CN101240283A - Rice blast bacterium and sporulation correlated gene and application - Google Patents
Rice blast bacterium and sporulation correlated gene and application Download PDFInfo
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- CN101240283A CN101240283A CNA2007100085847A CN200710008584A CN101240283A CN 101240283 A CN101240283 A CN 101240283A CN A2007100085847 A CNA2007100085847 A CN A2007100085847A CN 200710008584 A CN200710008584 A CN 200710008584A CN 101240283 A CN101240283 A CN 101240283A
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Abstract
Rice blast bacterium and gene associated with spore are provided. The rice blast bacterium spore gene MGO08093.4 is obtained by separation and appraising in T-DNA insertion mutation. The gene affects the forming of conidia of rice blast bacterium and does not affect differentiation forming of conidia. The spore germination and appressorium forming are delayed. It does not affect infestation ability. The gene is mainly associated with the spore confirmed in gene out-knocking and function complementing, and the gene does not affect appressorium forming and pathogenicity. The gene can be used as molecule target of newly pesticides design.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to the Pyricularia oryzae gene relevant, produce knocking out, have complementary functions and using of spore gene with producing spore.
Background technology
Infecting the rice blast that paddy rice causes by Pyricularia oryzae Magnaporthe grisea (Herbert) Barr (asexual generation Pyricularia grisea) is one of most important disease on the paddy rice, and sexual generation is the heterothallic ascomycetes of monoploid.Because the importance of this disease and to the operability of Pyricularia oryzae tradition, molecular genetic, it has become the research plant pathogenic fungi and the host makes comparatively one of ideal modular system mutually.The molecular biology of rice blast pathogenic and with paddy rice host's mutual do research be the key of seeking new diseases prevention approach.
Pyricularia oryzae is survived the winter on sick straw, sick paddy with mycelia and conidium, becomes the first source of infecting in next year.The mycelia that survives the winter in the sick straw, 1 year day in spring samming stable when rising to 20 ℃, if meet rainfall, just can produce conidium.Conidium borrows air-flow to propagate into rice field (rice seedling bed or Honda), after the strain of spore contact rice, sprouts under suitable temperature and humidity conditions, and directly invades from the blade epidermis, also can invade from wound, then causes and just infects.The conidium that disease portion produces can borrow the air-flow propagation to infect again again.Under favourable condition, superinfection can be infected repeatedly.Can as seen, spore plays important effect infecting of Pyricularia oryzae in the circulation, quantity that spore and spore produce what produce, and has directly influenced developing of disease under field conditions (factors).Hamer etc.
[24]Adopt the phenotype complementary method to identify several conidium mutant (SMO) first, and cloned this gene, but this mutant just produces the conidium of abnormal morphology, produce spore quantity and do not change.Shi and Leung are by chemical induction or insert the gene locuss that the method for suddenling change has been identified 6 each steps of control product spore approach: CON1, CON2, CON4, CON5, CON6 and CON7.The muton of each gene all exists unusual on product spore process or spore shape.Wang Gang etc. utilize a mutant H6 of rice blast fungus 131 strains system to identify a control and produce the gene of spore and growth, and this mutant produces spore to be reduced and poor growth, infer that this gene is in the upstream that the gene of spore and mycelial growth is produced in control.In addition, it is even more important to the adjusting of the adjusting comparison conidia germination of formation of Pyricularia oryzae appressorium and function thereof to studies show that cAMP and map kinase approach are regulated two signal pathways.
