CN105483106A - Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1 - Google Patents
Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1 Download PDFInfo
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Abstract
The invention provides a botrytis cinerea gene BcFch1 relative to pathogenicity and application of the botrytis cinerea gene BcFch1, and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcFch1 sourced from botrytis cinerea and used for controlling pathogenicity is shown in SEQ ID No:1, and comprises 1409 nucleotides. The amino acid sequence of protein coded by the gene BcFch1 is shown in SEQ ID No:2, and comprises 435 amino acids. The gene BcFch1 can be applied to the field of gene engineering of plant botrytis cinerea-resisting gray molds. Deletion, mutation or modification is conducted on the protein coded by the gene BcFch1 used for controlling the pathogenicity of botrytis cinerea to ensure that the pathogenicity of protein is flawed, and the flawed protein can be applied to design and screening of antifungal medicaments while serving as a target.
Description
Technical field
The invention belongs to technical field of microbial genetic engineering, be specifically related in plant protection art, control the gene of epiphyte pathogenic and the application of coded protein thereof.
Background technology
Ash arrhizus bacteria (Botrytiscinerea), usually also known as doing Botrytis cinerea, belongs to Ascomycota (Ascomycota) fungi, is the pathogenic bacteria of gray mold, can infects 200 various plants, comprise nearly all vegetables and fruit tree.Host all can fall ill from seedling stage, the phase of bearing fruit to storage period, and each position of plant all can be infected by ash arrhizus bacteria, and the classical symptom of leaf portion morbidity shows as " V " shape scab, and flower portion main manifestations is rotten and tune withers, and fruit main manifestations is for rotting and coming off.The generation of disease and spread and there is close relationship with the humidity of environment, temperature, at 20 DEG C-23 DEG C, occurs serious when relative humidity more than 90%.Therefore, gray mold belongs to low temperature and high relative humidity type disease, very easily occurs in rainy season or Protected production, and the annual financial loss caused because of this disease is up to 100-1000 hundred million dollars in the world.Because host range is extensive, in production, harm is serious, and add associated molecule investigative technique maturation, ash arrhizus bacteria has become one of most important model plant pathogenic fungi, is subject to extensive research.
Ash arrhizus bacteria is typical necrotrophic pathogenic fungi, multiple virulence factor can be generated participate in causing a disease, mainly comprise cell wall degradation enzyme, at, toxin, plant hormone, the enzyme of opposing defense enzymes, tiny RNA and small-molecule substance etc., these factors mutually cooperate and enable ash arrhizus bacteria kill host cell, and decompose dead host tissue as nutrition.Under natural condition, botrytis cinerea mainly with conidium as infecting the First aggression of host and infecting source again.Ash arrhizus bacteria is often attached on plant invalid body with mycelium, conidium or sclerotium, or survives the winter in soil and get over the summer, becomes the primary infection inoculum of next Growing season.When condition is suitable for, sclerotial germination aerial mycelium and conidiophore, and produce a large amount of conidiums.Ripe conidium can be propagated by wind, rainwater, the general water of filling and farming operation etc.Under low temperature and high relative humidity condition, conidia germination forms germ tube, and germ tube end expands slightly to develop into appressorium or formed further and infects the Infection structures such as pad, mainly invades from the floral organ of decaying, wound and necrotic tissue.
When the ash arrhizus bacteria conidium of high density infects host, rapidly, the appressorium now formed mainly through germ tube top invades in morbidity; Reduce with spore concentration, the ratio that invaded by germ tube top is reduced, and onset speed is also corresponding delays 1-4 days, now mainly through the appressorium that become by hyphal development or infect pad and invade.After ash arrhizus bacteria invades host cell, directly will face the challenge of hostile environment in host tissue, pathogenic bacteria must adjust rapidly, suppress the defensive raction of plant on the one hand, want physics, chemical environment in active adaption host cell on the other hand, accomplish these two aspects, ash arrhizus bacteria is just expected to successfully phytoparasite.To a great extent, ash arrhizus bacteria is the pathways metabolism by changing self, and the relevant effector (as toxin) of secretion realizes above-mentioned target, but participate in the gene of respective process, the molecular mechanism of albumen and meta-bolites and regulation and control thereof still knows little.This field is furtherd investigate, qualification ash arrhizus bacteria is in order to adapt to the key factor of host's environment, not only contribute to disclosing the pathogenic molecular mechanism of this necrotrophic pathogenic fungi of ash arrhizus bacteria, also likely therefrom finding can as the protein of mycocide action target, and theory and technology basis established by the efficient medicament preventing and treating gray mold and other similar disease for exploitation.
