CN110183521A - Application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity - Google Patents

Abstract

The present invention discloses a kind of application of rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity, belongs to plant genetic engineering field.The present invention is conducted into rice blast fungus protoplast by building gene knockout carrier；Gene M oRMD1 is knocked out from rice blast fungus using methods of homologous recombination, obtains knockout mutations body Δ MoRMD1；Obtained rice blast fungus knockout mutations body existing defects in terms of note fields, and MoRMD1 plays an important role to the integrality for maintaining cell wall.Pathogenicity shows that the missing of MoRMD1 significantly reduces the virulence of rice blast fungus, and obvious scab cannot be formed on rice leaf.The invention demonstrates that MoRMD1 is rice blast fungus note fields, maintains cell wall integrity and pathogenic necessary.Our research facilitates the pathogenic molecular mechanism for deeply illustrating rice blast fungus, provides target gene to develop effective fungicide.

Description

Application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity

Technical field

The invention belongs to plant genetic engineering field, in particular to a kind of rice blast fungus gene M oRMD1 is caused in regulation rice blast fungus Application in sick power.

Background technique

Rice blast caused by rice blast fungus (Magnaporthe oryzae) is a kind of important disease in Rice Production.The whole world Every year because of about 66,000,000,000 dollars of rice economic loss caused by rice blast, the rice of loss can provide food for 60,000,000 people.Rice blast Bacterium specifically includes that (1) conidium is propagated with wind and rain to the infection processs of rice, and is adhered to rice leaf surface；(2) mitogenetic Spore germination forms germ tube；(3) germ tube is differentiated to form appresorium；(4) appresorium, which is differentiated to form, infects nail；(5) nail is infected to penetrate Host cell, and formed in host cell and infect mycelia, it is extended in iuntercellular.Wherein, rice blast fungus successfully infects rice Critical process be to be formed Infection structure-appresorium of eggcase, the turgescence generated after appresorium is mature can make to infect nail Rice cuticula is penetrated, to successfully infect rice cell.When rice blast fungus cannot form complete appresorium, pathogenicity is aobvious It writes and weakens.

DUF structural domain (a domain of unknown function) refers to a kind of egg for not containing known specific function White structural domain is the general designation of agnoprotein functional domain.In Pfam database, there are about 3000 DUF protein families.True In core biology, about contain 1500 DUFs.Many DUFs have well-conserved, thus it is speculated that play in the biological function of fungi Important function, but its concrete function is unknown.

Summary of the invention

In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of rice blast fungus gene M oRMD1 Application in regulation rice blast fungus pathogenicity.

Sporulation protein RMD1 is the protein (Protein of a prediction in rice blast fungus Predicted), it is named as MoRMD1；Through uniprotKB database (https: //www.uniprot.org/uniprot/) point Analysis, contains DUF155 structural domain.But the specific biological function of rice blast fungus MoRMD1 is not known at present.By Experiments have shown that the missing of MoRMD1 significantly reduces the virulence of rice blast fungus, obvious scab cannot be formed on rice leaf.

The purpose of the invention is achieved by the following technical solution:

The present invention provides a kind of application of rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity.

Further, application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus growth and development.

Further, application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus attachment spore is formed.

Further, the rice blast fungus gene M oRMD1 is maintaining the application in rice blast fungus cell wall integrity.

The present invention provides a kind of application of rice blast fungus gene M oRMD1 in prevention and treatment in the rice blast as caused by rice blast fungus.

The present invention provides a kind of application of rice blast fungus gene M oRMD1 as the target of the drug for control of plant disease, The plant disease is the rice blast as caused by rice blast fungus.

The present invention provides a kind of method for treating the plant rice blast as caused by rice blast fungus again, comprising blocking or inhibiting rice blast The expression (such as utilizing the antisense RNA of the gene or siRNA etc.) of gene M oRMD1 in bacterium.

Block or inhibit rice blast fungus in gene M oRMD1 expression medicament (such as using the gene antisense RNA or SiRNA etc.) application in medicine preparation, the drug is for controlling the plant rice blast as caused by rice blast fungus.

Wherein, the rice blast fungus gene M oRMD1, amino acid sequence is as shown in SEQ ID NO:2, or such as SEQ Amino acid sequence shown in ID NO:2 still has control rice by what one or more amino acid substitutions, insertion, missing obtained The analog of pest bacterium pathogenicity function；

The rice blast fungus gene M oRMD1, nucleotides sequence are classified as one of following A, B, C:

A, the DNA sequence dna of amino acid sequence shown in SEQ ID NO:2 is encoded；

B, the DNA sequence dna as shown in SEQ ID NO:1；

C, the above A and B still has the function of control rice blast fungus pathogenicity by what base insertion, missing or replacement obtained Analog；

Knockout carrier, recombinant bacterium containing above-mentioned rice blast fungus gene M oRMD1 also belong to the present invention in the application of above-mentioned aspect Protection scope.

