacterium to obtain a mutant strain with a gene deletion;
in the embodiment of the inventionBcSfp1The culture method of the anaplerotic mutant or the over-expression strain comprises the following steps:
1. construction of a vector containing the Botrytis cinereaBcSfp1A gene complement vector or an overexpression vector;
2. transforming the complement vector or the over-expression vector into agrobacterium to obtain a transformant;
3. and transforming the transformant into a receptor bacterium to obtain a gene complement mutant or an over-expression strain.
Example 1,BcSfp1Correlation analysis of genes
Botrytis cinereaBcSfp1The open reading frame of the gene consists of 793 nucleotides, including 3 exons, and the length of the coding region is 681 nucleotide. The encoded protein product consists of 226 amino acids. For a pair ofBcSfp1The gene is predicted and analyzed in the Botrytis cinerea genome network http:// furgi.BcSfp1The gene number is BCIN01G 01160, NCBI protein blast results show that the amino acid sequence comprises 5 functional domains, namely GATase1_PfpI_2 (6-197), glxA (2-196), DJ-1_PfpI (64-183), ftrA (122-183) and not_thiJ (64-178), and the prediction map is shown in figure 1.
、BcSfp1Knock-out and genetic complementation of genes
1) Knock-out vector construction
Primers were designed using DNAMAN and synthesized by Shanghai Biotechnology Inc.
By using primersBcSfp1-UP-F (5'-GGCGGCCTCGAGACAGCGAATGGTGTGATAATCAG-3') andBcSfp1UP-R (5'-ATGAGCTCGATGAAGGTAATTCAGAAGGTAGAAAT-3') amplification Using genomic DNA of Botrytis cinerea Strain B05.10 as templateBcSfp1A fragment of about 1000bp upstream of the gene; by using primersBcSfp1-DN-F (5'-ACGCGTCGACTCTCGGAGGAGTGAAGGGGCGT-3')BcSfp1DN-R (5'-AACTGCAGAATCCACCACAGTATCTTGGTTACGAT-3') an approximately 1000bp fragment downstream of the BcSfp1 gene was amplified using genomic DNA of Botrytis cinerea strain B05.10 as template. Reaction system (25 μl): takara PrimerSTAR Max DNA Polymerase (2×) 12.5 μl; 1. Mu.L of each of the upstream and downstream primers (10. Mu.M); 1. Mu.L of template DNA; ddH 2 O9.5. Mu.L. Amplification procedure: (1) 98 ℃ for 10s; (2) 53℃for 15s; (3) 10s at 72℃the 1-3 steps were cycled 35 times. The PCR products were recovered and purified using a Takara gel recovery kit, and the upstream and downstream fragments were ligated with pXEH vector using XhoI and SacI and SalI and PstI, respectively, to construct knockout vectors, as shown in FIG. 2.
2) Transformation of Botrytis cinerea
A. Cultivation of Agrobacterium tumefaciens AGL-1
Picking up carriers containing binary elementsBcSfp1Is inoculated into LB liquid medium containing 50 mu g/mL kanamycin and 25 mu g/mL rifampicin, and is shake-cultured for 36 h to OD 600 =0.8 or so. Preparation of IM liquid Medium (100 mL: 90 mL ddH) 2 O;1 mL of 50% glycerol; 1 mL of 20% glucose; 4 mL of 1M MES;2 mL M-N buffer;1 mL Tracelelement; 80 mu L K-buffer;250 μL of 20% NH 4 NO 3 ；100μL1%CaCl 2 ；1 mL 0.01% FeSO 4 The method comprises the steps of carrying out a first treatment on the surface of the 200. Mu.L 19.2% As), first at 90 mL ddH 2 O is added with FeSO 4 Mixing with As medicine, collecting shaking bacterial liquid 5ml in 15ml centrifuge tube, centrifuging at 4000 rpm for 5 min, discarding supernatant, addingIM culture medium (FeSO is not added) 4 As) 5ml, blow down, spin at 4000 rpm, centrifuge for 5 min, discard supernatant, add FeSO 4 As in IM culture medium, 5ml is added into a 15ml centrifuge tube, blown down to an empty bottle, and shaken by a shaker at 28 ℃ for 4 hours. The remaining IM liquid medium was placed in a refrigerator at 4 ℃.
