CN106520786B - One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application - Google Patents
One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application Download PDFInfo
- Publication number
- CN106520786B CN106520786B CN201610962073.8A CN201610962073A CN106520786B CN 106520786 B CN106520786 B CN 106520786B CN 201610962073 A CN201610962073 A CN 201610962073A CN 106520786 B CN106520786 B CN 106520786B
- Authority
- CN
- China
- Prior art keywords
- bcpck1
- gene
- arrhizus bacteria
- ash arrhizus
- spore
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application microorganism belonging to genus gene engineering technology field, regulation spore germination, spore output and pathogenic gene BcPCK1 provided by the invention from ash arrhizus bacteria, its DNA sequence dna is made of as shown in SEQ ID No:1 2644 nucleotide；The protein of the BcPCK1 coded by said gene of offer, amino acid sequence are made of as shown in SEQ ID No:2 560 amino acid；BcPCK1 gene can be applied in plant botrytis resistant genetic engineering field；Regulation spore germination, spore output and the encoded protein of pathogenic gene BcPCK1 from ash arrhizus bacteria can be used as target and apply in design and screening botrytis resistant bacterium medicament.
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 400 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, irrigation water 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 Pck1 is a kind of phosphoenolpyruvate carboxykinase (phosphoenolpyruvatecarboxyk
Inase, PEPCK), it participates in catalysis oxaloacetic acid and generates phosphoenolpyruvate, be second rate-limiting enzyme of gluconeogenesis approach,
By analyzing the pathogenic function of its encoding gene BcPCK1, the gene is evaluated in ash arrhizus bacteria and regulates and controls spore germination, spore output
And the effect of pathogenic course, be 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 regulation spore germination, spore output and pathogenic gene and its coding
Protein.
Regulation spore germination, spore output and pathogenic gene provided by the present invention derive from ash arrhizus bacteria, entitled
BcPCK1, DNA sequence dna is as shown in SEQ ID No:1.The DNA sequence dna is BcPCK1 gene open reading frame, by 2644 cores
Thuja acid composition is located between 5 ' the 1st to 277 nucleotide in end of SEQ ID No:1 wherein including 4 exons, the
Between 327 to 844 nucleotide, between the 895th to 1446 nucleotide, between the 1891st to 2226 nucleotide,
The coding section length of composition adds up to 1683 nucleotide.
The present invention provides the protein of BcPCK1 coded by said gene, amino acid sequence, should as shown in SEQ ID No:2
Sequence is made of 560 amino acid.
Regulation spore germination, spore output and pathogenic gene BcPCK1 from ash arrhizus bacteria can be applied to Genes For Plant Tolerance ash
Mildew genetic engineering field.
Regulation spore germination, spore output and the encoded protein of pathogenic gene BcPCK1 from ash arrhizus bacteria can
It is applied to the design and screening of botrytis resistant bacterium medicament as target.
Present invention demonstrates that the missing or mutation of BcPCK1 gene, cause ash arrhizus bacteria pathogenicity to significantly reduce, explanation
BcPCK1 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., BcPCK1 gene provided by the present invention is: the egg that the expression of the gene is encoded with it
White matter product can be used as important candidate targets site, the design and screening for botrytis resistant bacterium medicament.
Ash arrhizus bacteria bacterial strain B05.10 used in the present invention is bought from U.S.'s genetic of fungi material collection
(Fungal Genetics Stock Center, FGSC), other staff such as need the bacterial strain, can buy and obtain from the collection
, relevant information is as follows: collection address: Fungal Genetics Stock Center, Department of Plant
Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center,
Manhattan,KS 66506 USA；Network address:http://www.fgsc.net/scripts/ StrainSearchReturnPage.asp? OrgID=23812；Strain number: FGSC 10317.