The present invention is conducted into rice blast fungus protoplast by building gene knockout carrier；It will using methods of homologous recombination Gene M oRMD1 is knocked out from rice blast fungus, obtains knockout mutations body Δ MoRMD1；The mutant is deposited in terms of note fields In defect.To Pathogenicity, the result shows that, knockout mutations body Δ MoRMD1 cannot form obvious scab on rice leaf.On Stating test proves, rice blast fungus MoRMD1 gene is the pathogenic related gene of rice blast fungus.

The present invention has the following advantages and effects with respect to the prior art:

Rice blast fungus gene M oRMD1 provided by the invention contains DUF155 structural domain, but it is in the biological function of rice blast fungus It does not know.Test proves that the encoding gene MoRMD1 of albumen MoRMD1 is by hygromycin phosphotransferase gene (hph) and fluorescence After protein gene (SGFP) displacement, obtained rice blast fungus knockout mutations body existing defects in terms of note fields, and MoRMD1 plays an important role to the integrality for maintaining cell wall.Pathogenicity shows that the missing of MoRMD1 significantly reduces rice The virulence of pest bacterium cannot form obvious scab on rice leaf.The invention demonstrates that MoRMD1 is rice blast fungus note fields, dimension Hold cell wall integrity and pathogenic necessary.Our research facilitates the pathogenic molecular mechanism for deeply illustrating rice blast fungus, Target gene is provided to develop effective fungicide.

Detailed description of the invention

Fig. 1 is the building schematic diagram of rice blast fungus MoRMD1 gene knockout carrier.

Fig. 2 is the PCR amplification of the gene M oRMD1 of the hygromycin resistant transformed specific item in part；Wherein, M:Marker5000；Swimming Road 1: rice blast fungus wild type WT；Swimming lane 2-6: transformant 2,12,20,197,323.

Fig. 3 is the PCR amplification of the hygromycin resistant transformed sub- A-hph gene in part；Wherein, M:Marker5000；Swimming lane 1: PCT74 plasmid；Swimming lane 2-6: transformant 2,12,20,197,323.

Fig. 4 is positive transformant Southern blot analysis (using target gene as probe)；Wherein, swimming lane 1: rice blast fungus Wild type WT；Swimming lane 2-6: transformant 2,12,20,197,323.

Fig. 5 is positive transformant Southern blot analysis (using hph as probe)；Wherein, swimming lane 1-5: transformant 2, 12,20,197,323.

Fig. 6 is colonial morphology of the rice blast fungus knockout mutations body Δ MoRMD1 on YGA culture medium.

Fig. 7 is rice blast fungus knockout mutations body Δ MoRMD1 compared with the sporulation quantity of wild type；Note: product spore culture medium used is Tomato oat medium.

Fig. 8 is the measurement of rice blast fungus knockout mutations body Δ MoRMD1 cell wall integrity.

Fig. 9 is the conidial sprouting of rice blast fungus knockout mutations body Δ MoRMD1；Wherein, the scale size in figure is 10 μm。

Figure 10 is the pathogenic analysis of rice blast fungus knockout mutations body Δ MoRMD1.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels And material.

Embodiment 1

1, experimental material

1.1 strains testeds and plant

Rice blast fungus microspecies are Guangdong Province's dominant races ZC₁₃, it is that long-grained nonglutinous rice strain CO39 (is free of known blast resisting for examination rice Gene).

1.2 host strains and plasmid vector

Cloning vector is pMD18-T vector, and gene knockout carrier is filamentous fungi expression vector pCT74.

2, experimental method

The building of 2.1 rice blast fungus knockout carriers

The building schematic diagram of rice blast fungus MoRMD1 gene knockout carrier is as shown in Figure 1.In the upstream of MoRMD1 gene under Swim the sequence that each selection length scale is about 1500bp or so, and design primer (table 1).

The amplimer of table 1 rice blast fungus MoRMD1 DNA homolog arm A segment and homology arm B segment

Primer	Primer sequence 5 ' -3 '	Restriction enzyme site
			MoRMD1-AF	GGGGTACCGAATGGACCTCGGCCTGTAT	Kpn I
MoRMD1-AR	CCGCTCGAGTGTTCATCGGTCGTGCGAA	Xho I
			MoRMD1-BF	CGGAATTCGCATGGAATATCATCAGGGCG	EcoR I
MoRMD1-BR	GCTCTAGAGCGCTGGATGAAGCAGAATC	Xba I

Rice blast fungus genomic DNA is extracted using fungal DNA extraction kits method (OMEGAFungal DNAKit)；With the base Because group DNA is template, PCR amplification is carried out with primer MoRMD1-AF and MoRMD1-AR, obtains the homology arm A piece of MoRMD1 gene Section (MoRMD1-A)；PCR amplification is carried out with primer MoRMD1-BF and MoRMD1-BR, obtains the homology arm B piece of MoRMD1 gene Section (MoRMD1-B).