B. Spore-forming culture of botrytis cinerea
Activating strain B05.10, inoculating the bacterial cake to PDA culture medium (20% of potato is boiled and filtered, 2% of glucose and 1.5% of agar) for two days, placing in 25 deg.C incubator, culturing for 10-15 days, washing out spores with IM liquid culture medium, observing with microscope, and regulating spore concentration to 5×10 with hemocytometer 5 /mL。
C. Co-culture of Agrobacterium tumefaciens and conidium of Botrytis cinerea
Mixing the agrobacteria liquid induced in the IM liquid culture medium with the botrytis cinerea spores with the adjusted concentration in equal volume, taking 100 mu L of the mixture to be coated on the IM solid culture medium with the glass paper, coating 6 plates, sealing the plates, and culturing for 2 days at the temperature of 28 ℃ in a dark place. After the co-cultivation, the cellophane was transferred to PDA medium containing 100. Mu.g/mL hygromycin and cultivation was continued under the same conditions. After 4-7 days, the expanded colonies were picked up and subjected to secondary screening on screening media containing the same antibiotics.
D. Verification of transformants
Use of hygromycin primersBcSfp1The gene primers were screened for transformants by PCR amplification. The amplification results were determined to be consistent with the following resultsBcSfp1Gene deletion mutant:
the inner primers HpTa (5'-GTCGTTTGACAAGATGGTTCA-3') and HpTb (5'-CGTCTGCTGCTCCATACAA-3') of the hygromycin resistance gene can be amplified to a 993bp fragment (consistent with the plasmid positive control band size); spd1 gene primers (5'-ATGGCTCCAATCAATTTTGG-3') and (5'-TCAAGCTTCAACAATATGGTGA-3') did not amplify bands (amplified bands were present in wild type strain) (FIG. 3).
Screening from transformants to 4 independent strainsBcSfp1Gene deletion mutant: deltaBcSfp1-1、ΔBcSfp1-3、ΔBcSfp1-12 and deltaBcSfp1-19 (as shown in fig. 3) for subsequent functional analysis.
、BcSfp1Role of gene in growth and development process of botrytis cinerea
B05.10BcSfp1、△BcSfp1C, activating a filter paper sheet stored at the temperature of minus 80 ℃ on a PDA culture medium, reversely culturing the filter paper sheet in a 25 ℃ incubator for 2 d, then, using a puncher to punch out 6 mm uniform bacterial cakes, inoculating the bacterial cakes on a new PDA flat plate, reversely culturing the filter paper sheet in the 25 ℃ incubator, measuring the colony diameter every 12 h, and photographing and recording. Result discovery deltaBcSfp1The hyphal growth rate was faster than that of wild-type and complement (as shown in FIG. 4). It was shown that in the wild-type strain,BcSfp1negative regulation of hypha growth, and accelerated hypha growth after knocking out the gene.
Is H 2 O 2 Knock-out mutant deltaBcSfp1With wild strain B05.10 and complement deltaBcSfp1Oxidative stress analysis of-C
B05.10BcSfp1、△BcSfp1C, preserving filter paper sheets at the temperature of minus 80 ℃ to activate on a PDA culture medium, inversely culturing the filter paper sheets at the temperature of 25 ℃ in an incubator for 2 d, using a puncher to punch out 6 mm uniform bacterial cakes, inoculating the bacterial cakes on a new CM flat plate containing 0 mM, 2.5 mM, 5 mM and 10 mM, inversely culturing the filter paper sheets at the temperature of 25 ℃, measuring the bacterial colony diameter every 12 h, and photographing and recording. The colony morphology of 48H is shown in figure a, and different concentrations H are calculated by a calculation formula of the relative Inhibition Rate of the stress on the growth of the gray mold (Inhibition Rate (%) = (Dck-Dt)/Dck ×100% Dck: the colony diameter of the control; dt: the colony diameter of the sample) 2 O 2 For the mutant deltaBcSfp1With wild strain B05.10 and complement deltaBcSfp1-bacteriostasis rate of C and generating a histogram (as shown in fig. 5 b, C). The results indicate that when knocked outBcSfp1After the gene, deltaBcSfp1For H 2 O 2 Reduced adaptation of (c), descriptionBcSfp1Is involved in the stress response of gray mold bacteria to oxidative stress.
