CN105483138B - One kind ash arrhizus bacteria gene BcAtm1 relevant to pathogenicity and its application - Google Patents
One kind ash arrhizus bacteria gene BcAtm1 relevant to pathogenicity and its application Download PDFInfo
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- CN105483138B CN105483138B CN201610044656.2A CN201610044656A CN105483138B CN 105483138 B CN105483138 B CN 105483138B CN 201610044656 A CN201610044656 A CN 201610044656A CN 105483138 B CN105483138 B CN 105483138B
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Abstract
One kind ash arrhizus bacteria gene BcAtm1 relevant to pathogenicity and its application microorganism belonging to genus gene engineering technology field, control mycelia growth provided by the invention from ash arrhizus bacteria and pathogenic gene BcAtm1, its DNA sequence dna is made of as shown in SEQ ID No:1 2203 nucleotide；The protein of the BcAtm1 coded by said gene of offer, amino acid sequence are made of as shown in SEQ ID No:2 715 amino acid；BcAtm1 gene can be applied in plant botrytis resistant genetic engineering field；By the way that ash arrhizus bacteria control mycelia growth and pathogenic PROTEIN B cAtm1 are lacked, are mutated or modified, and make its pathogenicity that defect occur, can be used as target and applied in design and screening antifungal medicine.
Description
Technical field
The invention belongs to technical field of microbial genetic engineering, and in particular to epiphyte pathogenic is controlled in plant protection art
The application of gene and its coding protein.
Background technique
Ash arrhizus bacteria (Botrytis cinerea) is usually also referred to as Botrytis cinerea, belongs to Ascomycota (Ascomycota)
Fungi is the pathogen of gray mold, can infect 200 various plants, including almost all of vegetables and fruit tree.Host from seedling stage,
Period can fall ill to storage period, moreover, each position of plant can be infected by ash arrhizus bacteria, the classical symptom table of leaf portion morbidity
It is now " V " shape scab, flower portion is mainly shown as that rotten and tune withers, and fruit, which is mainly shown as, to rot and fall off.The generation of disease and
There are close relationships for the humidity of sprawling and environment, temperature, occur at 20 DEG C -23 DEG C, 90% or more relative humidity serious.Cause
This, gray mold category low temperature and high relative humidity type disease easily occurs in rainy season or Protected production, in the world every year because of the disease
Caused economic loss is up to hundred million dollars of 100-1000.It is endangered since host range is extensive, in production seriously, along with related point
Sub- investigative technique is mature, and ash arrhizus bacteria has become one of most important model plant disease fungus, studied extensively.
Ash arrhizus bacteria is typical necrotrophic disease fungus, produces a variety of virulence factors and participates in causing a disease, main to wrap
Cell wall degradation enzyme, cutinase, toxin, plant hormone, enzyme, tiny RNA and the small-molecule substance of resisting defense enzymes etc. are included,
These factors, which cooperate, enables ash arrhizus bacteria to kill host cell, and decomposes dead host tissue as nutrition.It is natural
Under the conditions of, botrytis cinerea mostly infects source as the First aggression for infecting host and again using conidium.Ash arrhizus bacteria often with mycelium,
Conidium or sclerotium are attached on plant invalid body, or overwintering in the soil and more summer, become the First aggression of next Growing season
Source.When condition is suitable for, sclerotial germination aerial mycelium and conidiophore, and generate a large amount of conidium.Mature
Conidium can be propagated by wind, rainwater, the general water of filling and farming operations etc..Under the conditions of low temperature and high relative humidity, conidium
Sprouting forms germ tube, and germ tube end expands to develop into appresorium or be further formed slightly infects the Infection structures such as pad, mainly
From floral organ, wound and the necrotic tissue intrusion to decay.
When the ash arrhizus bacteria conidium of high concentration infects host, morbidity rapidly, mainly passes through germ tube top shape at this time
At appresorium intrusion；It reduces, is reduced by the ratio that germ tube top invades, onset speed also accordingly delays with spore concentration
1-4 days, at this time mainly by by hyphal development at appresorium or infect pad intrusion.It, will after ash arrhizus bacteria invades host cell
The challenge of hostile environment in host tissue can be directly faced, pathogen must adjust rapidly, on the one hand inhibit the anti-of plant
Imperial reaction, on the other hand wants physics, chemical environment in active adaption host cell, accomplishes these two aspects, ash arrhizus bacteria is just expected to
Successfully parasitic plant.Largely, ash arrhizus bacteria is the metabolic pathway by changing itself, and secrete correlation effect because
Sub (such as toxin) participates in gene, albumen and the metabolite of respective process and its molecule of regulation come realizing above-mentioned target
Mechanism is still known little.The field is furtherd investigate, identifies key factor of the ash arrhizus bacteria to adapt to environment in host,
It not only facilitates and discloses the pathogenic molecular mechanism of this necrotrophic disease fungus of ash arrhizus bacteria, it is also possible to which therefrom discovery can
Using the protein as fungicide action target, the efficient medicament for exploitation prevention and treatment gray mold and other similar diseases establishes reason
By and technical foundation.