Detailed description of the invention
Fig. 1 is the structure domain analysis schematic diagram of BcPck1 protein
Wherein: PEPCK is the conserved domain of phosphoenolpyruvate carboxykinase；
Fig. 2 is knockout strategy (carrying out gene replacement by homologous recombination) schematic diagram of ash arrhizus bacteria BcPCK1 gene
Wherein: WT is wild-type strain B05.10, and PXEH-KO is knockout carrier
Fig. 3 is Semiquatitative RT-PCR assay electrophoresis of the BcPCK1 gene in wild type, deletion mutant and genetic complement bacterial strain
Figure
Wherein: pair of primers PCKU, PCKD and another pair for being designed using BcPCK1 gene cDNA do the primer of internal reference
Actin-U, Actin-D carry out semiquantitive PCR simultaneously；WT is wild-type strain B05.10, and Δ Bcpck1 lacks for BcPCK1 gene
Mutant is lost, Bcpck1-C is the complementing strain that complete BcPCK1 gene is transferred on the basis of BcPCK1 deletion mutant body；
Fig. 4 is the deletion mutant and genetic complement bacterial strain spore germination rate of wild-type strain B05.10, BcPCK1 gene
Statistical chart
Wherein: taking concentration respectively is 1 × 105/ mL conidium mixes with PDB solution 1:1, and drop 5ul mixed liquor is in load glass
On piece cultivates 2h, 4h, 6h in 20 DEG C of constant temperature respectively, respectively at micro- sem observation, counts germination rate using blood counting chamber；***
Indicate extremely significant in the horizontal upper difference of p < 0.001.
Fig. 5 is the deletion mutant of BcPCK1 gene and the production spore photo of wild-type strain and complementing strain
Fig. 6 is the deletion mutant of BcPCK1 gene and the sporulation quantity statistical chart of wild-type strain and complementing strain
Wherein: * * * indicates extremely significant in the horizontal upper difference of p < 0.001.
Fig. 7 be BcPCK1 gene deletion mutant compared with the pathogenicity of wild-type strain and complementing strain photo
Wherein: selected host is tomato leaf, using the method for Isolated leaf inoculation spore.Inoculation is commented after 3 days
Valence；
Fig. 8 is that the quantitative analysis that the mutant of BcPCK1 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；* * indicates extremely significant in the horizontal upper difference of p < 0.001.
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 BcPCK1 gene of embodiment
The open reading frame of ash arrhizus bacteria BcPCK1 gene is made of 2644 nucleotide, includes 4 exons, code area
CDNA overall length is 1683 nucleotide, and the protein product of coding is made of 560 amino acid, structure domain analysis discovery,
BcPck1 protein function structural domain is very conservative (see Fig. 1).
The knockout of 2 BcPCK1 gene of embodiment
1) building of knockout carrier
Using primer PCK-UP-F (5'-AGATCTCGGAGCATGTGAATGCCAAGT-3') and PCK-UP-R (5'-
GAGCTCAGCCGCCGTCGTTCGTAAAT-3'), using the genomic DNA of ash arrhizus bacteria bacterial strain B05.10 as template amplification
BcPCK1 upstream region of gene 555bp segment, using PCK-DN-F (5'-GTCGACAAGAAATACCAGTCGGAAGCTA-3') with
PCK-DN-R (5'-CTGCAGGAAAGCCAAACCTACAAAGTTC-3') expands ash arrhizus bacteria BcPCK1 downstream of gene 710bp piece
Section, reaction system are as follows: 10mmol/L dNTP Mixture, 0.5 μ L；10 × PCRbuffer, 2.5 μ L；Each 1 μ L of upstream and downstream primer
(10μmol/mL)；Template DNA, 1 μ L；Ex-Taq, 0.2 μ L (5U)；ddH2O, 18.8 μ L；Amplification program are as follows: 94 DEG C of initial denaturations 3
Minute, it 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.Above-mentioned two segment DNAs amplified production is cloned into carrier T respectively, then uses Bgl II, Sac I digestion respectively
The good upstream homologous fragment of digestion is cloned into the pXEH carrier with same enzyme digestion by plasmid containing fragment upstream and pXEH carrier
On；Plasmid after verifying is correct, then with Sal I, Pst I digestion containing segments downstream and the pXEH for being connected with upstream homologous fragment are carried
Body, then the good downstream homologous fragment of digestion is cloned on the pXEH carrier for being connected with upstream homologous fragment with same enzyme digestion,
It is built into knockout carrier PXEH-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 PXEH-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
BcPCK1 deletion mutant body: the primer c (5'-ATGACTTCAATGTCCCGCTAA- except the homology arm of upstream on genome
It can 3') be expanded with the primer d (5'-ACAGACGTCGCGGTGAGTTCA-3') of hygromycin gene pairing to expected size
Recombinant fragment；And code area primer a (5'-CCGAGGGTATGACTTCAGGTA-3') and b (5'-
TTGTGGTGCTCAATCTTCTCA-3') without amplified band (wild-type strain is amplifiable to arrive 0.81kb segment).As a result, from conversion
BcPCK1 deletion mutant body Δ Bcpck1 is screened in son to analyze for follow-up function.
