CN106754434A - Mutants which had K2e12 of Pyricularia oryzae and application thereof - Google Patents
Mutants which had K2e12 of Pyricularia oryzae and application thereof Download PDFInfo
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Abstract
It is Magnaporthe oryzae K2e12 the invention discloses a kind of mutants which had K2e12 of Pyricularia oryzae；Its preserving number is：CCTCC NO:M 2016107.The mutants which had K2e12 of the Pyricularia oryzae is MGG_07012 gene deletion strains.Its purposes is：Make plant not pathogenic；Or by the use of K2e12 albumen expression or modification as target, screen antimycotic medicine.
Description
Technical field
The present invention relates to a kind of mutant K2e12 of Pyricularia oryzae, and its make plant it is not pathogenic on purposes, belong to
Microorganism and microbe application field.
Background technology
Drug targets are pharmaceutically-active objects, typically with important physiology or pathologic function, can be with medicine phase
With reference to and produce the large biological molecule of pharmacological action.The screening and functional study of drug targets are to find special, efficient, hypotoxicity
The premise of medicine.In disease fungi, drug targets should this possess following characteristics：They should be that thalli growth is musted first
The gene or gene outcome and with highly conserved and special sequence for needing, so can just filter out to thalline high special
To human body or the harmless medicine of other hosts；Secondly, drug targets should be disease fungus growth and survive it is essential, extremely
It is few most important during host is infected；The biochemical function of last drug targets will confirm, it is necessary to have with reference to small molecule
The chemical characteristic of compound.The antifungal drug of application is broadly divided into three classes by its target spot at present：Act on fungal cell membrane
Antifungal drug, the antifungal drug for acting on nucleic acid synthesis and the antifungal drug for acting on Cell wall synthesis.First kind medicine
The representative of thing target is 14 α of the key enzyme lanosterol-demethylase in fungal cell membrane synthesis, uses azole antifungal
Medicine can suppress the activity of the enzyme, so as to block fungal cell membrane synthesis, cause fungi to rupture dead.The generation of Equations of The Second Kind medicine
Table 5-flurocytosine (5-FC) is into can be converted 5- fluorine deoxidation bird pyrimidine monophosphates so as to suppress thymidylic acid after fungal cell
The synthesis of synthase activity and then influence DNA.3rd class target is represented as β -1,3- glucan synthases, as fungal cell
The synzyme of the peculiar polysaccharide of wall is the comparatively ideal target spot of an analogy.
In recent years, with genomics, proteomics, bioinformatics fast development, new drug targets screening
Technology continuously emerges, such as DNA chip technology, protein biochip technology, computer aided drug design technology, combinatorial chemistry skill
Art, chemical genetics technology.These technologies substantially increase the efficiency of drug targets screening, accelerate the speed of new drug development.
Further confirmation is also needed to from the potential drug target for filtering out to drug targets are eventually become.The confirmation of current drug targets
Main to use the technologies such as gene knockout, RNAi and antibody capture technology, wherein gene knockout has what other methods hardly matched
Direct and validity, is still to confirm the most strong means of drug targets at present.High flux gene Knockout is in pathogenic
Using in fungi makes it possible by directly knocking out genescreen drug targets.
Protein kinase is the enzyme of a class catalytic proteins phosphorylation reaction, and it is by the γ-phosphate group ATP or GTP
It is transferred on the amino acid residue of substrate protein to activate substrate.In eukaryotic, about 30% protein has covalently
The phosphate group of connection, it can be seen that the key player that protein kinase is played the part of in vital movement.In fact protein kinase is participated in
Almost all of cell processes, such as cell metabolism, transcription, cell cycle, the framework construction of cell, apoptosis, differentiation, signal are passed
Lead, iuntercellular interaction, cell and extracellular matrix interact etc..Protein kinase is widely present in eucaryote, from low
Protozoan to high mammal, have protein kinase from lower algae to higher plant and in fungi.Due to egg
The member of white kinases is numerous, and they are classified as protein kinase superfamily by Hanks and Hunter.
The catalyst structure domain of eukaryotic protein kinase is made up of 250-300 amino acid residue, and its amino acid sequence is protected
Keep, 12 function subdomains can be divided into again.Hanks and Hunter compares to the amino acid sequence of 370 kinds of protein kinases of the mankind,
Compared with relying on cAMP protein kinase α catalytic subunits (PKA-C α), find the Gly50 and Gly52 of subdomain I, subdomain II
Lys72, the Glu91 of subdomain III, the Asp171 of the B of subdomain VI, the Asp184 and Gly186 of subdomain VII, the Clu208 of subdomain VIII are sub-
The Asp220 and Gly225 in domain Ⅸ, the Ary280 of subdomain Ⅺ are guarded in 95% sequence.Wherein VIB, VIII and IX are whole
Guarded very much in protein kinase superfamily, and the difference of different type protein kinase is concentrated mainly on VI, VIII and Ⅸ subdomain.Albumen
The function of kinase catalytic core includes：With reference to orientation ATP or GTP, the process has bivalent cation (Mg2+Or Mn2+) participate in；
With reference to orientation protein or polypeptide；Catalysis ATP or GTP γ-phosphate group is transferred on receptor protein amino acid residue.
Protein kinase can be respectively serine (serine), threonine with the 9 of phosphorylated substrate albumen kinds of amino acid residues
(threonine), tyrosine (tyrosine), cysteine (cysteine), arginine (arginine), lysine
(lysine), aspartic acid (aspartate), glutamic acid (glutamate) and histidine (histidine).According to its catalysis
The difference of Substrate residues can be divided into 5 classes, and wherein serine and Serineprotein kinase and LCK is most
Two main classes.In the research of human kinase group, 6600 phosphorylation sites on 2244 protein are analyzed, found
The serine (pSer) of phosphorylation, threonine (pThr) and tyrosine (pTyr) site account for 86.4%, 11.8% respectively, and
1.8% also show this point.
Hanks and Hunter is made that outstanding contributions in the research of protein kinase, and they are for the first time in molecular phylogeny
Generating layer face describes human kinase protein superfamily, has established the basis that descendant studies Protein Kinase Classification.Manning exists
On the basis of Hanks and Hunter researchs, by all of kinases by group, family, three kinds of hierarchical classifications of subfamily and by the mankind's
Protein Kinase Classification expands to 9 groups, 134 families, 201 subfamilies, and this is the research mankind and model organism associated kinase
Function and evolutionary relationship provide convenience.The classification Main Basiss of protein kinase are the sequence similarity degrees of catalyst structure domain,
The similarity and biochemical function of sequence and structure beyond catalyst structure domain are also the important evidence of classification.According to Manning
Classification, 518 kinds of protein kinases of the mankind are divided into the protein kinase (ePK) of " tradition " and the protein kinase (aPK) of " atypia "
Two major classes.EPK contains 478 kinds of protein kinases, and 9 groups are divided into again：AGC, CAMK, CK1, CMGC, RGC, STE, TK, TKL,
Other；APK contains 40 kinds of protein kinases, is divided into 8 groups：Alpha, PIKK, PDHK, RIO, A6, ABC1, BRD, Other.By
Do not have clear and definite similitude with ePK in sequence in aPK, but aPK has the function of protein kinase, therefore individually it is divided into one
Class.
The content of the invention
The technical problem to be solved in the present invention is to provide mutants which had K2e12 of a kind of Pyricularia oryzae and application thereof.
In order to solve the above-mentioned technical problem, the present invention provides a kind of mutants which had K2e12 of Pyricularia oryzae, is
Magnaporthe oryzae K2e12 (Pyricularia oryzae Magnaporthe oryzae K2e12)；Its preserving number is：CCTCC
NO:M 2016107。
As the improvement of the mutants which had K2e12 of Pyricularia oryzae of the invention：The mutants which had of the Pyricularia oryzae
K2e12 is MGG_07012 gene deletion strains.
The present invention also provides the purposes of the mutants which had K2e12 of above-mentioned Pyricularia oryzae simultaneously：For infecting plant, make
Plant is not pathogenic；The disease is rice blast.
As the improvement of the purposes of the mutants which had K2e12 of Pyricularia oryzae of the invention：The plant is paddy rice or big
Wheat；Paddy rice is, for example, Oryzae sativa cv.CO-39；Barley is, for example, Hordeum vulgare cv.Golden
promise。
The present invention also provides another purposes of the mutants which had K2e12 of above-mentioned Pyricularia oryzae simultaneously：Using K2e12
The expression or modification of albumen screen antimycotic medicine as target.
Bacterial strain Magnaporthe oryzae K2e12 of the invention, its preservation information is specific as follows：
Depositary institution：China typical culture collection center；Preservation address：Wuhan, China Wuhan University；Preservation date：
On March 14th, 2016, preservation title：Magnaporthe oryzae K2e12；Preserving number：CCTCC NO:M 2016107.
Pyricularia oryzae mutants which had K2e12 involved in the present invention, is specifically MGG_07012 deletion mutants
Body, more precisely compared with Pyricularia oryzae wild-type strain Guy11, differences of the K2e12 in genomic DNA level is
K2e12 is MGG_07012 gene deletion strains without MGG_07012 genes, i.e. Pyricularia oryzae mutants which had K2e12.In egg
Difference on white level is that K2e12 is free of MGG_07012 albumen, and the difference on expression is MGG_07012 genes
Do not expressed in K2e12.
In sum, bacterial strain Magnaporthe oryzae K2e12 belong to fungi from the mutant of Pyricularia oryzae
Boundary, Ascomycota, the pathogenic effects of the bacterial strain are lost.
Brief description of the drawings
Specific embodiment of the invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that double PCR verifies mutant result figure；
A in Fig. 1 represents first round PCR result：By the primer K2e12innerF/innerR of verifying purpose gene and interior
Ginseng gene β-tubulin checkings primer tubulinF+tubulinR is added in same reaction system and enters performing PCR, through agarose
Result as shown in Figure 1A occurs after electrophoresis, only has a band in K2e12 mutant, and there are two in wild type Guy11
Band；B in Fig. 1 represents the second wheel PCR results, and band as shown in Figure 1B, has one homologous to put after electrophoresis in K2e12 mutant
The band for changing, and there is no band in wild type Guy11.The result of two-wheeled PCR shows that Pyricularia oryzae MGG_07012 genes exist
Lacked in K2e12.
Fig. 2 is the comparison diagram of K2e12 and wild-type strain Guy11,
A is colonial morphology of 2 kinds of bacterial strains after 25 DEG C of light culture 7d on CM culture mediums；
B is 2 kinds of liquid CM cultures of bacterial strain, K2e12 and wild type Guy11 indifferences；
C is that 2 kinds of bacterial strains form of mycelia and spore on CM solid mediums compares；Compared with wild type Guy11,
K2e12 aerial hyphaes are sparse, are nearly free from conidium.
Fig. 3 is the pathogenic testing experiment of Pyricularia oryzae mutants which had and wild-type strain on barley isolated blade,
Figure below of Fig. 3 be K2e12 to the pathogenic forfeiture of barley leaves, there is no visible scab；By contrast, the upper figure of Fig. 3 is vaccinated with open country
The larger diffusivity black scab of generation area at inoculation of raw type bacterial strain, and scab quickly can diffuse to week along vein
The healthy area enclosed, scab middle section gradually rots, and there is substantial amounts of mycelia；Therefore learn：Bacterial strain K2e12 is to barley
Pathogenic forfeiture.
Fig. 4 is the pathogenic testing experiment of Pyricularia oryzae mutants which had and wild-type strain on rice seedlings, under Fig. 4
Figure is that K2e12 loses pathogenic to paddy rice, without any scab of generation；By contrast, the upper figure of Fig. 4 is wild-type strain in water
Typical fusiformis scab is produced on rice；Therefore learn：Pathogenic forfeitures of the bacterial strain K2e12 to paddy rice.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
With reference to above-mentioned accompanying drawing, specific embodiment of the invention is described in detail.
Remarks explanation：Following all of culture mediums are used at the sterilizing for being preceding required to carry out in a conventional manner HTHP
Reason (121 DEG C, 0.24MPa, 20 minutes).
The structure of embodiment 1, knockout carrier pKO1B-K2e12
Use ClonExpressTMMultiSOne Step Cloning Kit (Novi praises company) carries out knockout carrier
Build：The upstream and downstream piece of amplifying target genes is distinguished first with primer K2e12upF+upR and K2e12downF+downR
Section, length is about 1.5k；Then expand glufosinate resistance gene with primer BarF+BarR (primer sequence is as follows).Followed by
The pKO1B linear carriers that PCR obtains three fragments and obtains through XbaI and HindIII double digestions are reassembled into mesh ground by the kit
Carrier pKO1B-K2e12.
Primer sequence：
K2e12upF:5’-GGTACCCGGGGATCCTCTAGACCGTAAGTTCAGGGTCATCATC-3’
K2e12upR:5’-CTCCTTCAATATCATCTTCTAGATGCGGCGATGTCTCC-3’
K2e12downF:5’-TGCCCGTCACCGAGATTTAGTCGTCTGCGGAGTCTGTC-3’
K2e12downR:5’-ACGACGGCCAGTGCCAAGCTTATGAAGAGTTGGTGGGATGAC-3’
BarF:5’-AGAAGATGATATTGAAGGAG-3’
BarR:5’-CTAAATCTCGGTGACGGGCA-3’。
The acquisition of embodiment 2, Pyricularia oryzae mutants which had
Agrobacterium (Agrobacterium tumefaciens) strains A GL1 and plasmid pCAMBIA1300 etc. routine things are equal
Can obtain by conventional methods.Agrobacterium is general, and in 28 DEG C of dark culturings, the minimal medium of growth is LB fluid nutrient mediums,
Preserved at -70 DEG C in 4% glycerine.
CM (Complete Medium) culture medium：20 × Nitrate salts 50ml, 1000 × Trace Elements
1ml, D-glucose (glucose) 10g, Peptone (peptone) 2g, Yeast extract (yeast extract) 1g,
Casamino acid (casamino acid) 1g, 1000 × Vitamin solution 1ml, pH value is adjusted with 1mol/L NaOH
To 6.5, plus distilled water is to 1L.
CM solid mediums are addition agar powder 12g on the basis of above-mentioned CM (Complete Medium) culture medium.
The configuration of 20 × Nitrate salts (1000ml)：NaNO3120g, KCl 10.4g, MgSO4.7H2O 10.4g,
KH2PO430.4g, water is settled to 1L.
The configuration of 1000 × Trace Elements (100ml)：ZnSO4.7H2O 2.2g, H3BO31.1g, MnCl2.4H2O
0.5g, FeSO4.7H2O 0.5g, CoCl2.6H2O 0.17g, CuSO4.5H2O 0.16g, Na2MoO4.5H2O 0.15g,
Na4EDTA5g, water is settled to 100ml.
The configuration of 1000 × Vitamin solution (100ml)：Biotin 0.01g, Pyridoxin 0.01g,
Thiamine 0.01g, Riboflavin 0.01g, PABA (p-aminobenzonic acid) 0.01g, Nicotinic
Acid 0.01g, water is settled to 100ml.
Water agar：Distilled water adds 1.5% agar powder, and (i.e. agar powder and the mass ratio of distilled water is 1.5：98.5),
Autoclaving, for single spore separation.
LB fluid nutrient mediums (Luria-Bertani)：Tryptone (tryptone) 10g, Yeast extract (yeast
Extract) 5g, NaCl (sodium chloride) 10g, plus deionized water is adjusted to pH7.5, autoclaving to 1000ml with NaOH solution.
LB solid mediums：Tryptone (tryptone) 10g, Yeast extract (yeast extract) 5g, NaCl
(sodium chloride) 10g, agar powder 12g, plus deionized water are adjusted to pH7.5, autoclaving to 1000ml with NaOH solution.
Agrobacterium inducing culture AIM is formulated：0.8ml 1.25K-Phosphate-buffer pH 4.8 (use KH2PO4With
K2HPO4Prepare), 20ml MN-buffer (30g/l MgSO4·7H2O, 15g/l NaCl, 1L H2O2), 1ml 1%CaCl2·
2H2O (w/v), 10ml 0.01%FeSO4(w/v), 5ml spore elements (100mg/l ZnSO4·7H2O,100mg/l
CuSO4·5H2O,100mg/l H3BO3,100mg/l Na2MoO4·2H2O, 1L H2O2) (filtration sterilization), 2.5ml 20%
NH4NO3(w/v), 10ml 50%glycerol (v/v), 40ml 1M MES pH 5.5 (adjust pH value) with NaOH solution, and 20%
glucose(w/v):10ml, solid medium is added to add 5ml, add water to 1L in fluid nutrient medium, solid medium adds 1.5% fine jade
Cosmetics, (200mM AS- are prepared with dimethyl sulfoxide (DMSO) DMSO).
Above-mentioned all of culture medium is required to carry out the sterilization treatment (121 DEG C, 0.24MPa, 20 minutes) of HTHP.
In laboratory conditions, Pyricularia oryzae mutant of the invention can be obtained according to Agrobacterium-medialed transformation method
Bacterial strain.It is specific as follows：
First, prepared by Agrobacterium competence：The knockout carrier pKO1B-K2e12 that will be built is transformed into agriculture by freeze-thaw method
Bacillus strain AGL1.
Agrobacterium strains are activated on LB flat boards, 28 DEG C of shaken overnight trainings in 5mL LB fluid nutrient mediums are then gone to
Support；This nutrient solution of transferase 12 mL to equipped with 50mL LB fluid nutrient mediums triangular flask in 28 DEG C of shaken cultivations to OD600 0.5~
1.0；Place on ice and stop growing, 4 DEG C of 3000g are centrifuged 5min；The precipitation 20mM CaCl of 1mL precoolings2Solution suspension, packing
To in the Eppendorf centrifuge tubes for precooling, every pipe 0.1mL；
Plus 1 μ g carrier (knockout carrier pKO1B-K2e12) DNA in above-mentioned centrifuge tube, freezed in liquid nitrogen；37℃
Water-bath 5min thaws；Add 1mLLB fluid nutrient mediums, 28 DEG C of jog culture 2-4h；By cultured bacterium solution be applied to containing card that
On the LB flat boards of mycin (containing 50 μ g/ml), 30min is placed in front, after bacterium solution is cultured base absorption completely, is inverted culture dish,
28 DEG C of culture 2-4d.
Secondly, the T-DNA conversions of Pyricularia oryzae：Selected from the LB flat boards (containing 50 μ g/ml kanamycins) of above-mentioned culture
Agrobacterium single bacterium colony is inoculated in 5ml LB fluid nutrient mediums (containing 50 μ g/ml kanamycins), 200rpm/min, and 28 DEG C are overnight trained
Support；200-400 μ l nutrient solutions were transferred in induced fluid culture mediums (AIM) of the 5ml containing 50 μ g/ml kanamycins in second day,
OD values about 0.15,28 DEG C of 5~6 hours of culture make OD600 reach 0.5~0.6；With 10ml sterile purified waters from culture about 10
Lower Pyricularia oryzae wild type conidium is washed on it CM flat boards, is counted with blood counting chamber after three layers of lens wiping paper filtering, be used in combination
Sterilized water dilution spore concentration is 106Individual/ml；Take the cultured Agrobacterium AGL-1 of 100 μ l (containing carrier) bacterium solutions and 100 μ l are dilute
The rice blast pathogen conidiospore suspension mixing released, mixed liquor (200 μ l) is uniformly applied to the nitrocellulose filter on AIM flat boards
Surface, containing 200 μM of acetosyringones (AS, acetosyringone) or without AS as control, 22 DEG C co-culture 48 to AIM flat boards
Hour；Then nitrocellulose filter is transferred on DCM selection flat boards of the Glufosinate-ammoniumpesticideng with antibiotic, is contained in selective medium
300 μ g/ml glufosinate-ammoniums, 400 μ g/ml cephalosporins (cefotaxime), 60 μ g/ml streptomysins (streptomycin), will be flat
Ware cultivates transformant appearance at being placed in 28 DEG C.
Knockout carrier pKO1B-K2e12 is transferred into Agrobacterium, then carries out Agrobacterium-medialed transformation.Agriculture bacillus mediated
The transformant that conversion is obtained extracts postgenome and need to carry out two-wheeled PCR checkings：The first round is double PCR, by verifying purpose gene
Primer K2e12innerF/innerR and reference gene β-tubulin checking primer tubulinF+tubulinR be added to together
Enter performing PCR in one reaction system, result as shown in Figure 1A occurs after agarose electrophoresis, only one in K2e12 mutant
Band, and have two band in wild type Guy11；Then the second wheel PCR is carried out, the primer of wheel PCR is K2e12longF+
LbarR, band as shown in Figure 1B, there is a band for homologous replacement after electrophoresis in K2e12 mutant, and in wild type Guy11
There is no band.The result of two-wheeled PCR shows that Pyricularia oryzae MGG_07012 genes have been lacked in K2e12.
Pyricularia oryzae MGG_07012 genes, encode 548 amino acid, containing protein kinase domain, belong to AGC groups, change
Group includes relying on family (PKA and PKG), protein kinase C family, receptor,β kinases (β ARK), the core of cyclic nucleotide
Tang Ti S6 families, the catalytic site of these kinases is that the neighbouring serine of substrate basic amino acid (Lys and Arg) and threonine are residual
Base.The substrate of PKA, PKG, S6 is from N-terminal to there is specific alkaline amino acid residue phosphoric acid binding site.The substrate phosphorus of PKC and RAC
There is alkaline amino acid residue on polyadenylation sites both sides.And the receptor kinase (β ARK) of G-protein coupling is conversely, its substrate catalysis
Location proximate is sour environment.Pyricularia oryzae mutant K2e12, is in Pyricularia oryzae wild-type strain Guy11, using careless ammonium
Phosphine resistant gene displacement MGG_07012 gene gained, therefore in mutant K2e12, MGG_07012 genes are lacked, it is no to be somebody's turn to do
The expression of gene, while the albumen of the gene is not present.Mutant K2e12 has aobvious with Pyricularia oryzae wild-type strain Guy11
Difference is write, the mycelia of mutant K2e12 is thin, is nearly free from conidium, the pathogenic forfeiture to paddy rice and barley.Compare
Compared with, in Pyricularia oryzae wild-type strain Guy11, MGG_07012 gene normal expressions, the aerial hyphae of bacterial strain Guy11 is dense
It is close, substantial amounts of conidium can be produced, there is very strong pathogenecity to paddy rice and barley.
The present invention obtains knock out mutants body 3, and wherein 1 (K2e12) is carried out into preservation, and preservation information is specific such as
Under：
Depositary institution：China typical culture collection center；Preservation address：Wuhan, China Wuhan University；Preservation date：
On March 14th, 2016, preservation title：Magnaporthe oryzae K2e12；Preserving number：CCTCC NO:M 2016107.
Primer sequence：
K2e12innerF:5’-CACACTGCTAAATGCCGAGA-3’
K2e12innerR:5’-GCACAGTGTTGCTGTGGACT-3’
tubulinF:5’-AGCCCTACAACGCTACCC-3’
tubulinR:5’-CGAGCACAGACAGCGAGT-3’
K2e12longF:5’-GAACAAGTGGCGTACAGACG-3’
K2e12LbarR:5’-GTCCTCGTTCCTGTCTGCTAAT-3。
The morphologic observation of embodiment 3, Pyricularia oryzae mutants which had K2e12：
Colonial morphology, color, growth rate and the sporulation quantity of the mutants which had are observed on CM culture mediums.
Observation result (Fig. 2) display：Mutants which had K2e12 is and wild after 25 DEG C of light dark culture 7d on CM culture mediums
Type bacterial strain is compared, and aerial hyphae is sparse, bacterium colony Dark grey (Fig. 2A).Mutants which had K2e12 is cultivated in liquid CM culture mediums,
Similar with wild-type strain, mycelia is fluffy, and liquid is in yellow green (Fig. 2 B).Mutant K2e12 and wild-type strain Guy11 produces spore
There were significant differences for ability, and mutant K2e12 is nearly free from conidium, and wild-type strain Guy11 generations are substantial amounts of mitogenetic
Spore (Fig. 2 C).
Remarks explanation：Light dark culture be 12 hours optical cultures (light intensity 3000lux), 12 hours light cultures, below
Together.
Embodiment 4, Pyricularia oryzae mutants which had K2e12 infect pathogenic forfeiture to barley leaves：
Barley (ZJ-8) light dark nursery 6d, the first piece leaf of clip surface nondestructive, for inoculation test.It is 25 DEG C, solid in CM
Body culture medium glazing/light culture Pyricularia oryzae wild type Guy11 and mutants which had K2e12, beating with 0.5cm bores after 7 days
Hole device is beaten and takes bacteria cake, by pure culture biscuits involvng inoculation in vitro barley leaves, 3 bacteria cakes of every leaf, 3 repetitions, 25 DEG C, 12/12h
Light dark moisturizing culture, observes and records incidence after 4d, experiment is repeated 3 times.
Observation result (Fig. 3) display：Pyricularia oryzae mutants which had K2e12, can not to the pathogenic forfeiture of barley leaves
See scab；By contrast, wild-type strain produces the larger diffusivity black scab of area at inoculation, and scab can be quick
The healthy area of surrounding is diffused to along vein, scab middle section gradually rots, and there is substantial amounts of mycelia.
The pathogenic forfeiture of embodiment 5, Pyricularia oryzae mutants which had K2e12 to paddy rice：
Paddy rice (CO-39) light dark nursery 14d；The Pyricularia oryzae of CM flat boards culture 10 days, washes spore and is sterilized with three layers
Lens wiping paper is filtered, and spore suspension to concentration 1 × 10 is diluted with 0.2% gelatin5Individual/ml；With miniaturised nebuliser by 2-3ml spores
The uniform spraying of liquid, per basin paddy rice equivalent spore liquid, often processes 3 repetitions on rice seedlings；22 DEG C, dark moisturizing 48h, then
Inoculation seedling is placed under 26 DEG C of illumination conditions and is cultivated；After 5-7 days, incidence is observed and records, experiment is repeated 3 times.
Observation result (Fig. 4) display：Pyricularia oryzae mutants which had K2e12 loses pathogenic to paddy rice, appoints without producing
What scab；By contrast, wild-type strain produces typical fusiformis scab on paddy rice.
Embodiment 6, by the use of MoK2e12 albumen expression or modification as target, screen antimycotic medicine：
The promoter of the downstream gene by MoK2e12 gene regulations is built into fusion protein carrier with fluorescin GFP,
It is transferred in Pyricularia oryzae by the method for embodiment 2, then the rice of the GFP expression under the promoter regulation of downstream gene
Seasonal febrile diseases bacterium transformant bacterial strain mass propgation in liquid complete medium, some aliquots are divided into after collecting mycelia, are added in every portion
Enter compound to be screened or drug candidate continued in complete medium culture a few hours to a couple of days, take mycelia in fluorescence microscope
The green fluorescence expression of the GFP of lower observation mycelia.If MoK2e12 albumen is suppressed and lost activity by drug candidate,
The ability of regulation and control to the promoter expression of downstream gene is lost, GFP albumen is not expressed so as to mycelia loses green fluorescence, illustrates this
Candidate agent has the effect for suppressing MoK2e12 albumen.What is obtained is sharp again with the drug candidate for suppressing MoK2e12 protein functions
With the method for embodiment 3, candidate agent is added in the spore liquid of inoculation, determine wild type Pyricularia oryzae in this candidate agent
To the pathogenic either with or without decrease of paddy rice in the presence of under conditions of, the anti-mycotic efficiency of compound is further determined that.Work as measurement result
During for wild type rice blast bacterial strain to the pathogenic decrease of paddy rice, illustrate that drug candidate has obvious anti-mycotic efficiency, can be with
As the source of antifungal drug；When measurement result is that wild type rice blast bacterial strain is not changed in or strengthens to the pathogenic of paddy rice
When, illustrate that candidate agent does not have anti-mycotic efficiency, it is impossible to as antifungal drug.
In sum, Pyricularia oryzae mutant K2e12 has lacked Disease-causing gene due to meeting, can by designing and screening
The compound of the expression, shearing and its expression of encoding proteins of the gene is destroyed, or design and screening can be to the amino of the albumen
The compound that acid sequence is modified, so as to develop new antifungal drug, or the mutant can exist as biocontrol bacterial strain
Developed and applied on preventing and treating Pyricularia oryzae, therefore, its gene is expected to as drug targets, and its bacterial strain is expected to as biocontrol microorganisms
Agent.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
The all deformations directly derived or associate, are considered as protection scope of the present invention.
<110>Zhejiang University
<120>Mutants which had K2e12 of Pyricularia oryzae and application thereof
<160> 6
<210> 1
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<223>Primer K2e12upF
<400> 1
ggtacccggg gatcctctag accgtaagtt cagggtcatc atc 43
<210> 2
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>Primer K2e12upR
<400> 2
ctccttcaat atcatcttct agatgcggcg atgtctcc 38
<210> 3
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>Primer K2e12downF
<400> 3
tgcccgtcac cgagatttag tcgtctgcgg agtctgtc 38
<210> 4
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<223>Primer K2e12downR
<400> 4
acgacggcca gtgccaagct tatgaagagt tggtgggatg ac 42
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer BarF
<400> 5
agaagatgat attgaaggag 20
<210> 6
<211> 20
<212> DN A
<213>Artificial sequence
<220>
<223>Primer BarR
<400> 6
ctaaatctcg gtgacgggca 20
Claims (5)
1. the mutants which had K2e12 of Pyricularia oryzae, it is characterised in that：It is Magnaporthe oryzae K2e12；Its preservation
Number it is：CCTCC NO:M 2016107.
2. the mutants which had K2e12 of Pyricularia oryzae according to claim 1, it is characterised in that：The Pyricularia oryzae it is prominent
Variant strain K2e12 is MGG_07012 gene deletion strains.
3. the purposes of the mutants which had K2e12 of Pyricularia oryzae as claimed in claim 1 or 2, it is characterised in that：Make plant not
Cause a disease；The disease is rice blast.
4. the purposes of the mutants which had K2e12 of Pyricularia oryzae according to claim 3, it is characterised in that：The plant
It is paddy rice or barley.
5. the purposes of the mutants which had K2e12 of Pyricularia oryzae as claimed in claim 1 or 2, it is characterised in that：Utilize
The expression or modification of K2e12 albumen screen antimycotic medicine as target.
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| CN109293756A (en) * | 2018-10-23 | 2019-02-01 | 北京农学院 | The albumen of one control rice rice blast fungus sporulation quantity and infection ability |
| CN112553085A (en) * | 2020-11-27 | 2021-03-26 | 云南农业大学 | Non-toxic rice blast strain for preventing and controlling rice blast and application thereof |
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| CN102304528A (en) * | 2011-06-09 | 2012-01-04 | 华南农业大学 | Magnaporthe oryzae avirulence gene AvrPik/kp/km/kh/7 and application thereof |
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| CN112553085A (en) * | 2020-11-27 | 2021-03-26 | 云南农业大学 | Non-toxic rice blast strain for preventing and controlling rice blast and application thereof |
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