Ash arrhizus bacteria BcAtm1 is a kind of ABC protein called membrane transporters (ATP-binding cassette transporter),
Its function is not yet identified, by analyzing the pathogenic function of BcAtm1 gene, is evaluated the gene and is developed and cause in ash arrhizus bacteria
The effect of sick process is conducive to identify potential prevention and treatment target, for screening novel antifungal medicament.
Summary of the invention
The purpose of the present invention is intended to provide a kind of protein for controlling mycelia growth and pathogenic gene and its coding.
Control mycelia provided by the present invention growth and pathogenic gene derive from ash arrhizus bacteria, entitled BcAtm1,
DNA sequence dna is as shown in SEQ ID No:1.The DNA sequence dna is BcAtm1 gene open reading frame, is made of 2203 nucleotide,
Wherein include 2 exons, is located between 5 ' the 1st to 2062 nucleotide in end of SEQ ID No:1 and the 2118th
To between 2203 nucleotide, the coding section length of composition adds up to 2148 nucleotide.
The present invention provides the protein of BcAtm1 coded by said gene, amino acid sequence, should as shown in SEQ ID No:2
Sequence is made of 715 amino acid.
The growth of control mycelia and pathogenic gene BcAtm1 from ash arrhizus bacteria can be applied to plant botrytis resistant gene
Engineering field.
To from ash arrhizus bacteria control mycelia growth and the encoded protein of pathogenic gene BcAtm1 lacked,
Mutation or modification, and make its pathogenicity that defect occur, it can be used as target and applied in design and screening antifungal medicine.
Present invention demonstrates that the missing or mutation of BcAtm1 gene, cause ash arrhizus bacteria pathogenicity to significantly reduce, explanation
BcAtm1 gene is that ash arrhizus bacteria causes gene necessary to crops gray mold.Therefore, screening can prevent the gene expression
The compound of expression, modification and positioning with its protein can effectively control the generation of gray mold, so that it is new to facilitate exploitation
One important use of type fungicide, i.e., BcAtm1 gene provided by the present invention is: the egg that the expression of the gene is encoded with it
Expression, modification and the positioning of white matter product can be used as important candidate targets site, be used for antifungal medicine (especially anti-ash
Mildew bacterium medicament) design and screening.
Detailed description of the invention
Fig. 1 is the structure domain analysis schematic diagram of BcAtm1 protein
Wherein: Atm1 is the conserved domain of one kind ABC protein called membrane transporters；
Fig. 2 is knockout strategy (carrying out gene replacement by homologous recombination) schematic diagram of ash arrhizus bacteria BcAtm1 gene
Wherein: WT is wild-type strain B05.10, and pAtm1-ko is knockout carrier, and BcAtm1-KO lacks for BcAtm1 gene
Mutant is lost, a, b, c, d are the primer for verifying mutant and complementing strain；
Fig. 3 is the PCR verifying electrophoretogram of BcAtm1 deletion mutant body and genetic complement bacterial strain
Wherein: a, b, c, d are the primer, and Fig. 2 is seen in corresponding position；M1 is BcAtm1 deletion mutant body；M1/Atm1
For the complementing strain for being transferred to complete BcAtm1 gene on the basis of mutant M1；
Fig. 4 is that the deletion mutant of BcAtm1 gene and the cultural characteristic of wild-type strain B05.10 compare photo
Wherein: used medium PDA, 20 DEG C of cultures, observation in 3 days is taken pictures after inoculation；
Fig. 5 be BcAtm1 gene deletion mutant compared with the pathogenicity of wild-type strain and complementing strain photo
Wherein: selected host is Kidney bean, using the method for Isolated leaf inoculation spore.Inoculation is evaluated after 3 days；
Fig. 6 is that the quantitative analysis that the mutant of BcAtm1 gene infects Lesion size produced by host with control strain is illustrated
Figure
Wherein: inoculation method is same as above, and inoculation measured calculating to leaf spot lesion area after 3 days, is converted into relatively large
It is small.* is indicated in the horizontal upper significant difference of p < 0.01.
Specific embodiment
It in order to better describe the present invention, is further described below by specific embodiment, following embodiments
In method be unless otherwise instructed conventional method.