The genetic complement of 3 BcPCK1 deletion mutant body of embodiment
Using primer C-F (5'-AAAGATCAAAGGATCGAATTCCAATGTCCCGCTAAATGTCA-3') and C-R (5'-
CCGGGTACCGAGCTCGAATTCCCGATTGCTAGACTGGTGG-3'), ash arrhizus bacteria BcPCK1 full length gene 4672bp is expanded
(including promoter, open reading frame and terminator), is cloned into EcoR I of pXEB carrier (containing glufosinate resistance gene)
Point is built into genetic complement carrier PXEH-KO-C.Carrier confirms no amino acid mutation through sequence verification.Using as previously described
Agrobacterium-medialed transformation method, screened using 400 μ g/mL glufosinate-ammoniums, which be transferred to BcPCK1 gene
In deletion mutant genome, genetic complement bacterial strain Bcpck1-C is obtained.Select the BcPCK1 base of ash arrhizus bacteria bacterial strain B05.10
Because of the primer PCKU (5'-CGAAGGTAAGGACGGAGACG-3') and PCKD (5'- of cDNA genome design
CAGAGCCGAATTGGATAGCG-3') and wild-type strain B05.10 another pair is the primer Actin-U (5'- of internal reference
CATGGCTGGTCGTGATTTGA-3'), Actin-D (5'-GAGGATTGACTGGCGGTTTG-3') carries out sxemiquantitative simultaneously
PCR, electrophoresis result meet expected (see Fig. 3): not expanding in BcPCK1 deletion mutant body Δ Bcpck1-1, Δ Bcpck1-2
Increase shaping band, the brightness that wild type and complementing strain are expanded close to internal reference illustrates: in BcPCK1 deletion mutant body
BcPCK1 gene is knocked really, and complementing strain additionally possesses the subsequent BcPCK1 gene being transferred to, because of BcPCK1 gene
There is no the transcription of BcPCK1 gene in deletion mutant, and complementing strain and wild-type strain have the normal of BcPCK1 gene to turn
Record.
Effect of the 4 BcPCK1 gene of embodiment during the conidia germination of ash arrhizus bacteria
Taking concentration respectively is 1 × 105/ mL conidium mixes with PDB solution 1:1, drop 5ul mixed liquor on glass slide,
2h, 4h, 6h are cultivated respectively in 20 DEG C of constant temperature, count germination rate respectively at micro- sem observation；It has been observed that BcPCK1 gene when 2h
Deletion mutant conidia germination rate is less than 30%, and wild-type strain and complementing strain are close to 80%；BcPCK1 base when 4h
Because deletion mutant conidia germination rate is less than 60%, and wild-type strain and complementing strain are already up to 100%；Until 6h
BcPCK1 deletion mutant body conidia germination rate just reaches 100%.Above studies have shown that BcPCK1 gene has regulation
The effect of conidia germination (see Fig. 4).
Effect of the 5 BcPCK1 gene of embodiment in terms of ash arrhizus bacteria produces spore
10 μ L strain to be tested PDB spore suspensions (1 × 10 are taken respectively6ml-1) it is seeded in the center of PDA culture medium, 20 DEG C
Dark culturing one week (see Fig. 5), spore is collected, micro- sem observation is counted using haemocytometer.Analysis finds, BcPCK1 base
Because deletion mutant sporulation quantity only has 10% or so of wild-type strain, and complementing strain and wild-type strain are close, show
BcPCK1 gene has the function of conidium yield (see Fig. 6).
Effect of the 6 BcPCK1 gene of embodiment at the pathogenic aspect of ash arrhizus bacteria
Using Isolated leaf inoculation method, the pathogenicity situation of change of BcPCK1 deletion mutant body is evaluated.It is trained from greenhouse
Acquire mature leaf on feeding tomato plant, be horizontally arranged container in, take 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, after 3 days evaluate strain to be tested pathogenicity.Experimental result shows,
BcPCK1 deletion mutant body loses pathogenecity substantially, and small scab can only be found near vaccination.With this formation
Stark contrast, wild type can successfully infect tomato leaf, and spread to nearly half of blade face rapidly；Genetic complement bacterial strain
The pathogenicity of Bcpck1-C is back to normal, basically reaches wild-type levels (see Fig. 7).Meter is measured to leaf spot lesion area
It calculates, lesion area caused by discovery BcPCK1 deletion mutant body infects only has 25% or so caused by wild type (see Fig. 8).