Fch1 is a kind of ferrochelatase, participates in final step reaction (the 8th step) of catalysis protoheme route of synthesis.This albumen is extensively present in the biologies such as animal, plant, bacterium, fungi, and because protoheme can participate in multiple vital process as the prothetic group of numerous albumen, therefore, the Fch1 protein as one of catalysis heme synthetase class has important function.Fch1 protein exists equally in ash arrhizus bacteria, and function not yet obtains qualification, by analyzing the pathogenic function of ash arrhizus bacteria Fch1 encoding gene, evaluate this gene to grow and the effect of pathogenic course at ash arrhizus bacteria, be conducive to identifying potential control target, for screening novel fungicidal medicament.
Summary of the invention
Object of the present invention aims to provide and a kind ofly controls pathogenic gene and the protein of coding thereof.
Control pathogenic gene provided by the present invention derives from ash arrhizus bacteria, and name is called BcFch1, and its DNA sequence dna is as shown in SEQIDNo:1.This DNA sequence dna is BcFch1 gene open reading frame, be made up of 1409 Nucleotide, wherein comprise 3 exons, lay respectively at SEQIDNo:1 5 ' end the 1st to 267 Nucleotide between, between the 321st to 903 Nucleotide and between the 952nd to 1409 Nucleotide, the coding region length of composition adds up to 1308 Nucleotide.
The invention provides the protein of BcFch1 coded by said gene, its aminoacid sequence is as shown in SEQIDNo:2, and this sequence is made up of 435 amino acid.
Control pathogenic gene BcFch1 from ash arrhizus bacteria can be applicable to Genes For Plant Tolerance gray mold genetically engineered field.
Protein coded by control pathogenic gene BcFch1 from ash arrhizus bacteria is lacked, suddenlys change or modified, and makes its virulence generation defect, can be used as target and apply in design and screening antifungal medicine.
Present invention demonstrates that disappearance or the sudden change of BcFch1 gene, cause ash arrhizus bacteria virulence significantly to reduce, illustrate that BcFch1 gene is that ash arrhizus bacteria causes the necessary gene of farm crop gray mold.Therefore, screening can stop the compound of this genetic expression and its protein expression, modification and location, effectively can control the generation of gray mold, thus contribute to development of new sterilant, namely an important use of BcFch1 gene provided by the present invention is: the expression of the protein of the expression of this gene and its coding, modification and location, can as important candidate targets site, for design and the screening of antifungal medicine (particularly botrytis resistant bacterium medicament).
Accompanying drawing explanation
Fig. 1 is the domain analyses schematic diagram of BcFch1 protein
Wherein: Ferrochelatase is ferrochelatase structural domain;
Fig. 2 be ash arrhizus bacteria BcFch1 gene knock out strategy (carrying out gene replacement by homologous recombination) schematic diagram
Wherein: WT is wild type strain B05.10, pFch1-ko is knockout carrier, BcFch1-KO is BcFch1 deletion mutant body, and a, b, c, d are the primer for verifying mutant and complemented strain;
Fig. 3 is that the PCR of BcFch1 deletion mutant body and genetic complementation bacterial strain verifies electrophorogram
Wherein: a, b, c, d are the primer, and Fig. 2 is seen in corresponding position; M1 is BcFch1 deletion mutant body; M1/Fch1 is the complemented strain proceeding to complete BcFch1 gene on mutant M1 basis;
Fig. 4 is that the mutant of BcFch1 gene compares photo with the virulence of wild type strain
Wherein: selected host is tomato, adopt the method for Isolated leaf inoculation bacterium cake, inoculate and evaluate after 3 days; M1, M2 are the BcFch1 deletion mutant body that two strains independently obtain;
The mutant that Fig. 5 is BcFch1 gene and control strain infect host produce the quantitative analysis schematic diagram of Lesion size
Wherein: inoculation method is the same, inoculate, after 3 days, survey calculation is carried out to leaf spot lesion area, be converted into relative size.* represents significant difference in p<0.01 level.
Embodiment
In order to describe the present invention better, being further described below by specific embodiment, the method in following embodiment, if no special instructions, being ordinary method.
The correlation analysis of embodiment 1BcFch1 gene
The open reading frame of ash arrhizus bacteria BcFch1 gene is made up of 1409 Nucleotide, comprises 3 exons, and coding region cDNA total length is 1308 Nucleotide, and the protein of coding is made up of 435 amino acid.Get BcFch1 protein sequence to compare (http://blast.ncbi.nlm.nih.gov/Blast.cgi), find that Fch1 is extensively present in the cell biologicals such as animal, plant, fungus and bacterium.Domain analyses finds, BcFch1 protein comprises a conservative ferrochelatase structural domain (see Fig. 1).
Knocking out of embodiment 2BcFch1 gene
1) structure of knockout carrier
Adopt primers F ch1-UP-F (5'-CTCGAGAAGAGCGGCAAGCGTGGGA-3') and Fch1-UP-R (5'-GGTACCGGTACTCGAAAGGCAGACAGA-3'), with the genomic dna of ash arrhizus bacteria bacterial strain B05.10 for template amplification BcFch1 upstream region of gene 794bp fragment, Fch1-DN-F (5'-GGATCCGATGTCCTGGATGCAAGAGTGA-3') and Fch1-DN-R (5'-CTGCAGGTGGGTTGGTGTACGATATTCG-3') is adopted to increase ash arrhizus bacteria BcFch1 downstream of gene 767bp fragment, reaction system is: 10mmol/LdNTPMixture, 0.5 μ L, 10 × PCRbuffer, 2.5 μ L, the each 1 μ L of upstream and downstream primer (10 μm of ol/mL), template DNA, 1 μ L, Ex-Taq, 0.2 μ L (5U), ddH
2o, 18.8 μ L, amplification program is: 94 DEG C of denaturations 3 minutes, then (1) 94 DEG C, sex change 50 seconds, (2) 58 DEG C, anneal 50 seconds, (3) 72 DEG C, extend 60 seconds, (4) circulate 30 times, (5) 72 DEG C extend 10 minutes.Between XhoI, KpnI site above-mentioned two segment DNA amplified productions being successively cloned into pXEH carrier and between BamHI, PstI site, be built into knockout carrier pFch1-ko (see Fig. 2), and carry out sequence verification.
2) conversion of ash arrhizus bacteria
A. the cultivation of Agrobacterium
Picking contains the mono-bacterium colony of Agrobacterium tumefaciens strain Agl-1 of binary vector pFch1-ko, be seeded to the MM liquid nutrient medium (dipotassium hydrogen phosphate 0.205% containing 50 μ g/ml kantlex, 10 μ g/ml Rifampins, potassium primary phosphate 0.145%, sodium-chlor 0.015%, magnesium sulfate heptahydrate 0.05%, calcium chloride hexahydrate 0.01%, iron vitriol 0.00025%, ammonium sulfate 0.05%, glucose 0.2%) in, 250rpm, 28 DEG C of shaking culture 48h; 4000rpm, centrifugal 5 minutes, abandons supernatant, IM liquid nutrient medium (dipotassium hydrogen phosphate 0.205%, potassium primary phosphate 0.145%, sodium-chlor 0.015%, magnesium sulfate heptahydrate 0.05%, calcium chloride hexahydrate 0.01%, iron vitriol 0.00025%, ammonium sulfate 0.05%, glucose 0.2%, 200 μMs of AS, MES0.854%, glycerine 0.5%) resuspended, centrifugal 5 minutes of 4000rpm, abandons supernatant; IM substratum is resuspended, 28 DEG C, and 250rpm shaking culture 6h carries out pre-induced.
B. the product spore of ash arrhizus bacteria is cultivated
Select B05.10 bacterial strain, the spore that takes a morsel coats (the well-done filtration of potato 20% of PDA substratum, glucose 2%, agar 1.5%), put 28 DEG C of cultivation 8h and make spore fast-germination, be then transferred to 20 DEG C and cultivate 3-5 days, after treating that phage surface is covered by grey spore, by IM liquid nutrient medium scraping, collect spore, microscopic examination, utilize Hematocyte Counter to regulate spore concentration to be 1 × 10
6/ mL.
C. agrobacterium tumefaciens and ash arrhizus bacteria conidium Dual culture and transformant screening
Agrobacterium bacterium liquid and the mixing of ash arrhizus bacteria spore liquid equal-volume of 6h will be induced in advance in IM liquid nutrient medium, and add AS, make final concentration reach 500 μMs, mixing, then by 250 ~ 350 μ L/ wares, uniform application on the IM substratum being covered with glassine paper, 22 DEG C of dark culturing 48h; After Dual culture, glassine paper is transferred on the PDA substratum containing 100 μ g/mL Totomycin, continues under the same terms to cultivate.After 4 ~ 7 days, the bacterium colony of picking expansion is to containing in same antibiotic screening culture medium.
3) checking of deletion mutant
Two pairs of primers are selected to be screened transformant by pcr amplification.Amplification meets following result, is defined as BcFch1 deletion mutant body: the primer b (5'-ACAGACGTCGCGGTGAGTTCA-3') of the primer a (5'-TTGTTCGCTTCCTCTTCGTC-3') outside the homology arm of upstream on genome and hygromycin gene matches to increase to and expects the recombinant fragment of size (1.5kb); And coding region primer c (5'-GGGGATTTCTTGAGTAGATTATTTG-3') and d (5'-CATCACGGCGTAGACTGTAGC-3') are without amplified band (wild type strain can increase 0.8kb fragment).As a result, from transformant, 2 strain BcFch1 deletion mutant bodies are screened: M1 and M2 bacterial strain, for follow-up function analysis (see Fig. 3).
The genetic complementation of embodiment 3BcFch1 deletion mutant body
Adopt primer C-F (5'-GAATTCTTGTTCGCTTCCTCTTCGTC-3') and C-R (5'-CTGCAGGTGGGTTGGTGTACGATATTCG-3'), amplification ash arrhizus bacteria BcFch1 full length gene 3116bp (comprising promotor, open reading frame and terminator), first be cloned on pMD18-t carrier, and then subclone to pSULF carrier (containing chlorimuronethyl resistant gene) EcoRI and PstI site between, be built into genetic complementation carrier pFch1-ko-c.Carrier, through sequence verification, confirms do not have amino acid mutation.Adopt foregoing Agrobacterium-medialed transformation method, use 100 μ g/mL chlorimuronethyls to screen, this complementary fragment is proceeded in BcFch1 deletion mutant body M1 genome, obtain genetic complementation bacterial strain M1/Fch1.Once primer a and the b, c and the d that used when selecting mutant to verify carry out pcr amplification, result meets expection (see Fig. 3): identical with mutant M1, in complemented strain M1/Fch1, original BcFch1 gene is replaced by hygromycin gene HPH (primer a and b amplification are for positive), but additionally has a follow-up BcFch1 gene (coding region primer c and d amplification are similarly positive) proceeded to.
The effect of embodiment 4BcFch1 gene in ash arrhizus bacteria is pathogenic
Adopt Isolated leaf inoculation method, evaluate the virulence changing conditions of BcFch1 mutant.Gather mature leaf from the tomato plant of hot-house culture, in horizontal positioned container, use punch tool to beat and get test strains bacterium cake, face down left-hand thread on blade, 20 DEG C of moisturizing dark culturing, the virulence of 3 days postevaluation test strains.Experimental result shows, and BcFch1 mutant loses pathogenecity substantially, can only find small scab, and do not expand near vaccination.Form distinct contrast therewith, wild-type successfully can infect tomato leaf, and rapid spread to more than half blade face (see Fig. 4).The virulence of genetic complementation bacterial strain M1/Fch1 is normal, can return to wild-type levels.Carry out survey calculation to leaf spot lesion area, discovery BcFch1 mutant infects 20% (see Fig. 5) that the not enough wild-type of the lesion area caused causes.Above-mentioned incidence of leaf surface is carried out disinfection, is applied on PDA flat board after grinding, cultivate and count ash arrhizus bacteria bacterium colony number afterwards in 2 days.Research finds, the thalline that the blade that BcFch1 mutant infects almost is not lived after three days in inoculation, and can obtain more or less a hundred bacterium colony after the blade grinding that wild-type infects.This result of study shows, BcFch1 is a crucial Disease-causing gene, and it is necessary to be that ash arrhizus bacteria infects host, if the protein loss of activity of this gene or its coding, ash arrhizus bacteria infects losing the ability that host causes disease.
Sequence table
The sequence of SEQIDNo:2
(i) sequence signature: (A) length: 435 amino acid; (B) type: amino acid; (C) chain: strand.
(ii) molecule type: polypeptide
(iii) sequence description: SEQIDNo:2
Claims (4)
1., from a control pathogenic gene BcFch1 for ash arrhizus bacteria (Botrytiscinerea), it is characterized in that its DNA sequence dna is as shown in SEQIDNo:1.
2. the protein coded by the control pathogenic gene BcFch1 from ash arrhizus bacteria according to claim 1, is characterized in that its aminoacid sequence is as shown in SEQIDNo:2.
3. described in claim 1 from the application of control pathogenic gene BcFch1 in Genes For Plant Tolerance gray mold genetically engineered field of ash arrhizus bacteria.
4. lack from the protein coded by the control pathogenic gene BcFch1 of ash arrhizus bacteria, suddenly change or modify described in pair claim 2, make its virulence generation defect, as the application of target in design and screening antifungal medicine.
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| CN113684285A (en) * | 2021-04-25 | 2021-11-23 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof |
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| CN113684285A (en) * | 2021-04-25 | 2021-11-23 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof |
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