The correlation analysis of 1 BcAtm1 gene of embodiment
The open reading frame of ash arrhizus bacteria BcAtm1 gene is made of 2203 nucleotide, includes 2 exons, code area
CDNA overall length is 2148 nucleotide, and the protein product of coding is made of 715 amino acid, structure domain analysis discovery,
BcAtm1 protein includes the conserved domain Atm1 of an ABC protein called membrane transporters (see Fig. 1).
The knockout of 2 BcAtm1 gene of embodiment
1) building of knockout carrier
Using primer Atm1-UP-F (5'-GATCTTCACTAGTGGGAATTC GATGCGGACAATGAGACTGAA-3')
With Atm1-UP-R (5'-TTGGGTACCGAGCTCGAATTC GCTCTGGATAGCACTGCCTTT-3'), with ash arrhizus bacteria bacterial strain
The genomic DNA of B05.10 is template amplification BcAtm1 upstream region of gene 871bp segment, using Atm1-DN-F (5'-
AAAGATCAAAGGATCGTCGAC GGAGAAGGGTCAAGAACAAGAAG-3') and Atm1-DN-R (5'-
CTTGCATGCCTGCAGGTCGAC GGAATAGTATAAGCTCCAAGGCAC-3') amplification ash arrhizus bacteria BcAtm1 downstream of gene
683bp segment, reaction system are as follows: 10mmol/L dNTP Mixture, 0.5 μ L；10 × PCR buffer, 2.5 μ L；Upstream and downstream
Each 1 μ L of primer (10 μm of ol/mL)；Template DNA, 1 μ L；Ex-Taq, 0.2 μ L (5U)；ddH2O, 18.8 μ L；Amplification program are as follows: 94
It DEG C initial denaturation 3 minutes, then (1) 94 DEG C, is denaturalized 50 seconds；It (2) 58 DEG C, anneals 50 seconds；(3) 72 DEG C, extend 60 seconds；(4) it recycles
30 times；(5) 72 DEG C extend 10 minutes.By above-mentioned two segment DNAs amplified production be cloned into respectively the EcoR I site of pXEH carrier with
Sal I site is built into knockout carrier pAtm1-ko (see Fig. 2), and carries out sequence verification.
2) conversion of ash arrhizus bacteria
A. the culture of Agrobacterium
Picking contains the Agrobacterium tumefaciens strain Agl-1 single colonie of binary vector pAtm1-ko, is seeded to containing 50 μ g/ml cards
MM fluid nutrient medium (dipotassium hydrogen phosphate 0.205%, potassium dihydrogen phosphate 0.145%, sodium chloride of that mycin, 10 μ g/ml rifampins
0.015%, epsom salt 0.05%, calcium chloride hexahydrate 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%,
Glucose 0.2%) in, 250rpm, 28 DEG C of shaken cultivation 48h；4000rpm is centrifuged 5 minutes, abandons supernatant, IM fluid nutrient medium
(dipotassium hydrogen phosphate 0.205%, potassium dihydrogen phosphate 0.145%, sodium chloride 0.015%, epsom salt 0.05%, six water chlorinations
Calcium 0.01%, ferrous sulfate heptahydrate 0.00025%, ammonium sulfate 0.05%, glucose 0.2%, 200 μM of AS, MES 0.854%,
Glycerol 0.5%) it is resuspended, 4000rpm is centrifuged 5 minutes, abandons supernatant；IM culture medium is resuspended, and 28 DEG C, 250rpm shaken cultivation 6h, into
Row pre-induced.
B. the production spore culture of ash arrhizus bacteria
B05.10 bacterial strain is selected, a small amount of spore is taken to be coated on PDA culture medium (the well-done filtering of potato 20%, glucose
2%, agar 1.5%), set 28 DEG C of culture 8h and enable spore fast-germination, be then transferred to 20 DEG C and cultivate 3-5 days, to phage surface
After the covering of grey spore, with IM fluid nutrient medium scraping, spore is collected, micro- sem observation is adjusted using haemocytometer
Spore concentration is 1 × 106/mL。
C. Agrobacterium tumefaciems and the co-cultivation of ash arrhizus bacteria conidium and transformant screening
The Agrobacterium bacterium solution for inducing 6h in advance in IM fluid nutrient medium and ash arrhizus bacteria spore liquid are mixed in equal volume, added
Enter AS, final concentration is made to reach 500 μM, mix, then press 250~350 μ L/ wares, is uniformly applied to the IM culture for being covered with glassine paper
On base, 22 DEG C of dark culturing 48h；After co-cultivation, glassine paper is transferred to the PDA culture medium containing 100 μ g/mL hygromycin
On, continue to cultivate under the same terms.On the bacterium colony that picking extends after 4~7 days to the screening and culturing medium containing same antibiotic.
3) verifying of deletion mutant
Two pairs of primers are selected to screen by PCR amplification to transformant.Amplification meets following result, is determined as
BcAtm1 deletion mutant body: the primer a (5'-TCCGCTCACTCCAACCAA-3') except the homology arm of upstream on genome
Primer b (5'-ACAGACGTCGCGGTGAGTTCA-3') pairing with hygromycin gene can be expanded to expected size
The recombinant fragment of (1.3kb)；And code area primer c (5'-GCTTATGGACTTACCAGGATTG-3') and d (5'-
TCGGAGGACCGTTGATTTT-3') without amplified band (wild-type strain is amplifiable to arrive 0.9kb segment).As a result, from transformant
In screen 1 plant of BcAtm1 deletion mutant body: M1, for follow-up function analysis (see Fig. 3).
The genetic complement of 3 BcAtm1 deletion mutant body of embodiment
Using primer C-F (5'-AAAGATCAAAGGATCGAATTCAGCAAGCCATCCTTCCTATC-3') and C-R (5'-
CCGGGTACCGAGCTCGAATTCCATCTGGACCCATCACTACAT-3'), ash arrhizus bacteria BcAtm1 full length gene is expanded
3513bp (include promoter, open reading frame and terminator), is first cloned on pMD18-t carrier, be then subcloned again to
The EcoR I site of pXEB carrier (containing glufosinate resistance gene), is built into genetic complement carrier pAtm1-ko-c.Carrier warp
Sequence verification confirms no amino acid mutation.Using foregoing Agrobacterium-medialed transformation method, 200 μ g/mL grass are used
Ammonium phosphine is screened, which is transferred in BcAtm1 deletion mutant body M1 genome, obtains genetic complement bacterial strain
M1/Atm1.It selects the primer a once used when mutant verifying and b, c and d to carry out PCR amplification, as a result meets expected (see Fig. 3):
Identical as mutant M1, original BcAtm1 gene is replaced by hygromycin gene HPH and (draws in complementing strain M1/Atm1
Object a and b amplification are the positive), but additionally possess subsequent BcAtm1 gene (code area primer c and the d amplification knot being transferred to
Fruit is similarly positive).
Effect of the 4 BcAtm1 gene of embodiment in the mycelia growth course of ash arrhizus bacteria
Using plating method, the variation situation of the Relevant phenotypes such as the mycelia growth of BcAtm1 mutant is evaluated.Take 10 μ L
Strain to be tested PDB spore suspension (1 × 106ml-1) it is seeded in the center of PDA culture medium, 20 DEG C of dark culturings.It is observed after three days
It was found that the colonial morphology of mutant is normal, but the speed of growth significantly slows down, and bacterium colony size only has the half of wild type left
It is right.In genetic complement bacterial strain, mycelial growth rate is significantly speeded, substantially back to normal, it was demonstrated that BcAtm1 gene, which has, to be maintained
The ability of mycelia normal growth (see Fig. 4).
Effect of the 5 BcAtm1 gene of embodiment at the pathogenic aspect of ash arrhizus bacteria
Using Isolated leaf inoculation method, the pathogenicity situation of change of BcAtm1 mutant is evaluated.From the Kidney bean of hot-house culture
Mature leaf is acquired on plant, is horizontally arranged in container, is taken 15 μ L strain to be tested PDB spore suspensions (1 × 105ml-1) point connect
On blade face, 20 DEG C of moisturizing dark culturings evaluate the pathogenicity of strain to be tested after 3 days.Experimental result shows, BcAtm1 mutant
Substantially pathogenecity is lost, small scab can only be found near vaccination, and do not extend.Form distinct contrast with this
It is that wild type can successfully infect tomato leaf, and spread to nearly half of blade face rapidly；Genetic complement bacterial strain M1/Atm1's causes a disease
Power is back to normal, basically reaches wild-type levels (see Fig. 5).Calculating is measured to leaf spot lesion area, discovery BcAtm1 is prominent
Lesion area caused by variant infects only has 5% or so caused by wild type (see Fig. 6).The result of study shows that BcAtm1 is
One crucial Disease-causing gene is necessary to ash arrhizus bacteria infects host, if the protein of the gene or its coding is lost
Activity, ash arrhizus bacteria will lose the ability for infecting host and causing disease.
Claims (1)
1. one kind from ash arrhizus bacteria (Botrytis cinerea) control pathogenic geneBcAtm1In plant botrytis resistant
Application in genetic engineering field, the DNA sequence dna of the gene is as shown in SEQ ID No:1.
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