Specific PCR reaction system are as follows:

Template DNA	1.0μL
		MoRMD1-A/BF(20μmol/L)	1.0μL
MoRMD1-A/BR(20μmol/L)	1.0μL
		10×Taq Buffer(Mg2+plus)	5.0μL
dNTPs(2.5mmol/L)	4.0μL
		Taq(5U/μL)	0.5μL
ddH2O	37.5μL
		Total	50.0μL

PCR reaction condition are as follows: 94 DEG C of reaction 5min；94 DEG C of reactions 1min, 67 DEG C of reactions 1min, 72 DEG C of reaction 1min, altogether 35 circulations；72 DEG C of reaction 10min, i.e. acquisition pcr amplification product.With OMEGA Cycle Pure Kit kit, PCR is expanded Volume increase object carries out cleaning recycling.

The carrier T of 2.2 rice blast fungus MoRMD1 DNA homolog arms connects and conversion

With reference to pMD 18-T Vector Cloning Kit (TakaRa company) kit specification, by MoRMD1-A and MoRMD1-B is connect with carrier T respectively, obtains recombinant plasmid pMD18T-MoRMD1-A and pMD18T-MoRMD1-B.Specifically: 1 μ L pMD 18-T carrier is taken, the above-mentioned PCR recovery product of 4 μ L (MoRMD1 DNA homolog arm A or homology arm B segment) is separately added into With 5 μ L solution I, stayed overnight in 16 DEG C of connections.10 μ L connection products are taken to be added to 100 μ L Escherichia coli DH, 5 α competence In cell, 30min is placed on ice；Thermal shock 90s, cooled on ice 5min in 42 DEG C of water-baths；800 μ L LB liquid mediums are added, In 37 DEG C, 150rpm shaken cultivation 45min；It is centrifuged 5min then at 4000rpm, abandons supernatant, 100 μ L bacterium solutions and precipitating is stayed to mix, It is coated on LB solid medium (containing 50 μ g/mL Amp)；8~12h is cultivated at 37 DEG C.

Picking has the positive transformant of Amp resistance, extracts recombinant plasmid dna, and carry out sequencing identification.With Kpn I, Xho I carries out double digestion to pMD18T-MoRMD1-A and pCT74 carrier respectively, recycles A segment and pCT74 carrier.Use T4DNA A segment is connect by ligase with pCT74 carrier, converts 5 α of Escherichia coli DH；Obtain recombinant plasmid pCT74-MoRMD1-A.It presses Same program recycles B piece with EcoR I and Xba I double digestion pMD18T-MoRMD1-B and recombinant plasmid pCT74-MoRMD1-A Section and recombinant plasmid.B segment is connect with pCT74-MoRMD1-A with T4DNA ligase, converts 5 α of Escherichia coli DH；Through enzyme Identification is cut, gene knockout carrier pCT74-MoRMD1-KO is obtained.

The preparation of 2.3 rice blast fungus protoplasts

Rice blast fungus is inoculated into YPS culture medium (yeast extract 6g, caseinhydrolysate 6g, sucrose 10g, distilled water constant volume Into 1L), in 28 DEG C, 130rpm shaken cultivation 2d；200 mesh cell sieves of above-mentioned culture are filtered, grinds, takes appropriate mycelia Liquid is transferred in the YPS culture medium of 200mL, in 28 DEG C, 130rpm shaken cultivation 1d；Mycelia liquid, bacterium are filtered with 200 mesh cell sieves Silk is used aseptic water washing 2 times, and 0.8mol/L NaCl is rinsed 1 time, is weighed to wet mycelia.In enzyme solution and mycelia ratio (volume Mass ratio 10:1), it is added appropriate 10mg/mL lywallzyme enzyme solution, after 30 DEG C of enzymatic hydrolysis 3h, with sterilizing KIMTECH dust-free paper filtering, receives Collect digestion solution.In 4 DEG C, 3500rpm is centrifuged 10min；By precipitating be resuspended in 1.5~2mL pre-cooling STC (sorbierite containing 1.2M, 10mM Tris-HCl, pH 7.5,50mM CaCl₂), in 4 DEG C, 5000rpm is centrifuged 10min；Precipitating is resuspended in 1mL STC, Make protoplast final concentration of 1 × 10⁷A/mL.

