阅读说明：本技术 一种稻瘟病菌的致病因子或编码所述致病因子的基因的应用 (Application of pathogenic factor of rice blast fungus or gene for coding pathogenic factor ) 是由 时焕斌 寇艳君 邱结华 王聪聪 孟帅 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种稻瘟病菌的致病因子或编码所述致病因子的基因的应用。本发明通过基因敲除的方法获得了稻瘟病菌铁硫蛋白亚基MoRIP1的敲除突变体,揭示了稻瘟病菌铁硫蛋白亚基MoRIP1在稻瘟病菌菌丝生长产孢和致病中的重要生物学功能。本发明发现MoRIP1是一个新的潜在的药物靶点,为开发以线粒体复合体III为作用靶标的新型杀菌剂提供理论基础和重要参考。(The invention discloses a pathogenic factor of rice blast germ or application of a gene coding the pathogenic factor. The invention obtains the knockout mutant of the iron-sulfur protein subunit MoRIP1 of the rice blast fungus by a gene knockout method, and discloses the important biological functions of the iron-sulfur protein subunit MoRIP1in the growth sporulation and pathogenicity of the hypha of the rice blast fungus. The invention discovers that MoRIP1 is a new potential drug target, and provides a theoretical basis and important reference for developing a novel bactericide taking a mitochondrial complex III as an action target.)

1. The application of a pathogenic factor of rice blast fungus or a gene coding the pathogenic factor as a drug action target spot in screening of a drug for resisting the rice blast fungus is disclosed, wherein the pathogenic factor is the iron-sulfur protein subunit MoRIPl of the rice blast fungus.

2. The use of claim 1, wherein the amino acid sequence of the pathogenic agent is as set forth in SEQ ID No. 1.

3. The use according to claim 2, wherein the gene sequence encoding the pathogenic agent is as shown in SEQ ID No. 2.

4. The drug for resisting rice blast germs is characterized in that targets are pathogenic factors of the rice blast germs or genes encoding the pathogenic factors, and active ingredients of the drug for resisting the rice blast germs are at least one of the following substances:

(1) an inhibitor capable of inhibiting the pathogenic agent;

(2) a DNA or RNA sequence capable of reducing the expression level of said gene;

(3) a sequence capable of knocking out said gene.

5. The Pyricularia oryzae drug according to claim 4, wherein the sequence in which the gene can be knocked out is: and respectively inserting an upstream flanking sequence MoRIP1-up and a downstream flanking sequence MoRIP1-dn, which have the sequences shown in SEQ ID No.3 and SEQ ID No.4, into the vector plasmid to obtain the recombinant plasmid.

6. The drug against Pyricularia oryzae according to claim 5, wherein the vector plasmid is pFGL 821.

7. The drug against Pyricularia oryzae according to claim 5, wherein the plasmid is introduced into Pyricularia oryzae cells by an Agrobacterium transformation method.

8. Use of the Pyricularia oryzae drug according to any one of claims 4 to 7 for inhibiting Pyricularia oryzae.

9. Use of the Pyricularia oryzae drug according to any one of claims 4 to 7 for preventing or treating infection by Pyricularia oryzae of a crop.

10. The use of claim 9, wherein the crop is rice, millet, barley or wheat.

Technical Field

The invention relates to the technical field of biology, in particular to an application of a pathogenic factor of rice blast fungus or a gene coding the pathogenic factor.

Background

The rice is one of the most important grain crops in China, and the high and stable yield of the rice has important significance for guaranteeing economic development and social stability. The rice blast is one of the main diseases in rice planting, seriously threatening the production of rice, and is a fungal disease caused by ascomycete pyricularia oryzae (Magnaporthe oryzae). The disease occurs in different degrees every year in rice planting areas in China, the yield is generally reduced by 10-30%, and partial serious field grains are not harvested. Therefore, scientific prevention and control of the rice blast are important for guaranteeing the safety of the grains.

In China, the main medicaments for preventing and treating the rice blast comprise tricyclazole, Fushiyi, pyricularia grisea, iprobenfos, azoxystrobin and the like. Azoxystrobin belongs to strobilurin fungicides, and is registered to be used for preventing and treating rice blast and false smut. Researches report that the medicament can inhibit hypha growth, spore germination, sporulation and pathogenicity of rice blast fungi. The bactericide belongs to environment-friendly pesticides, has high bactericidal activity and wide disease prevention spectrum, and is safe to non-target organisms. The strobilurin fungicide is a bionic medicament, and acts on cytochrome bc in the electron transfer chain of fungi mitochondria₁Qo site of cytochrome b subunit of the complex blocks the transfer of electrons from cytochrome b subunit to cytochrome c₁The subunit transmits, thereby inhibiting the synthesis of ATP to play a role in sterilization. However, since the target cytochrome b subunit of this agent in fungi is encoded by mitochondrial genomic DNA, it is susceptible to mutation. With the wide use of azoxystrobin in the prevention and treatment of vegetable diseases in recent years, the drug resistance of some pathogenic bacteria such as phytophthora capsici and brown spot pathogen of cucumber is gradually revealed at present. In order to prevent the drug resistance problem of rice blast germs, the excavation of new drug targets has important significance for delaying the drug resistance of the medicaments.