The result of study shows that BcPCK1 is a crucial Disease-causing gene, is necessary to ash arrhizus bacteria infects host, if the base
The protein loss of activity of cause or its coding, ash arrhizus bacteria will lose the ability that host causes disease that infects substantially.
The sequence of SEQ ID No:1
(i) sequence signature: (A) length: 2644bp；(B) type: nucleotide；(C) chain: single-stranded
(ii) molecule type: DNA
(iii) sequence description: SEQ ID No:1
1 ATGTCGACCT CAAATATGAA TAAGAAGGCT TTCGATCCCA TCCAAAGAAC TGCATCTCCA
61 TATCCTGACA AGAGCAGTGT TGGTCCTTCG CACATTATTG CACAACACCA ACCTACAAAC
121 AACTATCATT CTTCATCACT CTCCACTCTC AAAATGGTTT CTACAAATCC AAGCGTCAAT
181 CGCACATGTC AGTATACCTT TCTTCATGCT CCTACTGCTT CAGCACTGCT TCAGACCTGC
241 CGCATTCGAC CTGGCATCTG ATGTTCGGGT CAGATTGGAT GTCGACGCAC CTGCGACTTT
301 AGCAGTCCGC CCACAGTCTT TCAGCTCGCA GCTCAATGAT GACGCTTCGC GATAGCTTTT
361 ATGCAACTTC ACTTTGCTAA CATTATCTTT TACAGCACTT CACCCAAGCG GTGTACAACC
421 ACAAGTTGAG CACACCGAGC TTGAGGAGGA ACTTCACGAG AACGCACATA TCGATTATGA
481 TCGTGTAGCT ATTGTGTGTA TCTCGCCCCC TCTTTGGATC TGCCAGCTGC TAACTCCTAT
541 TCTTTTCTAG GTTCCTAATC CAAGTGTCGC TGCCCTTTAC GAAGATGCAC TTGTTTACGA
601 AACTGGTTCT GCCATCACAT CTACCGGTGC TCTCACTGCA TACTCTGGAG CTAAGACAGG
661 TCGCTCGCCC TTAGACAAGA GAATTGTCAA AGAACCATCA TCTGAAAATG AGATTTGGTG
721 GGGACCAGTC AACAAGCCAA TGACACCTGA TGTAAGAACC TCTCTCTCTC TCTGTTTACA
781 TTCATTTCTT GCGTCTTTTA CTTTTTCCTT GGAAATGCAT TCAATACGTT TTCAGTCGAG
841 GAAATTTACA TTGGCTCTAC AACACTACGG ATTTTGAAAA CGGCAATCTC ATCTCAGCAT
901 TTTTGAGAGC ACATCTTCAC ATCTACAACT CATCACTTTC CTTTCTTCTT TACGCCAAAC
961 AATCATTCTA AGAGATTTCC CCGCACATTT CTACGTCATA AGCTCTTGGG CATCCTCGGC
1021 TTTATCTTTT TCCTTCCTTC CCCAAGCGAA AACGCGGGGT GCAGATAAAC CGATATTATC
1081 CCGATCTTGC TGCTATTAGT GATCAGCTAT CATATTCCCC CTTTACATGT CGCGTGCGAA
1141 CTTTATCTAT GAGTTTTTGA ATGTTGGAAA TTTAGTAGAC TAACATCAAT GATAGGTATG
1201 GAGAATCAAC CGTGAACGTG CAATTGATTA CTTAAACACG AGAAATCGTA TTTACGTCGT
1261 CGATGGTTTC GCAGGATGGG ACGAGAAATA CAGAATCAAG GTTCGAGTCG TATGCGCTCG
1321 TGCCTACCAC GCTCTTTTCA TGCGCAATAT GCTCATTAGA CCTGAACGCA AAGATCTCGA
1381 ACATTTCCAC CCAGATTATA CTATTTACAA CGCAGGATCT TTCCCTGCTA ACAGATACAC
1441 CGAGGGTATG ACTTCAGGTA CTTCAGTTGC TATCAACTTC GCAGCGAAGG AGATGGTTAT
1501 CTTGGGTACT GAGTACGCCG GAGAGATGAA GAAGGGTGTT TTTACAGTTC TCTTCTACGA
1561 GATGCCTGTC AAGCATAACG TTCTCACTCT TCACTCGTCA GCCAACGAAG GTAAGGACGG
1621 AGACGTCACT GTCTTCTTTG GTCTTTCGGG TACCGGTAAA ACAACACTTT CAGCAGACCC
1681 CAACCGTGCC TTGATTGGTG ACGACGAGCA CTGCTGGAGT GACCGTGGTG TTTTCAACAT
1741 TGAGGGTGGT GTAAGTGTTT CAGAATCTGC ATTACGTTGT ATCAAGCTAA CAATCTGCAG
1801 TGCTACGCTA AGTGCATCGG TCTTTCAGCA GAGAAGGAGC CTGATATCTT TGGCGCTATC
1861 CAATTCGGCT CTGTTCTTGA GAACGTTGTT TTTGACCCAG ACACTCGTCT TGTTGATTAT