The mensuration of Pyricularia oryzae genom sequence is finished, and this lays a good foundation for the function of studying its key gene from full genomic level with the functional genomics method.At present, the most direct easy method of studying some gene functions is to make up this gene mutation body, and will carry out the just essential mutant library of covering whole genome that makes up of functional genomics research as the basis.Agrobacterium tumefaciens (Agrobacterium tumefaciens) Ti-plasmids gene transformation system be on the present plant research at most, the clearest, the most sophisticated gene transformation of technological method of theoretical mechanism by way of; in addition; mutant to pathogenic molecular label insertion of morphing; can pass through the flanking sequence of TAIL-PCR, plasmid rescue and RT-PCR method amplifier molecule label; Blast is carried out in the order-checking back in full genome database; to being carried out real-time analysis and evaluation, for final clone's pathogenic related gene lays the foundation by the relevant Disease-causing gene of label.With this rice blast of setting up sudden change storehouse, will provide rapid and precise method for pathogenic and variation mechanism at full genomic level research rice blast fungus.
Summary of the invention
The purpose of this invention is to provide a Pyricularia oryzae and produce the relevant gene of spore, and the application of described gene in design pesticide molecule target spot.
The invention provides a Pyricularia oryzae and produce the relevant gene of spore, this gene directly influences conidial formation of Pyricularia oryzae.In the process of Pyricularia oryzae being identified and being analyzed, the mutant in the T-DNA mutant library that built up of random choose at first, screen the mutant that a sporulation quantity reduces, this mutant speed of growth does not change, sporulation quantity descends, sprout and appressorium formation delay, conidium form and conidiophore differentiation do not change; Under the same concentrations (1 * 10
5/ cm
2) inoculation do not have difference to the pathogenic and wild strain of susceptible variety.In the T-DNA mutant, the insertion owing to T-DNA under most of situation makes a certain gene inactivation, makes mutant show as a certain proterties defective.Therefore,, can confirm to be formed by the function of the gene of T-DNA label and sporulation quantity in this mutant relevant, and not influence that appressorium forms and pathogenic according to viewed mutation type surface.
Pyricularia oryzae and the relevant gene of product spore is characterized in that having the aminoacid sequence shown in nucleotide sequence shown in the SEQ ID1 and the SEQ ID2.Obtain the T-DNA flanking sequence (T-DNA flaking sequence) of mutant by the TAIL-PCR method, cloning and sequencing, the nucleotides sequence after the removal carrier sequence is listed in BLAST comparison in the Pyricularia oryzae genome database (www.broad.mit.edu/annotation/fungi/magnaporthe_grisea).Comparison result shows that its homologous sequence is the promoter region of MG08093.4.Therefore, can confirm MG08093.4 be one with produce the relevant gene of spore, i.e. nucleotide sequence shown in the SEQ ID1.
Pyricularia oryzae and the verification method that produces the spore genes involved is characterized in that may further comprise the steps: (1) knocks out the described gene fragment SEQID1 of claim 1 in the wild rice blast bacterial strain with the method for homologous recombination, then shows as sporulation quantity and reduces pathogenic decline; (2) adopt artificial constructed complementary carrier to transform to be knocked out and lack the rice blast bacterial strain of described SEQ ID1 gene fragment, then the complementary bacterial strain sporulation quantity of gained recovers.Concrete operation method: at first use the method for homologous recombination, the protoplast transformation that mediates by PEG replaces to the Totomycin transferase gene with MG08093.4.This gene defection type mutant also shows as sporulation quantity to be reduced, and the conidiophore differentiation is constant, and pathogenic aspect spore can be invaded but do not expanded, and does not form susceptible scab.Carry the complementary carrier that promotor is connected with this gene by making up gene, carry out the functional complementation experiment, can recover the sporulation quantity of T-DNA mutant and gene knockout mutant.By above verification method, the function of the gene M G08093.4 that affirmation is replaced is relevant with the product spore.
Pyricularia oryzae of the present invention and the relevant gene of product spore, its practical value is and can designs the molecular target of novel agrochemical according to the 26S Proteasome Structure and Function of its functional domain.By the homology comparison, MG08093.4 has the fungal transcriptional factor structure of a Zn (2) Cys (6).Transcription factor has outstanding influence power in protein families, can decide the dominant character of cell by regulate gene expression.The component composition of zinc finger property transcription factor family comprises the fragment that some are specific, or with the zinc indicator of specific dna sequence associating, can be used for screening the gene of further being regulated and control.Screening directly acts on the MG08093.4 gene or acts on and can both make the conidium of Pyricularia oryzae form ability drop with the medicament of its other genes of doing mutually, and further plays the effect of field control rice blast.
Embodiment
Embodiment 1: the foundation of Pyricularia oryzae T-DNA mutant library and sporulation quantity reduce the acquisition of mutant
The present invention screens in existing Pyricularia oryzae T-DNA mutant library, obtains the mutant strain T940001602-2 that a sporulation quantity significantly reduces.At first set up Pyricularia oryzae T-DNA mutant library: with agrobacterium strains AGL-1 (containing pBHt1), cultivate 48h for last 28 ℃ at minimum medium (containing Kan50 μ g/mL), it is centrifugal to draw an amount of culture, and wash twice with inducing culture, be resuspended in the inducing culture that contains Kan and Syringylethanone 28 ℃ of shaking culture 6h; Draw the conidial suspension (10 of above-mentioned culture of 100 μ L and 100 μ L Pyricularia oryzaes then
6Individual/as mL) to mix, be coated with on the cellulose membrane that is placed on the common substratum, cultivate 36h at 28 ℃.Wash film with the 2mL minimum medium, collect fungi and bacterial cultures, coat then on the selection culture medium flat plate that contains Totomycin and cefotaxime sodium, the single transformant of picking go to again on the rice bran substratum that contains Totomycin (rice bran 20g, agar 18g adds water to 1L, pH6.5) produce spore, monospore is separated on the yeast culture base (agar 15g adds water to 1L for starch 10g, yeast powder 2g) and preserves filter paper.
The picking mutant carries out the phenotype observation at random, each mutant and wild-type are grown on the yeast culture base earlier, punch covering with on the yeast starch culture-medium of mycelia with the punch tool of diameter 0.7cm, then the bacterium piece of circle is transferred and contain in the culture dish of rice bran substratum in diameter 9cm, spore 14d is produced in 28 ℃ of illumination, with 1mL sterilized water wash-out spore, blood counting chamber records the conidium number, is converted into the unit surface sporulation quantity again; Spore germination rate, appressorium form and rate of formation observational technique are on artificial hydrophobic surface (the Gelbond film of producing for U.S. BMA company), drip 20 μ L (10
4The spore suspension of individual spore/mL), 28 ℃ of spore germination rates of statistics when cultivating 8h down, 12h statistics appressorium rate of formation and whether observe the record form normal.More than experiment repeats results averaged equal 3 times.By above screening method, obtain the mutant strain T940001602-2 that sporulation quantity significantly reduces.This mutant sporulation quantity reduces, and spore germination rate and appressorium rate of formation postpone, but do not influence the pathogenic of this bacterium, 1 * 10
5/ cm
2Inoculum density under, also can form susceptible scab.
Extract this mutant gene group DNA, get about 8 μ g and carry out complete enzymolysis with restriction enzyme, (amplimer is hph-S:5 '-CGACAGCGTCTC CGACCTGA-3 ' with hygromycin gene; Hph-AS:5 '-CGCCCAAGCTGCATCATCGAA-3 ') carries out Southern hybridization for probe.The Southern results of hybridization shows that this mutant T-DNA inserts for single copy.With this mutant and GUY11 illumination face-off growth 30d on oat medium, formed thecaspore is separated, set up sexual filial generation colony.Analyze to the product spore shape of each offspring's bacterial strain of this colony with to hygromycin resistance, the result shows that the proterties segregation ratio of offspring's bacterial strain is 1: 1, illustrates that producing the spore proterties in this mutant is controlled by single-gene.
Embodiment 2: mutant T-DNA flanking sequence obtains and by the label assignment of genes gene mapping
Method by TAIL-PCR obtains the T-DNA flanking sequence of T940001602-2, and the primer is: LB1:5 '-GGGTTCCTATAGGGTTTCGCTCATG-3 '; LB2:5 '-CATGTGTTGAGCATATAAGAAACC CT-3 '; LB3:5 '-GAATTAATTCGGCGTTAATTCAGT-3 '; AD9:5 '-TCGTTCCGCA-3 '.Second and third step product of TAIL-PCR after 1.0% agarose electrophoresis, is reclaimed the specific fragment in the 3rd step product.The fragment that recovery is obtained is connected on the pGEM-T Easy Vector, clones.The positive transformant that the clone is obtained checks order, and after the measured sequence that arrives is removed carrier sequence and T-DNA sequence, compares at the Pyricularia oryzae database.Comparison result shows that this sequence is positioned at the promoter region of MG08093.4.Wherein MG08093.4 is the gene fragment shown in the SEQ ID1, has the fungal transcriptional factor structure of a Zn (2) Cys (6).
Embodiment 3: knocked out by label gene 5 ' end
Method with homologous recombination knocks out MG08093.4.At first make up and contain the upstream and downstream homologous fragment that will knock out gene to some extent and the recombinant plasmid of fungi screening mark.Design the primer of amplification upstream and downstream homologous fragment respectively, two segmental sizes are 500bp and 300bp.The Totomycin transferase gene that will contain promotor and terminator with restriction enzyme Sal I downcuts from the pCSN43 plasmid, behind the recovery purifying, this fragment is carried out external the connection with two other homologous fragment with the T4 ligase enzyme.Connecting product increases with segmental 3 ' the end primer of segmental 5 ' end primer in upstream and downstream.Amplified production is connected on the T-carrier, clones.Extract the plasmid DNA of cloning the positive transformant that obtains, carry out next step Pyricularia oryzae protoplast transformation.Extract the protoplastis of wild type strain, the method with the PEG mediation imports recombinant plasmid dna in the protoplastis, in the enterprising row filter growth of the substratum that contains Totomycin.The transformant that growth obtains carries out PCR and Southern hybridization checking.
The resulting positive of protoplasma knocks out transformant, shows as sporulation quantity and reduces, and appressorium forms proterties such as delay.Aspect pathogenic, show as and to invade but do not form susceptible scab.
Embodiment 4:T-DNA mutant with knock out mutant and have complementary functions
Because the T-DNA of T-DNA mutant is inserted in the promoter region of MG08093.4, the constructed complementary carrier of the present invention comprises the dna sequence dna of this gene 5 ' end and the preceding 2kb of gene.At first, complementary carrier pBARKS1 linearizing is carried out complete degestion with restriction enzyme Spe I to the pBARKS1 plasmid DNA and is carried out the dephosphorylation processing, behind the recovery endonuclease bamhi.DNA carries out the target gene fragment that pcr amplification obtains containing promoter region to the wild type gene group, also carries out carrying out external the connection with the carrier segments that reclaims with the T4 ligase enzyme after Spe I handles.Connect product and be transformed in the intestinal bacteria, the picking positive recombinant extracts plasmid and carries out next step protoplast transformation.Extract the protoplastis of T-DNA mutant and gene knockout mutant respectively, with the method for PEG mediation, recombinant plasmid dna is imported in the protoplastis equally, in the enterprising row filter growth of the substratum that contains weedicide.The positive transformant that obtains that screens carries out the phenotype analytical of sporulation quantity, appressorium rate of formation and aspect such as pathogenic equally.The result shows, no matter is the defective that the complementary transformant of T-DNA mutant or the complementary transformant that knocks out mutant can both recover sporulation quantity and other proterties.
Digestion with restriction enzyme system in the above embodiment and linked system are all operated according to the requirement in each enzyme product specification sheets.
Untitled.ST25
SEQUENCE?LISTING
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉Pyricularia oryzae and relevant gene and the application of product spore
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>897
<212>DNA
<213>Magnaporthe?grisea
<400>1
atggctactt?tcactgcact?gaatggcggc?tcgccaaagg?cattggacat?cgtgaatggc 60
ggtgttgcca?gcgagatgaa?gaggtccgga?tcagaggaac?tgccacgaac?cgaggctgtc 120
gcgccgagat?ctcagcgaga?acccaccaac?aacaaagcat?caagaccggc?agacgacgtg 180
agtccgtcta?ggacagcagc?tgctgagacc?gacagggaac?gtgcgaagca?aaatgcagcg 240
gaacttgatg?agcgggagcg?gcaaagggag?aaccgggcag?gtcccgcttc?aactgtcgac 300
cggccgcgtc?atccttcgac?cggcttttct?gatgtcgaca?gcggcgcact?aaaacgcaag 360
agatctaatt?caccagacac?ccgctcaacc?gcggaacccg?ctcatcgaga?aacgtccgaa 420
cccaaggaag?gtcaggcgca?atctcgcccc?cgctcacgtg?atcaaagcag?tttcagtccc 480
caaaagaacc?acgcgacgta?cgcagagacc?ggccacgaga?ggaggaacgg?gcaggagcaa 540
agcgtgagga?agtctctttt?tgggcctccg?ggagccaggg?atgattgggg?accaaactat 600
gagcaggccc?attctgcaat?tccaagctct?gccatcagcg?aggtagaccc?catgggtgac 660
agccttgcca?gggcgacgca?gcgcataagc?aatgacgatg?agagggccag?ccctgatgat 720
atggatgatt?cacccatgac?gccttaccca?agcggctcat?acaatgccga?acagcaacgc 780
gatgggctag?ttcattcgga?tcccaagaag?agaaagcgca?actttagcaa?tcggaccaag 840
acaggctgcc?ttacctgcag?gaaacgcaaa?aagaagtgtg?acgagggcca?aacctga 897
<210>2
<211>298
<212>PRT
<213>Magnaporthe?grisea
<400>2
Met?Ala?Thr?Phe?Thr?Ala?Leu?Asn?Gly?Gly?Ser?Pro?Lys?Ala?Leu?Asp
1 5 10 15
Ile?Val?Asn?Gly?Gly?Val?Ala?Ser?Glu?Met?Lys?Arg?Ser?Gly?Ser?Glu
20 25 30
Glu?Leu?Pro?Arg?Thr?Glu?Ala?Val?Ala?Pro?Arg?Ser?Gln?Arg?Glu?Pro
35 40 45
Thr?Asn?Asn?Lys?Ala?Ser?Arg?Pro?Ala?Asp?Asp?Val?Ser?Pro?Ser?Arg
50 55 60
Thr?Ala?Ala?Ala?Glu?Thr?Asp?Arg?Glu?Arg?Ala?Lys?Gln?Asn?Ala?Ala
65 70 75 80
Untitled.ST25
Glu?Leu?Asp?Glu?Arg?Glu?Arg?Gln?Arg?Glu?Asn?Arg?Ala?Gly?Pro?Ala
85 90 95
Ser?Thr?Val?Asp?Arg?Pro?Arg?His?Pro?Ser?Thr?Gly?Phe?Ser?Asp?Val
100 105 110
Asp?Ser?Gly?Ala?Leu?Lys?Arg?Lys?Arg?Ser?Asn?Ser?Pro?Asp?Thr?Arg
115 120 125
Ser?Thr?Ala?Glu?Pro?Ala?His?Arg?Glu?Thr?Ser?Glu?Pro?Lys?Glu?Gly
130 135 140
Gln?Ala?Gln?Ser?Arg?Pro?Arg?Ser?Arg?Asp?Gln?Ser?Ser?Phe?Ser?Pro
145 150 155 160
Gln?Lys?Asn?His?Ala?Thr?Tyr?Ala?Glu?Thr?Gly?His?Glu?Arg?Arg?Asn
165 170 175
Gly?Gln?Glu?Gln?Ser?Val?Arg?Lys?Ser?Leu?Phe?Gly?Pro?Pro?Gly?Ala
180 185 190
Arg?Asp?Asp?Trp?Gly?Pro?Asn?Tyr?Glu?Gln?Ala?His?Ser?Ala?Ile?Pro
195 200 205
Ser?Ser?Ala?Ile?Ser?Glu?Val?Asp?Pro?Met?Gly?Asp?Ser?Leu?Ala?Arg
210 215 220
Ala?Thr?Gln?Arg?Ile?Ser?Asn?Asp?Asp?Glu?Arg?Ala?Ser?Pro?Asp?Asp
225 230 235 240
Met?Asp?Asp?Ser?Pro?Met?Thr?Pro?Tyr?Pro?Ser?Gly?Ser?Tyr?Asn?Ala
245 250 255
Glu?Gln?Gln?Arg?Asp?Gly?Leu?Val?His?Ser?Asp?Pro?Lys?Lys?Arg?Lys
260 265 270
Arg?Asn?Phe?Ser?Asn?Arg?Thr?Lys?Thr?Gly?Cys?Leu?Thr?Cys?Arg?Lys
275 280 285
Arg?Lys?Lys?Lys?Cys?Asp?Glu?Gly?Gln?Thr
290 295
Claims (4)
- A Pyricularia oryzae with produce the relevant gene of spore, it is characterized in that having the nucleotide sequence shown in the SEQ ID1.
- 2. a kind of Pyricularia oryzae according to claim 1 and the relevant gene of product spore is characterized in that aminoacid sequence is shown in SEQ ID2.
- 3. Pyricularia oryzae and the verification method that produces the spore genes involved is characterized in that may further comprise the steps:(1) with the method for homologous recombination, the described gene fragment SEQ ID1 of claim 1 in the wild rice blast bacterial strain is knocked out, then show as sporulation quantity and reduce pathogenic decline;(2) adopt artificial constructed complementary carrier to transform to be knocked out and lack the rice blast bacterial strain of described SEQ ID1 gene fragment, then the complementary bacterial strain sporulation quantity of gained recovers.
- 4. utilize a kind of Pyricularia oryzae as claimed in claim 1 or 2 gene relevant to prepare novel agrochemical as the target spot gene with producing spore.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100085847A CN101240283A (en) | 2007-02-09 | 2007-02-09 | Rice blast bacterium and sporulation correlated gene and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100085847A CN101240283A (en) | 2007-02-09 | 2007-02-09 | Rice blast bacterium and sporulation correlated gene and application |
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| CN101240283A true CN101240283A (en) | 2008-08-13 |
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| CNA2007100085847A Pending CN101240283A (en) | 2007-02-09 | 2007-02-09 | Rice blast bacterium and sporulation correlated gene and application |
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| CN (1) | CN101240283A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112094852A (en) * | 2018-09-21 | 2020-12-18 | 华南农业大学 | Application of MODIP gene in regulation of growth and development of rice blast fungi and sporulation |
| CN112795578A (en) * | 2021-02-07 | 2021-05-14 | 沈阳农业大学 | Magnaporthe grisea MoPTEN gene and application thereof |
-
2007
- 2007-02-09 CN CNA2007100085847A patent/CN101240283A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094852A (en) * | 2018-09-21 | 2020-12-18 | 华南农业大学 | Application of MODIP gene in regulation of growth and development of rice blast fungi and sporulation |
| CN112094852B (en) * | 2018-09-21 | 2022-04-01 | 华南农业大学 | Application of MODIP gene in regulation of growth and development of rice blast fungi and sporulation |
| 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 |
| CN109293756B (en) * | 2018-10-23 | 2020-10-09 | 北京农学院 | Protein for controlling spore yield and infection capacity of rice blast fungus |
| CN112795578A (en) * | 2021-02-07 | 2021-05-14 | 沈阳农业大学 | Magnaporthe grisea MoPTEN gene and application thereof |
| CN112795578B (en) * | 2021-02-07 | 2022-06-10 | 沈阳农业大学 | Magnaporthe grisea MoPTEN gene and application thereof |
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Open date: 20080813 |