The invention with the publication number of CN110669115A discloses a pathogenic factor related to mitochondrion autophagy of rice blast fungus, a gene and application thereof. The gene related by the invention has slow growth, reduced sporulation amount, reduced formation of infected hyphae and greatly reduced pathogenicity on susceptible rice varieties due to gene deletion mutation caused by homologous recombination. Thus, the most important uses of the invention are: by applying the results, compounds capable of destroying the expression and shearing of the gene and the expression of the protein coded by the gene are designed and screened, or compounds capable of modifying the amino acid sequence of the protein are designed and screened, so that novel antifungal medicaments are developed. Meanwhile, because the gene family of the genes does not have homologous genes in plants, the influence of antifungal drugs developed by taking the genes as targets on host plants is small.

The invention patent application with publication number CN103146716A discloses a nucleotide sequence of a coding region of a novel gene Movma11 derived from Magnaporthe grisea and playing an important role in vegetative growth, spore production and anchorage pathogenicity and an amino acid sequence of a protein encoded by the same. Specifically, the invention discloses a fungal pathogenic gene Movma11 derived from rice blast fungus, wherein the nucleotide sequence of the gene Movma11 or the nucleotide sequence of a complementary chain of the gene Movma11 is SEQ ID NO: 1. the protein coded by the fungus pathogenic gene Movma11 has the sequence shown in SEQ ID NO: 3. The expression of the gene Movma11 is used as a target for designing and screening antifungal medicines, and the expression and modification of the protein are used as targets for designing and screening antifungal medicines.

Mitochondrial cytochrome bc₁The third important complex in the mitochondrial electron transport chain, which contains three core subunits, a cytochrome b subunit and a cytochrome c subunit₁Subunits and an iron-sulfur protein. The iron-sulfur protein is encoded by a nuclear gene, and the amino acid sequence is more stable than cytochrome b. The biological function of this subunit in fungi has not been reported.

Disclosure of Invention

The invention provides a pathogenic factor of rice blast germ or application of a gene coding the pathogenic factor.

An application of a pathogenic factor of rice blast fungus or a gene coding the pathogenic factor as a drug action target in screening a drug for resisting the rice blast fungus is disclosed, wherein the pathogenic factor is rice blast fungus iron sulfur protein subunit MoRIP 1.

The amino acid sequence of the pathogenic factor is shown in SEQ ID No. 1. The gene sequence for coding the pathogenic factor is shown as SEQ ID No. 2.

The invention also provides a rice blast resistance drug, the target point is a pathogenic factor of rice blast resistance or a gene for coding the pathogenic factor, and the active ingredient of the rice blast resistance drug is at least one of the following substances:

(1) an inhibitor capable of inhibiting the pathogenic agent;

(2) a DNA or RNA sequence capable of reducing the expression level of said gene;

(3) a sequence capable of knocking out said gene.

Preferably, the sequence capable of knocking out the gene is: and respectively inserting an upstream flanking sequence MoRIP1-up and a downstream flanking sequence MoRIP1-dn, which have the sequences shown in SEQ ID No.3 and SEQ ID No.4, into the vector plasmid to obtain the recombinant plasmid.

More preferably, the vector plasmid is pFGL 821. More preferably, the plasmid is introduced into cells of Pyricularia oryzae by Agrobacterium transformation.

The invention also provides application of the rice blast bacterium resistant medicament in inhibiting rice blast bacterium.

The invention also provides application of the rice blast germ resisting medicine in preventing or treating infection of rice blast germs of crops. Preferably, the crop is rice, millet, barley or wheat.

The invention obtains the knockout mutant of the iron-sulfur protein subunit MoRIP1 of the rice blast fungus by a gene knockout method, and discloses the important biological functions of the iron-sulfur protein subunit MoRIP1in the growth sporulation and pathogenicity of the hypha of the rice blast fungus. The invention discovers that MoRIP1 is a new potential drug target, and provides a theoretical basis and important reference for developing a novel bactericide taking a mitochondrial complex III as an action target.

Drawings

FIG. 1 is a graph showing the results of the knockdown of Pyricularia oryzae MoRIP1 resulting in growth retardation without conidium production, wherein (A) a wild-type strain,. DELTA.Morip 1 and a anaplerosis mutant strain, Morip1c, were photographed after being cultured on CM and OA medium plates for 7 days; (B) colony diameter statistics after 7 days of growth of the wild type strain, Δ Morip1 and anaplerotic mutant strain on CM and OA medium plates; (C) counting the colony sporulation amount of the wild type strain, the delta Morip1 and the anaplerotic mutant strain after growing for 7 days on a CM and OA culture medium plate; (D) the differentiation of conidiophores of the wild type strain, Δ Morip1 and the anaplerotic mutant strain was microscopically observed.