1921 GATGACTCAA CCTTGACTGA GAACACCCGT TGCGCATACC CAATTGAGTA CATTTCCAAC
1981 GCTAAGATTC CTTGTCTATC GACAAACCAC CCAAGCAACA TCATTCTTCT TACATGTGAT
2041 GCTCGTGGTG TCCTTCCACC CATTTCCAAG CTTGACTCCG CCCAAACCAT GTTCCATTTC
2101 ATCTCCGGTT ACACCTCAAA GATGGCCGGT ACCGAAGATG GTGTCACAGA GCCACAGGCT
2161 ACCTTCTCAT CTTGCTTCGC GCAACCTTTC TTGGCTTTAC ACCCAATGCG TTATGCTAAG
2221 ATGTTGGCTG AGAAGATTGA GCACCACAAG GCTAATGCTT GGTTGTTGAA CACTGGATGG
2281 GTTGGAGCTG GTGCTACCAC TGGTGGCAAG CGTTGCCCGT AAGTACAATT GGAATCTTCC
2341 GGAAAAACGC ATGCTAACAT GAACTAGATT GAAATACACT CGTGCTATTC TTGACGCAAT
2401 TCACTCTGGA GAGCTTGCAA AGGTTGAGTA TGAAAACTAC GAGACCTTCA ACCTCCAAGT
2461 ACCAAAGACT TGCACCAATG TTCCATCGGA ATTGTTGAAC CCAAGAAATG CATGGAGTCA
2521 AGGAGAGGAT AGCTTCAAAG CCGAGGTTAA CAAACTTGGT GGTCTCTTCG TTGAGAACTT
2581 TAAGAAATAC CAGTCGGAAG CTACTGAGGA TGTCATTGCA GCAGGGCCAG CTGTTAAGGT
2641 TTAA
The sequence of SEQ ID No:2
(i) sequence signature: (A) length: 560 amino acid；(B) type: amino acid；(C) chain: single-stranded
(ii) molecule type: polypeptide
(iii) sequence description: SEQ ID No:2
1 MVSTNPSVNR TSLHPSGVQP QVEHTELEEE LHENAHIDYD RVAIVPNPSV AALYEDALVY
61 ETGSAITSTG ALTAYSGAKT GRSPLDKRIV KEPSSENEIW WGPVNKPMTP DVWRINRERA
121 IDYLNTRNRI YVVDGFAGWD EKYRIKVRVV CARAYHALFM RNMLIRPERK DLEHFHPDYT
181 IYNAGSFPAN RYTEGMTSGT SVAINFAAKE MVILGTEYAG EMKKGVFTVL FYEMPVKHNV
241 LTLHSSANEG KDGDVTVFFG LSGTGKTTLS ADPNRALIGD DEHCWSDRGV FNIEGGCYAK
301 CIGLSAEKEP DIFGAIQFGS VLENVVFDPD TRLVDYDDST LTENTRCAYP IEYISNAKIP
361 CLSTNHPSNI ILLTCDARGV LPPISKLDSA QTMFHFISGY TSKMAGTEDG VTEPQATFSS
421 CFAQPFLALH PMRYAKMLAE KIEHHKANAW LLNTGWVGAG ATTGGKRCPL KYTRAILDAI
481 HSGELAKVEY ENYETFNLQV PKTCTNVPSE LLNPRNAWSQ GEDSFKAEVN KLGGLFVENF
541 KKYQSEATED VIAAGPAVKV
Sequence table
The sequence of SEQ ID No:1
(i) sequence signature: (A) length: 2644 bp；(B) type: nucleotide；(C) chain: single-stranded
(ii) molecule type: DNA
(iii) sequence description: SEQ ID No:1
1 ATGTCGACCT CAAATATGAA TAAGAAGGCT TTCGATCCCA TCCAAAGAAC TGCATCTCCA
61 TATCCTGACA AGAGCAGTGT TGGTCCTTCG CACATTATTG CACAACACCA ACCTACAAAC
121 AACTATCATT CTTCATCACT CTCCACTCTC AAAATGGTTT CTACAAATCC AAGCGTCAAT
181 CGCACATGTC AGTATACCTT TCTTCATGCT CCTACTGCTT CAGCACTGCT TCAGACCTGC
241 CGCATTCGAC CTGGCATCTG ATGTTCGGGT CAGATTGGAT GTCGACGCAC CTGCGACTTT
301 AGCAGTCCGC CCACAGTCTT TCAGCTCGCA GCTCAATGAT GACGCTTCGC GATAGCTTTT
361 ATGCAACTTC ACTTTGCTAA CATTATCTTT TACAGCACTT CACCCAAGCG GTGTACAACC
421 ACAAGTTGAG CACACCGAGC TTGAGGAGGA ACTTCACGAG AACGCACATA TCGATTATGA
481 TCGTGTAGCT ATTGTGTGTA TCTCGCCCCC TCTTTGGATC TGCCAGCTGC TAACTCCTAT
541 TCTTTTCTAG GTTCCTAATC CAAGTGTCGC TGCCCTTTAC GAAGATGCAC TTGTTTACGA
601 AACTGGTTCT GCCATCACAT CTACCGGTGC TCTCACTGCA TACTCTGGAG CTAAGACAGG