The conversion of 2.4 rice blast fungus protoplasts

Double digestion is carried out to knockout carrier pCT74-MoRMD1-KO with Kpn I and Xba I, obtains A-hph-sgfp-B piece Section.The A-hph-sgfp-B segment of 2 μ g and 200 μ L protoplasts are mixed, ice bath 20min；1mL PTC is added and converts buffer (60%PEG4000,50mM CaCl₂, 10mM Tris-HCl, pH7.5), it is placed at room temperature for 20min；3500rpm is centrifuged at 4 DEG C 10min；Fluid nutrient medium (yeast extract 6.0g, caseinhydrolysate 6.0g, sucrose 200.0g, distilled water constant volume are regenerated with 4mL It is resuspended and precipitates to 1L), in 28 DEG C, 16~18h of 100rpm shake culture.(the regeneration liquid training of 40mL regenerated solids culture medium is added Support in base containing 1.5% agar powder and 200 μ g/mL hygromycin), a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is mixed, in 28 DEG C of 3~4d of dark culturing；Picking hygromycin Resistant transformants, be transferred to containing 200 μ g/mL hygromycin YGA culture medium (5.0g containing yeast extract, anhydrous grape 22.0g, Agar powder 17.0g, distilled water are settled to 1L), in 28 DEG C of 3~4d of dark culturing.

The PCR of 2.5 rice blast fungus knockout mutations bodies verifies analysis

According to fungal DNA extraction kits method (OMEGA Fungal DNAKit) specification, it is positive to extract above-mentioned hygromycin The genomic DNA of transformant carries out PCR verifying analysis.Genetic fragment is carried out with primer MoRMD1-F/MoRMD1-R respectively The PCR amplification of MoRMD1；The PCR amplification analysis of genetic fragment A-hph is carried out with primer A-hph-F/A-hph-R.

MoRMD1-F:5 '-CCAATTCCAAGCGAACCGTC-3 ',

MoRMD1-R:5 '-ACGTCCAGTCGCTCGTTTAG-3 ',

A-hph-F:5 '-GCTCCTCGTCGTATCGTCTC-3 ',

A-hph-R:5 '-ACCGCAAGGAATCGGTCAAT-3 '；

PCR reaction system is as follows:

Template DNA	0.5μL
		MoRMD1-F/A-hph-F(20μmol/L)	0.5μL
MoRMD1-R/A-hph-R(20μmol/L)	0.5μL
		10×Taq Buffer(Mg2+plus)	2.5μL
dNTPs(2.5mmol/L)	2.0μL
		Taq(5U/μL)	0.25μL
ddH2O	18.75μL
		Total	25.0μL

PCR reaction condition are as follows: 94 DEG C of reaction 5min；94 DEG C of reactions 1min, 61 DEG C of reactions 1min, 72 DEG C of reaction 2min, altogether 32 circulations；72 DEG C of reaction 10min, obtain amplified production.

The Southern blot of 2.6 rice blast fungus knockout mutations bodies is analyzed

According to DIG High Prime DNALabeling and Detection Starter Kit I (Roche LOT28309220) specification carries out Southern blot hybridization.With primer MoRMD1-F/MoRMD1-R amplifying target genes Probe expands hph gene probe with hph-F/hph-R.

MoRMD1-F:5 '-CCAATTCCAAGCGAACCGTC-3 ',

MoRMD1-R:5 '-ACGTCCAGTCGCTCGTTTAG-3 ',

Hph-F:5 '-TGCTGCTCCATACAAGCCAA-3 ',

Hph-R:5 '-GACATTGGGGAGTTCAGCGA-3 '；

The PCR amplification system of DNA probe is as follows:

Template DNA	1.0μL
		MoRMD1-F/hph-F(20μmol/L)	1.0μL
MoRMD1-R/hph-R(20μmol/L)	1.0μL
		10×Ex Taq Buffer(Mg2+plus)	5.0μL
dNTPs(2.5mmol/L)	4.0μL
		Ex Taq(5U/μL)	0.5μL
ddH2O	37.5μL
		Total	50.0μL

PCR reaction condition are as follows: 94 DEG C of reaction 5min；94 DEG C of reactions 1min, 54 DEG C of reactions 1min, 72 DEG C of reaction 2min, altogether 32 circulations；72 DEG C of reaction 10min, obtain amplified production.

The Phenotypic Observation of 2.7 rice blast fungus knockout mutations bodies

(1) colony morphological observation and speed of growth measurement.Rice blast fungus wild type and knockout mutations body Δ MoRMD1 are inoculated with In on YGA culture medium (5.0g containing yeast extract, anhydrous grape 22.0g, agar powder 17.0g, distilled water are settled to 1L), in 28 DEG C It is cultivated under dark condition.Colony diameter is measured in 2d, 4d, 6d, 8d, 10d respectively, and observes rice blast fungus wild type and mutation The colonial morphology of body.