FIG. 2 is a graph showing the results of detection of rice blast pathogen pathogenicity loss caused by MoRIP1 knockout.

FIG. 3 is a graph showing the results of detection of the localization of MoRip1 to mitochondria at different stages of growth and development of Pyricularia oryzae.

FIG. 4 is a graph showing the results of detection of decrease in intracellular ATP levels due to MoRIP1 knock-out.

FIG. 5 is a graph showing the results of the sensitivity test of MoRIP1 participating in Magnaporthe grisea on azoxystrobin, wherein (A) the growth of wild type strain,. DELTA.Morip 1 and anaplerosis mutant strain, Morip1c, on plates of different azoxystrobin concentrations; (B) after 5 days of culture the colony diameter was measured and the sensitivity of the strain to different concentrations of azoxystrobin was calculated.

FIG. 6 is a graph showing the results of hydrogen peroxide-sensitive detection of Pyricularia oryzae caused by the deletion of MoRIP 1.

Detailed Description

The primers required are shown in Table 1 below.

TABLE 1

Primer name	Sequence (5 '-3')
		MoRIP1-upF	CCGGGGATCCTCTAGATCCTCGTGATAGCACTTACC
MoRIP1-upR	CTTCAATATACTCGACGAGCGGACCTTTCGTTTA
		MoRIP1-dnF	ATGCCGACCGGAACCAAATGGAGGGTGGTGTCTG
MoRIP1-dnR	GGCCAGTGCCAAGCTTTCTGGTTCTGGTCCGTGT
		HPH-F	GTCGAGTATATTGAAGGAGC
HPH-R	TGGTTCCGGTCGGCATCTAC
		MoRIP1inneryzF	CAGGGTGAGTTGGAGGAA
MoRIP1inneryzR	CGTAGTGAGATCCGTGGC
		MoRIP1upyzF	CGTGATTGTCTCGCTGTTG
HPH-yzR	ACCTCCACTAGCTCCAGCCAAG
		npRIP1C-F	AGAAACTCGAGAATTCCCTACCACAGCCTAACGG
npRIP1C-R	TACCGAGCTCGAATTCCCACAGTTGGTGCACTTGGC

Example 1: knockout of MoRIP1 gene and complementation of deletion mutant

The amino acid sequence of the rice blast fungus MoRIP7 coding protein is shown as SEQ ID No.1, and the nucleotide sequence of the rice blast fungus MoRIP1 gene is shown as SEQ ID No.2 (containing a non-coding region).

To reveal the biological functions of MoRIP1in the growth, development and pathogenicity of Magnaporthe grisea, we constructed deletion mutants of MoRIP1 gene using a strategy of homologous recombination.

(1) Firstly, a knockout vector of MoRIP1 is constructed: an upstream flanking sequence MoRIP1-up (SEQ ID No.3) and a downstream flanking sequence MoRIP1-dn (SEQ ID No.4) of about 1Kb of the MoRIP1 gene are respectively amplified by PCR (polymerase chain reaction) by using wild genomic DNA as an amplification template and using primers MoRIP1-upF/upR and MoRIP 1-DnF/DnR. The HPH resistance gene sequence (SEQ ID No.5) was PCR amplified using the primer HPH-F/R, and plasmid pFGL821(addge ID: 58223) containing the hygromycin resistance gene as a template. The upstream and downstream DNA sequences of MoRIP1, the HPH fragment of the resistance gene and the backbone plasmid pKOIB after the restriction enzymes XbaI and HindIII are cut are connected together by utilizing a commercial homologous recombinase kit, and finally, a knockout vector pKOIB-MoRIP1 is obtained.

(2) Agrobacterium mediated transformation of Pyricularia oryzae: the knockout carrier is transformed into agrobacterium strain AGL1, then conidium solution of agrobacterium and rice blast germ is mixed and evenly coated on an induction culture medium which is stuck with a nitrocellulose membrane, and the nitrocellulose membrane is transferred to a resistance plate containing hygromycin after 2 days. Four days later, transformants grew on the nitrocellulose membrane edge, and transformants were picked onto new resistant plates.

(3) And (3) knockout transformant verification: extracting transformant genome DNA, verifying the transformant by using an internal verification primer MoRIP1inneryz-F/R and a verification primer MoRIP1upyzF/HPH-R after homologous recombination, and finally obtaining a knockout mutant which is named as delta Morip 1.