661 TCGCTCGCCC TTAGACAAGA GAATTGTCAA AGAACCATCA TCTGAAAATG AGATTTGGTG
721 GGGACCAGTC AACAAGCCAA TGACACCTGA TGTAAGAACC TCTCTCTCTC TCTGTTTACA
781 TTCATTTCTT GCGTCTTTTA CTTTTTCCTT GGAAATGCAT TCAATACGTT TTCAGTCGAG
841 GAAATTTACA TTGGCTCTAC AACACTACGG ATTTTGAAAA CGGCAATCTC ATCTCAGCAT
901 TTTTGAGAGC ACATCTTCAC ATCTACAACT CATCACTTTC CTTTCTTCTT TACGCCAAAC
961 AATCATTCTA AGAGATTTCC CCGCACATTT CTACGTCATA AGCTCTTGGG CATCCTCGGC
1021 TTTATCTTTT TCCTTCCTTC CCCAAGCGAA AACGCGGGGT GCAGATAAAC
CGATATTATC
1081 CCGATCTTGC TGCTATTAGT GATCAGCTAT CATATTCCCC CTTTACATGT
CGCGTGCGAA
1141 CTTTATCTAT GAGTTTTTGA ATGTTGGAAA TTTAGTAGAC TAACATCAAT
GATAGGTATG
1201 GAGAATCAAC CGTGAACGTG CAATTGATTA CTTAAACACG AGAAATCGTA
TTTACGTCGT
1261 CGATGGTTTC GCAGGATGGG ACGAGAAATA CAGAATCAAG GTTCGAGTCG
TATGCGCTCG
1321 TGCCTACCAC GCTCTTTTCA TGCGCAATAT GCTCATTAGA CCTGAACGCA
AAGATCTCGA
1381 ACATTTCCAC CCAGATTATA CTATTTACAA CGCAGGATCT TTCCCTGCTA
ACAGATACAC
1441 CGAGGGTATG ACTTCAGGTA CTTCAGTTGC TATCAACTTC GCAGCGAAGG
AGATGGTTAT
1501 CTTGGGTACT GAGTACGCCG GAGAGATGAA GAAGGGTGTT TTTACAGTTC
TCTTCTACGA
1561 GATGCCTGTC AAGCATAACG TTCTCACTCT TCACTCGTCA GCCAACGAAG
GTAAGGACGG
1621 AGACGTCACT GTCTTCTTTG GTCTTTCGGG TACCGGTAAA ACAACACTTT
CAGCAGACCC
1681 CAACCGTGCC TTGATTGGTG ACGACGAGCA CTGCTGGAGT GACCGTGGTG
TTTTCAACAT
1741 TGAGGGTGGT GTAAGTGTTT CAGAATCTGC ATTACGTTGT ATCAAGCTAA
CAATCTGCAG
1801 TGCTACGCTA AGTGCATCGG TCTTTCAGCA GAGAAGGAGC CTGATATCTT
TGGCGCTATC
1861 CAATTCGGCT CTGTTCTTGA GAACGTTGTT TTTGACCCAG ACACTCGTCT
TGTTGATTAT
1921 GATGACTCAA CCTTGACTGA GAACACCCGT TGCGCATACC CAATTGAGTA
CATTTCCAAC
1981 GCTAAGATTC CTTGTCTATC GACAAACCAC CCAAGCAACA TCATTCTTCT
TACATGTGAT
2041 GCTCGTGGTG TCCTTCCACC CATTTCCAAG CTTGACTCCG CCCAAACCAT
GTTCCATTTC
2101 ATCTCCGGTT ACACCTCAAA GATGGCCGGT ACCGAAGATG GTGTCACAGA
GCCACAGGCT
2161 ACCTTCTCAT CTTGCTTCGC GCAACCTTTC TTGGCTTTAC ACCCAATGCG
TTATGCTAAG
2221 ATGTTGGCTG AGAAGATTGA GCACCACAAG GCTAATGCTT GGTTGTTGAA
CACTGGATGG
2281 GTTGGAGCTG GTGCTACCAC TGGTGGCAAG CGTTGCCCGT AAGTACAATT
GGAATCTTCC
2341 GGAAAAACGC ATGCTAACAT GAACTAGATT GAAATACACT CGTGCTATTC
TTGACGCAAT
2401 TCACTCTGGA GAGCTTGCAA AGGTTGAGTA TGAAAACTAC GAGACCTTCA
ACCTCCAAGT
2461 ACCAAAGACT TGCACCAATG TTCCATCGGA ATTGTTGAAC CCAAGAAATG
CATGGAGTCA
2521 AGGAGAGGAT AGCTTCAAAG CCGAGGTTAA CAAACTTGGT GGTCTCTTCG
TTGAGAACTT
2581 TAAGAAATAC CAGTCGGAAG CTACTGAGGA TGTCATTGCA GCAGGGCCAG
CTGTTAAGGT
2641 TTAA
The sequence of SEQ ID No:2
(i) sequence signature: (A) length: 560 amino acid；(B) type: amino acid；(C) chain: single-stranded
(ii) molecule type: polypeptide
(iii) sequence description: SEQ ID No:2
1 MVSTNPSVNR TSLHPSGVQP QVEHTELEEE LHENAHIDYD RVAIVPNPSV AALYEDALVY
61 ETGSAITSTG ALTAYSGAKT GRSPLDKRIV KEPSSENEIW WGPVNKPMTP DVWRINRERA
121 IDYLNTRNRI YVVDGFAGWD EKYRIKVRVV CARAYHALFM RNMLIRPERK DLEHFHPDYT