(2) conidial generation is observed with sprouting.By rice blast fungus be seeded to tomato oat medium (raw oat 40g, 1h is boiled with distilled water, 150mL Tomato juice, 0.06g calcium carbonate are added after filtering, 2.5%~3% agar powder uses distilled water It is settled to 1L), 28 DEG C of incubator cultures are placed in, for 24 hours 7~10d of illumination.3~5mL aseptic water washing conidium is added in every ware, It is filtered with 3~5 layers of KIMTECH dust-free paper, as conidial suspension.It is counted with blood counting chamber, counts sporulation quantity. Conidial suspension is inoculated on pvdf membrane, is sampled when 2h, 4h, 6h, 8h, 10h, with the mitogenetic spore of micro- sem observation The sprouting of son.

(3) cell wall integrity measures

By rice blast fungus wild type and knockout mutations body Δ MoRMD1 be seeded in respectively containing 0.005%SDS, 0.01%SDS, On 0.02%SDS and 200 μ g/mL Congo red YGA culture medium, after 28 DEG C of 10~14d of culture carton upside down culture, observation is knocked out The bacterium colony growing state of mutant Δ MoRMD1 and wild-type strain.

The pathogenic analysis of 2.8 rice blast fungus knockout mutations body △ MoRMD1

The the 4th or the 5th leaf for taking 4~5 leaf phase rice seedlings, with rice blast fungus wild type and knockout mutations body Δ MoRMD1 Conidial suspension (1~5 × 10⁵A/mL contains 0.25% polysorbas20) spray inoculation；It is placed under 28 DEG C of dark conditions For 24 hours, the incidence of rice leaf is observed after the dark 12h alternate culture of light 12h/, 5~7d.

3 results and analysis

The building of 3.1 rice blast fungus MoRMD1 gene knockout carriers

Using PCR amplification method, clone obtains MoRMD1 DNA homolog arm A and homology arm B segment respectively；By its respectively with Carrier T connection, inverted Escherichia coli, Amp resistance screening, plasmid are extracted to be identified with sequencing, obtains recombinant plasmid pMD18T- MoRMD1-A and pMD18T-MoRMD1-B.PMD18T-MoRMD1-A is connect with pCT74 plasmid, obtains recombinant plasmid pCT74- MoRMD1-A；It is subjected to double digestion with pMD18T-MoRMD1-B, identifies, obtains through DNA connection, Escherichia coli conversion, digestion Gene knockout carrier pCT74-MoRMD1-KO (Fig. 1).

The screening of 3.2 rice blast fungus knockout mutations bodies

3.2.1 the PCR verifying of genetic fragment MoRMD1

Using methods of homologous recombination, gene knockout carrier is converted into rice blast fungus protoplast, obtains 215 hygromycin sun Property transformant.Extraction through rice blast fungus genomic DNA, using MoRMD1 gene-specific primer, to 68 hygromycin therein Positive transformant has carried out PCR verifying analysis.The result shows that there are 63 positive transformants amplifiable to target gene fragment, say This bright 63 transformants still contain MoRMD1 gene；There are 5 transformants not expand to MoRMD1 gene, Preliminary Identification this 5 Transformant is positive transformant (Fig. 2).

3.2.2 the PCR of genetic fragment A-hph is verified

Using above-mentioned 5 without expanding the transformant genomic DNA to MoRMD1 gene as template, A-hph specificity is utilized Primer carries out PCR amplification；The result shows that above-mentioned arrive 5 transformants of MoRMD1 gene without amplification, about 1500bp has been arrived in all amplifications Target fragment, further illustrate that this 5 transformants are positive transformant (Fig. 3).

3.2.3 the Southern blot verifying of knockout mutations body

5 positive transformants for expanding no amplification to MoRMD1 gene to A-hph gene have carried out Southern Blot analysis.The result shows that being hybridized using target gene as probe, 5 transformants do not have hybridising band (Fig. 4).With Hph is that probe is hybridized, it is found that 5 transformants have single copy band (Fig. 5) occur.Above-mentioned test proves this 5 conversions Son is positive transformant.

The colonial morphology and growth rate of 3.3 rice blast fungus knockout mutations bodies are analyzed

2 knockout mutations bodies (△ MoRMD1-197 and △ MoRMD1-323) is randomly selected, is inoculated in YGA culture medium, Its growing state is observed in different time respectively.The result shows that compared with rice blast fungus wild type, knockout mutations body △ The colonial morphology of MoRMD1 does not have significant difference (Fig. 6).

The production spore and conidia germination of 3.4 rice blast fungus knockout mutations bodies are observed

Knockout mutations body △ MoRMD1-197 and △ MoRMD1-323 are inoculated in tomato oat medium, cultivate 14d Afterwards, sporulation quantity analysis is carried out.The result shows that there was no significant difference for the sporulation quantity of rice blast fungus wild type and mutant Δ MoRMD1 (Fig. 7).