、BcSfp1Role of gene in pathogenicity of Botrytis cinerea
Inoculating spore liquid: culturing each strain under the same culture condition to produce spores,collecting and concentrating spore liquid, diluting the spore liquid obtained by centrifugation to 2.5X10 by using a volume buffer 5 And (3) fully and uniformly mixing the two parts per mL, sucking 4 mu L of spore liquid by using a liquid-transferring gun, dripping the spore liquid to two sides of the leaves of the host plant with the same batch size and good state, carrying out moisturizing culture on the inoculated host, observing the disease condition, and photographing and recording. We explored the differences in virulence of wild type, knockdown and complement to arabidopsis host plants by spore fluid vaccination experiments (shown in figure 6). The results indicate that when knocked outBcSfp1The pathogenic ability of Botrytis cinerea to host plants is obviously enhanced later, which indicatesBcSfp1Is involved in regulating the pathogenic process of Botrytis cinerea on host plants.
Knock-out mutant deltaBcSfp1ROS detection of Arabidopsis leaves inoculated with wild type Strain B05.10 as host plant
DAB (3, 3' -diaminobenzodine) staining was performed on the gray mold infected Arabidopsis leaves and bean leaves, and the DAB staining solution was required to be protected from light. Preparing a sample: with a fungus cake or spore liquid (2.5X10) 5 individual/mL) infects plant leaves, leaf samples are taken at different times of infestation; preparation of EP tube: wrapping the EP pipes by using tin paper, and placing the blades in the EP pipes with proper specifications, wherein the blades in each EP pipe cannot be placed too much; dyeing: pouring DAB staining solution into the EP tube to ensure that the blade is buried in the staining solution and can be fully stained, putting a sample into a shaking table, and vibrating at a low speed of 100 rpm for 4-5 h; bleaching: after the dyeing is finished, the dyeing liquid is poured off, and the leaves are eluted by using bleaching liquid (alcohol: acetic acid: glycerol=3:1:1); decoloring: sealing the sample, placing into boiled water, and decolorizing for 15 min. This bleaches chlorophyll, leaving a brown precipitate formed by the reaction of DAB with hydrogen peroxide. The decoloring time can be properly adjusted according to the appearance of the blade until the blade is completely free of chlorophyll; photographing: the leaves are then removed to become visual leaves, the brown stained parts and the uncolored parts can be seen, the photographing can be directly performed, and the microstructure of the sample can be photographed. B05.10 and DeltaABcSfp1At 48H of infection of Arabidopsis leaves 2 O 2 The dyeing result shows that the same timeΔBcSfp1More H is accumulated at the site of WT infection 2 O 2 (as shown in fig. 7). Analytical data relating to this experiment were derived from at least three independent experiments, each of which included three biological replicates. All quantitative analyses used relative quantification, i.e. the control group was set to 1. Significance analysis was performed by SPSS software using the Duncan method of single factor analysis of variance, significance results: p is p<0.05 Indicated by "; p is p<0.0 1, indicated by "×".
The above examples demonstrate thatBcSfp1The deletion of the gene leads to the enhancement of the pathogenicity of the botrytis cinerea and the reduction of the adaptability of the botrytis cinerea to oxidative stress, which indicatesBcSfp1The gene participates in the pathogenic process of the gray mold on the host plant and plays an important role in the growth and development of the gray mold and the stress response of the gray mold.
The non-pathogenic botrytis cinerea of the plant disease-resistant inducer prepared by the invention isBcSfp1The gene overexpression body is used for inducing the plants to generate immunity to the botrytis cinerea.
The invention is described inBcSfp1After the gene is introduced into plants, plant varieties which are remarkably resistant to gray mold diseases and oxidation stress are obtained.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.
Claims (3)
1.BcSfp1Application of gene in preparing medicine for preventing and treating botrytis cinerea, said medicine is prepared from plant materialBcSfp1The pathogenicity of gray mold bacteria is regulated and controlled by genes,BcSfp1the gene number is BCIN01G 01160; the medicine comprisesBcSfp1Gene promoters or containBcSfp1An over-expression vector for the gene.
2. The use according to claim 1, wherein:BcSfp1the gene and/or the coded protein regulate the oxidation stress resistance of the botrytis cinerea.
3. The use according to claim 1, wherein:BcSfp1the activity and/or the expression quantity of the gene or/and the coded protein in the botrytis cinerea are increased, so that the adaptability of the botrytis cinerea to oxidative stress is improved.
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