181 IYNAGSFPAN RYTEGMTSGT SVAINFAAKE MVILGTEYAG EMKKGVFTVL FYEMPVKHNV
241 LTLHSSANEG KDGDVTVFFG LSGTGKTTLS ADPNRALIGD DEHCWSDRGV FNIEGGCYAK
301 CIGLSAEKEP DIFGAIQFGS VLENVVFDPD TRLVDYDDST LTENTRCAYP IEYISNAKIP
361 CLSTNHPSNI ILLTCDARGV LPPISKLDSA QTMFHFISGY TSKMAGTEDG VTEPQATFSS
421 CFAQPFLALH PMRYAKMLAE KIEHHKANAW LLNTGWVGAG ATTGGKRCPL KYTRAILDAI
481 HSGELAKVEY ENYETFNLQV PKTCTNVPSE LLNPRNAWSQ GEDSFKAEVN KLGGLFVENF
541 KKYQSEATED VIAAGPAVKV
Claims (1)
- Application of the 1.BcPCK1 gene in reduction ash arrhizus bacteria is pathogenic, which is characterized in that especially by knockout BcPCK1 base Because pathogenic to reduce, the DNA sequence dna of the BcPCK1 gene is as shown in SEQ ID No:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610962073.8A CN106520786B (en) | 2016-11-04 | 2016-11-04 | One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610962073.8A CN106520786B (en) | 2016-11-04 | 2016-11-04 | One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106520786A CN106520786A (en) | 2017-03-22 |
| CN106520786B true CN106520786B (en) | 2019-09-20 |
Family
ID=58326134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610962073.8A Expired - Fee Related CN106520786B (en) | 2016-11-04 | 2016-11-04 | One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106520786B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154454A (en) * | 2015-10-19 | 2015-12-16 | 吉林大学 | Pathogenicity-related Botrytis cinerea gene BcArs2 and application thereof |
| CN105483146A (en) * | 2016-01-22 | 2016-04-13 | 吉林大学 | Botrytis cinerea gene BcAls1 relative to pathogenicity and application of botrytis cinerea gene BcAls1 |
-
2016
- 2016-11-04 CN CN201610962073.8A patent/CN106520786B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154454A (en) * | 2015-10-19 | 2015-12-16 | 吉林大学 | Pathogenicity-related Botrytis cinerea gene BcArs2 and application thereof |
| CN105483146A (en) * | 2016-01-22 | 2016-04-13 | 吉林大学 | Botrytis cinerea gene BcAls1 relative to pathogenicity and application of botrytis cinerea gene BcAls1 |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION：G2Y9Z4;UniProtKB;《UniProt》;20111116;G2Y9Z4 * |
| Genome Update of Botrytis cinerea Strains B05.10 and T4;Martijn Staats等;《Eukaryotic Cell》;20121130;第11卷(第11期);1413-1414 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106520786A (en) | 2017-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105154454B (en) | Pathogenicity-related botrytis cinerea gene BcArs2 and application thereof | |
| CN105154453A (en) | Pathogenicity related botrytis cinerea gene BcSep4 and application thereof | |
| CN105483146B (en) | One kind ash arrhizus bacteria gene BcAls1 relevant to pathogenicity and its application | |