The cell wall integrity of 3.5 rice blast fungus knockout mutations bodies measures

Knockout mutations body △ MoRMD1-197 and △ MoRMD1-323 are inoculated in SDS containing various concentration and Congo red YGA culture medium observes its colonial morphology.The result shows that Δ MoRMD1 mutant is to 0.02% compared with wild type SDS and 200 μ g/mL are Congo red more sensitive, illustrate after knocking out MoRMD1, affect the integrality (Fig. 8) of rice blast fungus cell wall.

Conidium is inoculated on pvdf membrane, 28 DEG C of incubator cultures are placed in；It is seen respectively in different time sampling It examines.The result shows that rice blast fungus wild type conidium can normally generate germ tube and attachment spore；But knockout mutations body Δ MoRMD1 points Complete appresorium (8h) (Fig. 9) cannot be formed after raw spore germination.

The pathogenic analysis of 3.6 Pyricularia oryzae knockout mutations bodies

Rice blast fungus wild type and knockout mutations body Δ MoRMD1 conidium are distinguished into Inoculated Rice excised leaf, after 7d It is observed.The result shows that occurring obvious scab on rice leaf after the inoculation of rice blast fungus wild type；And knockout mutations body △ After MoRMD1 inoculation, rice leaf fails to form obvious scab (Figure 10).After the result illustrates knockout MoRMD1 gene, rice blast fungus Pathogenicity is decreased obviously.

Therefore, gene provided by the invention can be used for control of plant disease, especially by the rice blast caused by rice blast fungus Disease.In addition, gene provided by the invention can be used as the target of the drug for control of plant disease.Those skilled in the art can be with The introduction and enlightenment of this specification that follows up, exploitation is for controlling plant diseases, particularly the drug of rice blast.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Agricultural University Of South China

<120>application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2072

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>nucleotide sequence of rice blast fungus gene M oRMD1

<400> 1

atggccgaag ccgtgacgga aacctctccg ctgctatcga cggtgtcgga gccgaccact 60

accccggaat tccctcgtcc caattccaag cgaaccgtca ccttcaatcc gaaccctgtc 120

acgcgaacca tcgagcccga acgacaacct ctccgccgcc gcatttccac aaacatcgat 180

gtcgctcgcg catctgggca gaggtcgcct ggcggctcgg gtccgcccac ccccacgagc 240

tcaataagca ccggtagtct cgccggtgct ccgccaatgc tggtcgcact caactctaaa 300

cttcgccgcc gcaactccca tggaggtaca cagggttcca tgccgggctc aaacaaccta 360

ctaccagccc cgggtggtgt gggtctcgtc gccggggccg gtcacctgcc caagattggg 420

ccccagcgta gcaccaagaa ggcgcagaag ctcaagctgc ttcccacgcc tgaacttgcc 480

gacgatggtg acgatgacca agatgaggag agcggacgtg aggtctacag ccagtacacg 540

cgtatcaagg accccaatgc ccgacgagat gccgccaggc taggcaaggc cgatcgcgac 600

cgtctgccgc gcgtcacggc gtactgcacc gccaacaaat accagatgga tggcctgatg 660

cactggctga aggggagcag gagcagagca aagggagcga accccaagct cgtggacgag 720

tgcatataca cgccgtacca gtataaagac acgccaccac cgcccccagg acgtggtgcg 780

aggattcgca gagttgccag tgctcagaat gctgccgacg ctgccgagga tgccgcgagg 840

gctgcggtca acgctgatgg catcctgggg tctgaggtaa ccagtgactc accggagccg 900

atgtctaccc aacagaggag acactcgact ggtgatgtcg aggccacccc accccgattc 960

agccaggagg acctgataga tctggagcca gaggctatct ctgatgtgca agatggacga 1020

agcgaggcac gcatagagag cctcggagaa ggagaccacg cagaaatatc aagcagcccg 1080

caagaacgtg accacgagca tggtcacgac catgatcaat cccccagctc ggcagaccat 1140

cgggatgaaa tgatcagcac gcaccacgag cccatcaacg agcgacctgc cgattttgat 1200

atcgaggtac acaccccgga ggttttcctc tttgactacg gcgtggtggt gatatggggc 1260

atgacgatgg cacaggagcg gaggtttctc aaggaaattg ccaagtttga aaccgagaaa 1320

ctggcgaccg aggaggtcga gactgaacac ttcaactttt actacacccg cgagtatcag 1380

cccaggatat acaacgactt tatcaccctc cgtgacaagc acaactacat gaccaagctg 1440

gccatttcac atgcgctggc acagagtgtc aaggttagta ctctcactct ttggctgttt 1500

tgccatgcgt tctatttcgg tgggcacttc taacacgaac tagacgtctc ttttcgagga 1560

gctcatcgcc tctaccgtcg acacgtgcaa ggacattccg acgcagattg ctttgaccgg 1620

caagatcaat cttagcagga cacagatcaa catgcagatc ggcgagcttt tcattctgag 1680

gatcagtatt cacctcaacg gctccgtcct tgacacgcca gagcttttct gggtcgagcc 1740

tcaactcgag ccgctgtacg ccgctgtgcg gtcctaccta gagatggacc agcgagttgg 1800

cctgctaaac gagcgactgg acgtcattgc ggacttgctt gctgtgctca aagaccaact 1860

gagtcatggt catggtgaga aactcgagtg gattggtaag tttgcttcca ttctttcgcg 1920

cgcccagtcc cttgcgcctt tgaacttgtt ctatggtatc atgaatactg accagcatcc 1980

tttgggttta cacagttatt gtattgattg cagccgaaat ccttgttgct gctgttaaca 2040

tagttgtaga tttatatgcg ggcgtcgact ag 2072

<210> 2

<211> 633

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>amino acid sequence of rice blast fungus gene M oRMD1

<400> 2

Met Ala Glu Ala Val Thr Glu Thr Ser Pro Leu Leu Ser Thr Val Ser

1 5 10 15

Glu Pro Thr Thr Thr Pro Glu Phe Pro Arg Pro Asn Ser Lys Arg Thr

20 25 30

Val Thr Phe Asn Pro Asn Pro Val Thr Arg Thr Ile Glu Pro Glu Arg

35 40 45

Gln Pro Leu Arg Arg Arg Ile Ser Thr Asn Ile Asp Val Ala Arg Ala

50 55 60

Ser Gly Gln Arg Ser Pro Gly Gly Ser Gly Pro Pro Thr Pro Thr Ser

65 70 75 80

Ser Ile Ser Thr Gly Ser Leu Ala Gly Ala Pro Pro Met Leu Val Ala

85 90 95

Leu Asn Ser Lys Leu Arg Arg Arg Asn Ser His Gly Gly Thr Gln Gly

100 105 110

Ser Met Pro Gly Ser Asn Asn Leu Leu Pro Ala Pro Gly Gly Val Gly

115 120 125

Leu Val Ala Gly Ala Gly His Leu Pro Lys Ile Gly Pro Gln Arg Ser

130 135 140

Thr Lys Lys Ala Gln Lys Leu Lys Leu Leu Pro Thr Pro Glu Leu Ala

145 150 155 160

Asp Asp Gly Asp Asp Asp Gln Asp Glu Glu Ser Gly Arg Glu Val Tyr

165 170 175

Ser Gln Tyr Thr Arg Ile Lys Asp Pro Asn Ala Arg Arg Asp Ala Ala

180 185 190

Arg Leu Gly Lys Ala Asp Arg Asp Arg Leu Pro Arg Val Thr Ala Tyr

195 200 205

Cys Thr Ala Asn Lys Tyr Gln Met Asp Gly Leu Met His Trp Leu Lys

210 215 220

Gly Ser Arg Ser Arg Ala Lys Gly Ala Asn Pro Lys Leu Val Asp Glu

225 230 235 240

Cys Ile Tyr Thr Pro Tyr Gln Tyr Lys Asp Thr Pro Pro Pro Pro Pro

245 250 255

Gly Arg Gly Ala Arg Ile Arg Arg Val Ala Ser Ala Gln Asn Ala Ala

260 265 270

Asp Ala Ala Glu Asp Ala Ala Arg Ala Ala Val Asn Ala Asp Gly Ile

275 280 285

Leu Gly Ser Glu Val Thr Ser Asp Ser Pro Glu Pro Met Ser Thr Gln

290 295 300

Gln Arg Arg His Ser Thr Gly Asp Val Glu Ala Thr Pro Pro Arg Phe

305 310 315 320

Ser Gln Glu Asp Leu Ile Asp Leu Glu Pro Glu Ala Ile Ser Asp Val

325 330 335

Gln Asp Gly Arg Ser Glu Ala Arg Ile Glu Ser Leu Gly Glu Gly Asp

340 345 350

His Ala Glu Ile Ser Ser Ser Pro Gln Glu Arg Asp His Glu His Gly

355 360 365

His Asp His Asp Gln Ser Pro Ser Ser Ala Asp His Arg Asp Glu Met

370 375 380

Ile Ser Thr His His Glu Pro Ile Asn Glu Arg Pro Ala Asp Phe Asp

385 390 395 400

Ile Glu Val His Thr Pro Glu Val Phe Leu Phe Asp Tyr Gly Val Val

405 410 415

Val Ile Trp Gly Met Thr Met Ala Gln Glu Arg Arg Phe Leu Lys Glu

420 425 430

Ile Ala Lys Phe Glu Thr Glu Lys Leu Ala Thr Glu Glu Val Glu Thr

435 440 445

Glu His Phe Asn Phe Tyr Tyr Thr Arg Glu Tyr Gln Pro Arg Ile Tyr

450 455 460

Asn Asp Phe Ile Thr Leu Arg Asp Lys His Asn Tyr Met Thr Lys Leu

465 470 475 480

Ala Ile Ser His Ala Leu Ala Gln Ser Val Lys Thr Ser Leu Phe Glu

485 490 495

Glu Leu Ile Ala Ser Thr Val Asp Thr Cys Lys Asp Ile Pro Thr Gln

500 505 510

Ile Ala Leu Thr Gly Lys Ile Asn Leu Ser Arg Thr Gln Ile Asn Met

515 520 525

Gln Ile Gly Glu Leu Phe Ile Leu Arg Ile Ser Ile His Leu Asn Gly

530 535 540

Ser Val Leu Asp Thr Pro Glu Leu Phe Trp Val Glu Pro Gln Leu Glu

545 550 555 560

Pro Leu Tyr Ala Ala Val Arg Ser Tyr Leu Glu Met Asp Gln Arg Val

565 570 575

Gly Leu Leu Asn Glu Arg Leu Asp Val Ile Ala Asp Leu Leu Ala Val

580 585 590

Leu Lys Asp Gln Leu Ser His Gly His Gly Glu Lys Leu Glu Trp Ile

595 600 605

Val Ile Val Leu Ile Ala Ala Glu Ile Leu Val Ala Ala Val Asn Ile

610 615 620

Val Val Asp Leu Tyr Ala Gly Val Asp

625 630

<210> 3

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-AF

<400> 3

ggggtaccga atggacctcg gcctgtat 28

<210> 4

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-AR

<400> 4

ccgctcgagt gttcatcggt cgtgcgaa 28

<210> 5

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-BF

<400> 5

cggaattcgc atggaatatc atcagggcg 29

<210> 6

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-BR

<400> 6

gctctagagc gctggatgaa gcagaatc 28

<210> 7

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-F

<400> 7

ccaattccaa gcgaaccgtc 20

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> MoRMD1-R

<400> 8

acgtccagtc gctcgtttag 20

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> A-hph-F

<400> 9

gctcctcgtc gtatcgtctc 20

<210> 10

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> A-hph-R

<400> 10

accgcaagga atcggtcaat 20

<210> 11

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> hph-F

<400> 11

tgctgctcca tacaagccaa 20

<210> 12

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223> hph-R

<400> 12

gacattgggg agttcagcga 20

Claims (10)

1. application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus pathogenicity, it is characterised in that:

The rice blast fungus gene M oRMD1, amino acid sequence is as shown in SEQ ID NO:2, or such as SEQ ID NO:2 Shown in amino acid sequence by what one or more amino acid substitutions, insertion, missing obtained still there is control rice blast fungus to cause The analog of sick power function.

2. application according to claim 1, it is characterised in that:

Application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus growth and development.

3. application according to claim 2, it is characterised in that:

Application of the rice blast fungus gene M oRMD1 in regulation rice blast fungus attachment spore is formed.

4. application according to claim 2, it is characterised in that:

The rice blast fungus gene M oRMD1 is maintaining the application in rice blast fungus cell wall integrity.

5. application of the rice blast fungus gene M oRMD1 described in claim 1 in prevention and treatment in the rice blast as caused by rice blast fungus.

6. application of the rice blast fungus gene M oRMD1 described in claim 1 as the target of the drug for control of plant disease, It is characterized by: the plant disease is the rice blast as caused by rice blast fungus.

7. according to claim 1, application described in 2,3,4,5 or 6, it is characterised in that:

The rice blast fungus gene M oRMD1, nucleotides sequence are classified as one of following A, B, C:

A, the DNA sequence dna of amino acid sequence shown in SEQ ID NO:2 is encoded；

B, the DNA sequence dna as shown in SEQ ID NO:1；

C, the class still with control rice blast fungus pathogenicity function that the above A and B is obtained by base insertion, missing or replacement Like object.

8. a kind of method for treating the plant rice blast as caused by rice blast fungus, it is characterised in that: comprising blocking or right being inhibited to want The expression of rice blast fungus gene M oRMD1 described in asking 1.

9. the application of the medicament of the expression of rice blast fungus gene M oRMD1 described in blocking or inhibition claim 1 in medicine preparation, It is characterized by:

The medicament is the antisense RNA or siRNA of rice blast fungus gene M oRMD1 described in claim 1, and the drug is for controlling Make the plant rice blast as caused by rice blast fungus.

10. application according to claim 9, it is characterised in that:

The rice blast fungus gene M oRMD1, nucleotides sequence are classified as one of following A, B, C:

A, the DNA sequence dna of amino acid sequence shown in SEQ ID NO:2 is encoded；

B, the DNA sequence dna as shown in SEQ ID NO:1；

C, the class still with control rice blast fungus pathogenicity function that the above A and B is obtained by base insertion, missing or replacement Like object.