(4) Complementation of deletion mutant Δ Morip 1: to demonstrate that the phenotypic changes observed in strain Δ Morip1 were all due to deletion of the MoRIP1 gene, we constructed the complementation vector p822-npRIP 1C. The genomic sequence npRIP1C (SEQ ID No.6) of MoRIP1 was PCR amplified using the wild-type genomic DNA as template with primer npRIP1C-F/R and ligated into the pFGL822 (adddge ID: 58225) vector. After the sequencing is correct, the strain is transferred into agrobacterium AGL1, and then a deletion mutant delta Morip1 is transferred. The strain screened for phenotype complementation was designated Morip1 c.

Example 2: functional analysis of MoRIP1 on Magnaporthe grisea growth and sporulation

Wild-type WT,. delta. Morip1 and Morip1c strains were cultured on Complete Media (CM) plates for 7 days, stipes of 0.5CM diameter were punched at the edges of the colonies with a punch, the stipes were inoculated on CM and tomato oat media (OA) plates, 3 replicates, and the colony diameters were measured and photographed after 7 days of culture. After photographing, 3mL of sterile water was added to the plate, and the spore solution on the plate was collected with an applicator and the spore concentration was observed under a microscope. As shown in FIG. 1, the diameter of Δ Morip1 decreased compared to WT on both media, and the diameter of the complementation strain Morip1c did not differ from the wild type, indicating that MoRIP1 was involved in the hyphal growth of Pyricularia oryzae. The spore amounts of WT and Morip1c were also not significantly different on both media plates, but no conidia were observed in Δ Morip1, indicating that MoRIP1 is essential for sporulation of Pyricularia oryzae.

Example 3: action of MoRIP1 on pathogenicity of Magnaporthe grisea

Since the strain Δ Morip1 has no conidia, we used the method of inoculating the barley leaf ex vivo with stipe to judge the function of MoRIP1in pathogenicity. Wild-type WT,. delta. mori 1 and mori 1c strains were cultured on complete Culture Medium (CM) plates for 7 days, stipes with a diameter of 0.5CM were punched at the edges of colonies with a punch, the stipes were placed on barley leaves, kept moist and placed in a light incubator, and photographs were taken after 5 days. As shown in FIG. 2, brown lesions were observed on barley leaves inoculated with WT and Morip1c, but no lesions were produced by the inoculation of Δ Morip1, indicating that the pathogenicity of Pyricularia oryzae was completely lost after the knockout of MoRIP 1.

Example 4: positioning of MoRIP1in different growth and development stages of rice blast fungus and influence on mitochondrial function

In order to observe the localization condition of MoRIP1in different stages of rice blast germs, a fluorescent expression strain MoRIP1-mCherry of MoRIP1 is constructed. Wild genomic DNA is used as a PCR template, a DNA fragment is obtained by utilizing the amplification of a primer MoRip 1-mCheryF/R, and the fragment is connected into a pFGL 823-mCheryy vector to obtain a fluorescent expression vector pFGL823-MoRIP 1-mCheryy. pFGL823-MoRIP1-mCherry was then transformed into the mitoGFP strain using Agrobacterium-mediated transformation. Transformants with red fluorescence expression were screened and observed by confocal laser microscopy for localization of MoRip1in hyphae, spores, anchorage cell and infected hyphae. As shown in FIG. 3, in the hyphae, conidia, anchorage cells and infective hyphae, MoRip1 labeled with the red fluorescent protein mCherry was co-localized with the mitochondrial marker protein Mito-GFP all the time, indicating that MoRip1 was localized in mitochondria at different growth and development stages of Pyricularia oryzae.

Since the homologous gene Rip1 of MoRip1in yeast is cytochrome bc of mitochondrial electron transfer chain₁One core subunit of the complex, which mediates electron transfer from cytochrome b to cytochrome c₁Transport, in turn, affects membrane potential and intracellular ATP synthesis. We therefore examined whether intracellular ATP synthesis was affected following moip 1 deletion. WT, Δ Morip1, and Morip1c were cultured in liquid CM medium, mycelia were collected and ground with liquid nitrogen, and the contents of WT, Δ Morip1, and Morip1c were measured with an ATP detection kit. The results are shown in fig. 4, where ATP content in Δ Morip1 is significantly lower than that of WT and Morip1c, indicating that Morip1 is important for intracellular ATP synthesis.

Example 5: MoRIP1 involved in sensitivity of Magnaporthe grisea to azoxystrobin

Azoxystrobin is an important agent for controlling fungi and oomycetes, and the drug target is cytochrome b subunit which is coded by mitochondrial DNA. MoRip1 is the core subunit in the cytochrome bc1 complex, encoded by genomic DNA. Therefore, we tested the role of MoRIP1in the sensitivity of Pyricularia oryzae to azoxystrobin. After culturing WT,. DELTA.Morip 1 and Morip1c 7 on CM plates for a period of days, the stipe was punched with a punch, and the stipe was inoculated on CM plates containing azoxystrobin (10, 20, 40 and 100. mu.g/mL) at different concentrations to continue the culture, with the DMSO-added CM plates as controls, and the colony diameter was measured and the growth inhibition rate was calculated after culturing for 5 days in the dark. As shown in fig. 5, the inhibition rate of Δ Morip1 was significantly lower on azoxystrobin plates of different concentrations than WT and Morip1c, indicating that the absence of Morip1 resulted in enhanced resistance of pyricularia oryzae to azoxystrobin.

Example 6: MoRIP1 involved in oxidative stress response

We examined the sensitivity of WT, Δ Morip1 and Morip1c strains in oxidative stress, and punched out stipes at the edges of 7-day-old WT, Δ Morip1 and Morip1c colonies using a punch, inoculated the stipes on CM plates containing different concentrations of hydrogen peroxide (5 and 10mM) and continued the culture, and measured the colony diameter and calculated the growth inhibition rate after 5-day dark culture using CM plates without hydrogen peroxide as a control. As shown in FIG. 6, the inhibition rate of Δ Morip1 was significantly higher than that of WT and Morip1c under two different concentrations of hydrogen peroxide, indicating that the absence of MoRIP1 results in rice blast fungus being more sensitive to oxidative stress.

Sequence listing

<110> institute of Rice research in China

<120> use of a pathogenic factor of Pyricularia oryzae or a gene encoding the pathogenic factor

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 236

<212> PRT

<213> Magnaporthe oryzae (Magnaporthe oryzae)

<400> 1

Met Ala Pro Leu Thr Thr Ala Thr Arg Ala Ile Thr Arg Cys Ala Ala

1 5 10 15

Ser Pro Ser Lys Ala Val Thr Thr Ala Ala Arg Ala Leu Ser Thr Thr

20 25 30

Pro Ala Gln Asn Gly Ser Ser Gly Ala Ser Phe Ser Ser Pro Phe Arg

35 40 45

Gly Glu Gln Lys Ser Thr Gln Ile Pro Asp Phe Lys Lys Tyr Met Ala

50 55 60

Pro Lys Gly Ser Ser Thr Ser Asn Ala Val Phe Ser Tyr Phe Met Val

65 70 75 80

Gly Ala Met Gly Ala Ile Ser Ala Ala Gly Ala Lys Ser Thr Val Gln

85 90 95

Glu Phe Leu Val Asn Met Ser Ala Ser Ala Asp Val Leu Ala Met Ala

100 105 110

Lys Val Glu Val Asp Leu Thr Thr Ile Pro Glu Gly Lys Asn Val Ile

115 120 125

Ile Lys Trp Arg Gly Lys Pro Val Phe Ile Arg His Arg Thr Gly Arg

130 135 140

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

145 150 155 160

Glu Ala Asp Ser Asp Arg Val Lys Lys Pro Glu Trp Leu Val Met Leu

165 170 175

Gly Val Cys Thr His Leu Gly Cys Val Pro Ile Gly Glu Ala Gly Asp

180 185 190

Phe Gly Gly Trp Phe Cys Pro Cys His Gly Ser His Tyr Asp Ile Ser

195 200 205

Gly Arg Ile Arg Lys Gly Pro Ala Pro Leu Asn Leu Glu Ile Pro Ala

210 215 220

Tyr Asp Phe Pro Glu Asp Asp Lys Leu Val Ile Gly

225 230 235

<210> 2

<211> 1047

<212> DNA

<213> Magnaporthe oryzae (Magnaporthe oryzae)

<400> 2

atggcgccct tgacgaccgc cacccgcgca atcacgcgct gcgcagcgtc gccctccaag 60

gccgtgacta ccgccgcgcg cgccctgagc acgacgccgg cccagaacgg cagctcgggt 120

gccagcttct ccagcccctt ccgcggcgag cagaagtcga cccagatccc cgactttaag 180

aagtacatgg cccccaaggg ctcgtcgacc agcaacgccg tcttctcgta cttcatggtc 240

ggtgccatgg gtgccatctc ggccgccggt gccaagtcga ccgtccaggg tgagttggag 300

gaatctttcc ctcttgtgaa tactcttgaa ggatatgtgg atgtagtgtg ggatgggggg 360

aatacagtct ggagttaagg tttacgccaa ttgggagctt cttgtgactg acgactgtgt 420

gttctttttt cgtgcagagt tcctggtcaa catgtctgct tccgcggacg ttttggccat 480

ggccaaggtc gaggttgatc tgaccaccat ccccgagggc aagaacgtga gtgaccaccc 540

gcgcaatcta ctgtcttcga gtcgtcggaa aacctagtcg acacttgtga ttggtaacat 600

gtggtctgac tttttatctt ttctggattt aggtcatcat caagtggcgt ggaaagcccg 660

tcttcattcg ccaccgtacc ggccgtgaga ttgacgaggc gaacaagatc gacgttgcct 720

cattaagaga tcccgaggcc gacagcgacc gtgtcaagaa gcccgagtgg ctcgttatgt 780

tgggtaagtg gaattcctga tttgctttcc agcttccaag cgtcgatatc gtcgatcgac 840

atgcttactt tgatttctac gacaggtgtc tgcacacatc ttggttgtgt ccccattggc 900

gaggctggcg actttggcgg ttggttctgc ccctgccacg gatctcacta cgacatttcc 960

ggacgtataa ggaaaggacc tgctcccctg aacctcgaga ttcccgcgta cgacttcccc 1020

gaggacgaca agctggtcat tggttaa 1047

<210> 3

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<212> DNA

<213> Magnaporthe oryzae (Magnaporthe oryzae)

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cggtacatgg aaagctgatc gctgtggcgg atatcatgaa gtccatcaca ctcctcgagt 180

atattcctgg cgttggcaag tcagccaaga cgggaggcaa agacaaggcc accaggagtg 240

ataaggaagt agaggggtcg aagcaagcaa aacttgtcga ggtctgtcgt gattaccaag 300

caatgtggtc cacggctgta tcacacctcg agggagactc ttggatagta gctgatggtg 360

atggcaacct tgtcgtcctt ctgagaaaca ctgcaggtgt tacgctagag gataagcggc 420

gaatgcagat gacaagtgaa tttggcctgg gcgagtgtgt caacaagatc caaaaggtca 480

tggtcgagac aagtgcaaat gcgccaattg ttgccaaagc attcttgtct acggtaagag 540

ctctctatgc tcatttggcc cggtacctat gccgttttac tgacattttt tttttttgtt 600

ttccgcttag accgagggtt caatctacct attcggtact gttgcaccca agttccagag 660

cctgctcatg gactttcagg cgaacatgga agcacacgtg agctccccgc tcggagagct 720

tcagttcaac cagtggaggt cattccgcaa cccagagcga gagggagcag ggccagaaag 780

gtttctggat ggcgagttcc tcgaaatgtt tcttgacatg gaagagaata ctcaaatcga 840

tatttgccaa gggctgtctt atactgcgga ggatatgagg aatttgattg gggagatgaa 900

aaacatgcat tgattgtttg gagtacaagg gtaggacttg attgaatgat ggttcactct 960

agtcaaagag tcacatgtca tttcttcttt ttcaaactgg gcctgccaac gagttttgtt 1020

ttgaaggttg accttcgtga tggagctggt aatattagct accagcagat ctgataacag 1080

tgtagaagcg atcattacgg aatggacagg gcttggttga tgcaagttaa gactaaggta 1140

ctctcattgt aacagaacca tcacggcgtt cgcttatgta attcgactgt taccggggtt 1200

cgggttgctg gagccggatc ggattctctc gggataattc gcggtgatgt caaagctctc 1260

gccttcaacc aatcaaagcg taccaattag aagaaagcct caggcaattc ttcggcccag 1320

accaacgttt gcattggaaa agacctcaaa agattaccgc atcgtatcgt accgaaaacc 1380

accaccgacc gcccggcgac aggacacgct gaaatccctc cacaccctca atcggaacct 1440

ccccgtccac ctaattaatt cctaaacgaa aggtccgctc 1480

<210> 4

<211> 1238

<212> DNA

<213> Magnaporthe oryzae (Magnaporthe oryzae)

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aatggagggt ggtgtctggc agtggcgagg ggggcatcac ccgacacata gacgtctttt 60

acttgggcag agggaggttt tgctgccaag tttgggcgcg ctattacttt ttaaagcata 120

caacatttcc tttatagacg tcgtttgcgt ttgcgcggag aaagtgaaga gggtgttggt 180

ttccccagct cacgcatctg ctgtttgtac aaagaaaggt ccttatagag gcgtgaacgt 240

ttggagacta ggatcataaa gaacagtggc gtttgttgtt ttgtagtttg ctgcccctga 300

ctggatgctt ttgctttttg gtttgttgat ttggtgatat gccaagtgca ccaactgtgg 360

aggctgactc tgggatctcg gtgagtatat ggggtgacgg gcgcacattg tctgtcggct 420

ttgcaggtca tgtcgcggtt gtgatgagtg tataagatct tcttctcacg tggaaattat 480

aatgtctcgt ggcatatgaa ttttttggaa acagttgcgc gaagatcttg gtttcgagga 540

agccccaggc gtatatctag aagttgtatg tacagtcatt atcgtgctga ctggtggtgt 600

gtaacaaggg agacggtaaa aaaagttggg tcaccaaggt ctctgtcatc tgcacccaat 660

aatgttcgat tcattttctc ctccccaaga ctgaattcat ttgtggacaa ccgttggact 720

aattgtacgg acttgttgag gatagtctcg caaataacca tcaccctcaa gcagcccaga 780

tattttgaga tctaccaagt gacggagaat gcctagacct ggagacatca aagattacga 840

tttcacacaa gtaaacctcg gaacaagcat ccttaggcac ccaagaatcc taaaatttgg 900

tgccggagag cttgatgtat ggatctgaaa aagattcctt ggaataacaa aatttatatg 960

tgaacaaaag ccaaaccggt cattctctgt atctttgcgt tggaagggct ccttttgtaa 1020

gaagaatttg aaaacgtgag aaacatgggt cgacgtgctt ttatatcttt atattttcct 1080

tgcctacctt cctagatcca gaagtacttc gtagaagtat gcaaatattt ggctcgggta 1140

aactgtacgc cggcctcctg atacaatgta agaaccgttg gcttttctct ccagagtttg 1200

tcaacccatg accagacgtc acacggacca gaaccaga 1238

<210> 5

<211> 1410

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcgagtata ttgaaggagc actttttggg cttggctgga gctagtggag gtcaacaatg 60

aatgcctatt ttggtttagt cgtccaggcg gtgagcacaa aatttgtgtc gtttgacaag 120

atggttcatt taggcaactg gtcagatcag ccccacttgt agcagtagcg gcggcgctcg 180

aagtgtgact cttattagca gacaggaacg aggacattat tatcatctgc tgcttggtgc 240

acgataactt ggtgcgtttg tcaagcaagg taagtggacg acccggtcat accttcttaa 300

gttcgccctt cctcccttta tttcagattc aatctgactt acctattcta cccaagcatc 360

caaatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc 420

gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc 480

gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa 540

gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac 600

attggggagt ttagcgagag cctgacctat tgcatctccc gccgttcaca gggtgtcacg 660

ttgcaagacc tgcctgaaac cgaactgccc gctgttctac aaccggtcgc ggaggctatg 720

gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa 780

ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg 840

tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat 900

gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc 960

ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag 1020

gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg 1080

gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg 1140

ccacgactcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt 1200

gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc 1260

ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat 1320

ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca 1380

aagaaataga gtagatgccg accggaacca 1410

<210> 6

<211> 3629

<212> DNA

<213> Magnaporthe oryzae (Magnaporthe oryzae)

<400> 6

cctaccacag cctaacggct tgtgcagcat atttgtgaca tgcgaacact cgagtctaat 60

tcacggtgcc gaacgacgaa ttgtttattc ggcggtcacg gcacactcgg ctgcatatgt 120

ctgtccgttt gatacggcgg cattccgtga ttgtctcgct gttgcaaccg aatcagcaat 180

cgaccggaga atggaactga agatctcgcg catcgacagg cagcgccagt gccagatgat 240

gacacgtccc atgggcgaga atgtacgcag catagcatat tcgagcgctg ataaggtgtt 300

tggtctggga tgcatcagac gagtgctgtc cagaggtatc gaaaaagtat acggcacgtt 360

caaacttttt gacgaggtca tattcgagcc caagggcaat gtctttgcac tcgaagatgg 420

tgaggtgcca gaatgcgtca ctcgtgcacc gctactcgat agttatggcg agcaagcgga 480

gaggttcata gttggcacac ggtacctctc gggaactggg agtggtcatg gcggccgagt 540

ccttgttttc ggtgttgacg aaagccgttc gccgtacctc attcatgcac actctaccaa 600

aagcggctgc cggcgaatag ctaccatgga tgacgatctc ctcgtgatag cacttaccaa 660

gacggtggtt ctagtgaggt attcagagac atcaacaact tctgccaagt ttctcaaagt 720

ggcggcgttt caaacatcca gttatgccgt tgatgtgacg gtacatggaa agctgatcgc 780

tgtggcggat atcatgaagt ccatcacact cctcgagtat attcctggcg ttggcaagtc 840

agccaagacg ggaggcaaag acaaggccac caggagtgat aaggaagtag aggggtcgaa 900

gcaagcaaaa cttgtcgagg tctgtcgtga ttaccaagca atgtggtcca cggctgtatc 960

acacctcgag ggagactctt ggatagtagc tgatggtgat ggcaaccttg tcgtccttct 1020

gagaaacact gcaggtgtta cgctagagga taagcggcga atgcagatga caagtgaatt 1080

tggcctgggc gagtgtgtca acaagatcca aaaggtcatg gtcgagacaa gtgcaaatgc 1140

gccaattgtt gccaaagcat tcttgtctac ggtaagagct ctctatgctc atttggcccg 1200

gtacctatgc cgttttactg acattttttt tttttgtttt ccgcttagac cgagggttca 1260

atctacctat tcggtactgt tgcacccaag ttccagagcc tgctcatgga ctttcaggcg 1320

aacatggaag cacacgtgag ctccccgctc ggagagcttc agttcaacca gtggaggtca 1380

ttccgcaacc cagagcgaga gggagcaggg ccagaaaggt ttctggatgg cgagttcctc 1440

gaaatgtttc ttgacatgga agagaatact caaatcgata tttgccaagg gctgtcttat 1500

actgcggagg atatgaggaa tttgattggg gagatgaaaa acatgcattg attgtttgga 1560

gtacaagggt aggacttgat tgaatgatgg ttcactctag tcaaagagtc acatgtcatt 1620

tcttcttttt caaactgggc ctgccaacga gttttgtttt gaaggttgac cttcgtgatg 1680

gagctggtaa tattagctac cagcagatct gataacagtg tagaagcgat cattacggaa 1740

tggacagggc ttggttgatg caagttaaga ctaaggtact ctcattgtaa cagaaccatc 1800

acggcgttcg cttatgtaat tcgactgtta ccggggttcg ggttgctgga gccggatcgg 1860

attctctcgg gataattcgc ggtgatgtca aagctctcgc cttcaaccaa tcaaagcgta 1920

ccaattagaa gaaagcctca ggcaattctt cggcccagac caacgtttgc attggaaaag 1980

acctcaaaag attaccgcat cgtatcgtac cgaaaaccac caccgaccgc ccggcgacag 2040

gacacgctga aatccctcca caccctcaat cggaacctcc ccgtccacct aattaattcc 2100

taaacgaaag gtccgctccg aaaaaaaacc tttacgatgg cgcccttgac gaccgccacc 2160

cgcgcaatca cgcgctgcgc agcgtcgccc tccaaggccg tgactaccgc cgcgcgcgcc 2220

ctgagcacga cgccggccca gaacggcagc tcgggtgcca gcttctccag ccccttccgc 2280

ggcgagcaga agtcgaccca gatccccgac tttaagaagt acatggcccc caagggctcg 2340

tcgaccagca acgccgtctt ctcgtacttc atggtcggtg ccatgggtgc catctcggcc 2400

gccggtgcca agtcgaccgt ccagggtgag ttggaggaat ctttccctct tgtgaatact 2460

cttgaaggat atgtggatgt agtgtgggat ggggggaata cagtctggag ttaaggttta 2520

cgccaattgg gagcttcttg tgactgacga ctgtgtgttc ttttttcgtg cagagttcct 2580

ggtcaacatg tctgcttccg cggacgtttt ggccatggcc aaggtcgagg ttgatctgac 2640

caccatcccc gagggcaaga acgtgagtga ccacccgcgc aatctactgt cttcgagtcg 2700

tcggaaaacc tagtcgacac ttgtgattgg taacatgtgg tctgactttt tatcttttct 2760

ggatttaggt catcatcaag tggcgtggaa agcccgtctt cattcgccac cgtaccggcc 2820

gtgagattga cgaggcgaac aagatcgacg ttgcctcatt aagagatccc gaggccgaca 2880

gcgaccgtgt caagaagccc gagtggctcg ttatgttggg taagtggaat tcctgatttg 2940

ctttccagct tccaagcgtc gatatcgtcg atcgacatgc ttactttgat ttctacgaca 3000

ggtgtctgca cacatcttgg ttgtgtcccc attggcgagg ctggcgactt tggcggttgg 3060

ttctgcccct gccacggatc tcactacgac atttccggac gtataaggaa aggacctgct 3120

cccctgaacc tcgagattcc cgcgtacgac ttccccgagg acgacaagct ggtcattggt 3180

taaactggta gcaaacaaca catgtatcgc cacacttgaa actgtacgag tatggaacaa 3240

ggggagagag agagagagga aagaagaaaa aatggagggt ggtgtctggc agtggcgagg 3300

ggggcatcac ccgacacata gacgtctttt acttgggcag agggaggttt tgctgccaag 3360

tttgggcgcg ctattacttt ttaaagcata caacatttcc tttatagacg tcgtttgcgt 3420

ttgcgcggag aaagtgaaga gggtgttggt ttccccagct cacgcatctg ctgtttgtac 3480

aaagaaaggt ccttatagag gcgtgaacgt ttggagacta ggatcataaa gaacagtggc 3540

gtttgttgtt ttgtagtttg ctgcccctga ctggatgctt ttgctttttg gtttgttgat 3600

ttggtgatat gccaagtgca ccaactgtg 3629

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccggggatcc tctagatcct cgtgatagca cttacc 36

<210> 8

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttcaatata ctcgacgagc ggacctttcg ttta 34

<210> 9

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgccgaccg gaaccaaatg gagggtggtg tctg 34

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggccagtgcc aagctttctg gttctggtcc gtgt 34

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtcgagtata ttgaaggagc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tggttccggt cggcatctac 20

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cagggtgagt tggaggaa 18

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgtagtgaga tccgtggc 18

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cgtgattgtc tcgctgttg 19

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acctccacta gctccagcca ag 22

<210> 17

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agaaactcga gaattcccta ccacagccta acgg 34

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

taccgagctc gaattcccac agttggtgca cttggc 36