| CN110183521A (en) | Application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity | |
| CN105838642B (en) | A kind of actinomyces of cure plant disease of peanut and its application | |
| CN100491529C (en) | HrpN gene for encoding plant multifunction activity and broad spectrum resistance cell signal factor and its expression product and use | |
| CN108531407B (en) | Construction of strain incapable of producing aflatoxin and method for preventing and treating aflatoxin pollution | |
| CN106497943B (en) | One kind ash arrhizus bacteria gene BcSEP5 relevant to pathogenicity and its application | |
| CN108486149A (en) | A kind of application of cucumber CsWRKY50 genes in enhancing cucumber downy mildew resistance | |
| CN105483143B (en) | One kind ash arrhizus bacteria gene BcCpo1 relevant to pathogenicity and its application | |
| CN106520786B (en) | One kind ash arrhizus bacteria gene BcPCK1 relevant to pathogenicity and its application | |
| CN106399328B (en) | One kind ash arrhizus bacteria gene BcSEP6 relevant to pathogenicity and its application | |
| CN106497942B (en) | One kind ash arrhizus bacteria gene BcSEP3 relevant to pathogenicity and its application | |
| CN110358778A (en) | SlDALR2 gene is improving the application in tomato bacterial leaf spot resistance | |
| CN105483106B (en) | One kind ash arrhizus bacteria gene BcFch1 relevant to pathogenicity and its application | |
| CN105602970B (en) | One kind ash arrhizus bacteria gene BcPda1 relevant to pathogenicity and its application | |
| CN1858210A (en) | Plant multifunction activity and broad spectrum resistance cell signal factor encoding hrpN gene and its expession product and use | |
| CN109456985A (en) | One kind ash arrhizus bacteria gene BcMBF1 relevant to pathogenicity and its application | |
| CN109593769A (en) | Wild rice brand spores form related gene Itd1 and its application | |
| CN109868282B (en) | Pathogenicity-related botrytis cinerea gene BcEXO70 and application | |
| CN116179399A (en) | Rhizosphere growth promoting bacterium and application thereof | |
| CN108559753A (en) | The breeding method of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_17694 genes in stripe rust prevention | |
| CN105483147B (en) | One kind ash arrhizus bacteria gene BcUdc1 relevant to pathogenicity and its application | |
| CN105483144B (en) | One kind ash arrhizus bacteria gene BcAdh1 relevant to pathogenicity and its application | |
| CN105483138B (en) | One kind ash arrhizus bacteria gene BcAtm1 relevant to pathogenicity and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190920 Termination date: 20211104 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |