阅读说明：本技术 源于稻瘟病菌的效应子蛋白MoErs1及其编码基因与用途 (Effector protein MoErs1 derived from rice blast germ as well as coding gene and application thereof ) 是由 张正光 刘木星 郑小波 张海峰 刘昕宇 杨志香 于 2021-10-27 设计创作，主要内容包括：本发明公开了一种来源于稻瘟病菌的效应子蛋白及其编码基因与应用。该蛋白是具有序列表中的SEQ ID No.1的氨基酸序列的蛋白质；编码该蛋白的核苷酸序列如序列表中的SEQ ID No.2所示。实验证明,该蛋白编码基因的敲除导致稻瘟病菌致病力显著下降,且无法抑制水稻活性氧的积累。本发明对于揭示病原菌与寄主互作的分子机制,解析病原菌突破寄主防御、实现侵染致病的机理具有理论指导价值,同时可望基于该蛋白的表达与修饰设计和筛选新型低毒高效稻瘟病菌杀菌剂。(The invention discloses an effector protein derived from rice blast bacteria, and a coding gene and application thereof. The protein is a protein with an amino acid sequence of SEQ ID No.1 in a sequence table; the nucleotide sequence for coding the protein is shown as SEQ ID No.2 in the sequence table. Experiments prove that the pathogenicity of rice blast bacteria is obviously reduced due to the knockout of the protein coding gene, and the accumulation of active oxygen of rice cannot be inhibited. The invention has theoretical guidance value for revealing the molecular mechanism of the interaction between the pathogenic bacteria and the host, analyzing the mechanism that the pathogenic bacteria breaks through the host defense and realizes the infection pathogenesis, and simultaneously, the invention is expected to design and screen the novel low-toxicity high-efficiency rice blast germ bactericide based on the expression and modification of the protein.)

1. An effector MoErs1 protein derived from Pyricularia oryzae, which is characterized by having an amino acid sequence shown in SEQ ID No. 1.

2. A gene encoding the effector MoErs1 protein of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 2.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 2 or 3.

5. Use of the gene according to claim 2 or 3 for the preparation of transgenic plants, wherein the gene is overexpressed in plants by transgenic methods to produce plant varieties sensitive to Pyricularia oryzae.

6. The use of claim 5, wherein said plant is a monocot.

7. Use of the effector MoErs1 protein of claim 1 for designing and/or screening a fungicide for Pyricularia oryzae.

8. The use according to claim 7, for designing and/or screening a compound for a fungicidal agent against Pyricularia oryzae, targeting an effector MoErs1 protein.

9. The use of claim 8, wherein the small molecule compound is designed or predicted according to the crystal structure of the MoErs1 protein, the binding of the compound to the MoErs1 protein is verified by a microcalorimetric electrophoresis experiment, the inhibitory effect of the compound on the function of the MoErs1 protein is further determined, and the bacteriostatic effect of the compound is determined.

10. The use of claim 9, further comprising testing the designed or screened compound for control of rice blast.

Technical Field

The invention belongs to the field of plant pathology and molecular biology, and relates to an effector protein MoErs1 derived from rice blast germs, and a coding gene and application thereof.

Background

In the process of long-term co-evolution with pathogenic bacteria, multiple defense measures are developed step by step to resist the infection of rice blast germs, and the defense measures comprise the following steps: pathogen-associated molecular patterns (PAMPs) and pathogenic effector (effector) induced immune responses (ETIs). Wherein PTI is a basic immune response triggered by recognition of PAMPs released by pathogenic bacteria by pattern-recognition receptors (PRRs) on plant cell membranes. To successfully infect the host plant, the pathogen secretes effectors that interfere with this PTI response, inducing plant susceptibility (ETS). Subsequently, the plant develops a disease-resistant protein corresponding to the effector, and triggers a more vigorous and more effective defense response-ETI, by directly or indirectly recognizing the effector.

Pyricularia oryzae (A)Magnaporthe oryzae) The caused rice blast is the most important destructive fungal disease on rice, about 30 hundred million kilograms of grain loss is caused to China every year, and 6 million people can be maintained in the yield loss caused to the world. Because the pathogenic bacteria have high mutation speed and strong adaptability, the occurrence of the rice blast can not be completely controlled by the traditional disease-resistant variety cultivation and chemical control. Despite the excellent research progress in the biology of rice blast and its interaction with hosts, the disease remains a major threat to global food safety.

During the process of the military competition of rice blast and rice, the pathogenic bacteria can secrete a large number of effectors to enter rice cells to inhibit the PTI of the rice. For example, apoplast effectors Slp1 and MoAa91 secreted by magnaporthe oryzae can compete with the OsCEBiP protein for binding to chitin oligosaccharides, resulting in failure of OsCEBiP to normally recognize chitin oligosaccharides and ultimately suppression of rice immune response. Chitinases MoChi1 and MoChia1 secreted by rice blast germs have effector functions and can also combine with chitin, so that receptors OsMBL1 and OsCEBiP cannot recognize the chitin, and the immune response of hosts is avoided. Two strain-specific effectors Iug6 and Iug9 are identified in rice blast germ field strains 98-06 at the early stage, and the two effectors can inhibit the expression of defense response genes and interfere the immune response of a host. Mutants lacking both effector-encoding genes fail to inhibit the accumulation of reactive oxygen species at the site of infection. The non-toxic effector molecule AvrPiz-t also plays an important role in inhibiting the immune process of rice, and can inhibit the generation of flg22 and chitin-induced active oxygen and other defense reactions. It is now clear that AvrPiz-t can inhibit the process of PTI by more than one mechanism. AvrPiz-t interacts with two RING E3 ligase APIP6 and APIP10 in rice, affects the activity of E3 ligase, and promotes the degradation of protein. APIP10 promotes the degradation of NB-LRR resistance protein Piz-t in rice. In rice plants containing Piz-t, the gene APIP10 is knocked out or silenced, cell death is caused, and a large amount of Piz-t protein is accumulated, which indicates that E3 ligase APIP10 negatively regulates the expression of Piz-t. AvrPiz-t may also interact with the bZIP transcription factor APIP5, inhibiting its transcriptional activity and accumulating in large numbers during the necropsy vegetative phase. The APIP5 silences the plant to have cell death phenomenon, and the necrosis phenomenon is more serious when the plant expresses AvrPiz-t.

Although effectors secreted by Magnaporthe grisea play an important role in inhibiting host immunity, how to design and develop novel low-toxicity high-efficiency bactericides based on the protein structure of the effectors has not been reported. Therefore, the effector of the pathogenic bacteria and the action target thereof in the host plant are identified, and the interaction mechanism of the effector and the action target is analyzed, so that the pathogenic bacteria pathogenesis is known, and a new thought and a new guidance can be provided for the development of the novel bactericide target.

Disclosure of Invention

The invention aims to provide an effector protein MoErs1 derived from rice blast germ, and a coding gene and application thereof. The effector protein MoErs1 provided by the invention is specifically derived from pathogenic fungi magnaporthe grisea (Magnaporthe grisea)Magnaporthe oryzae) The amino acid sequence of the protein is SEQ ID No.1, and the nucleotide sequence of the gene is SEQ ID No. 2.

The invention firstly provides an effector MoErs1 protein derived from rice blast fungus, which is characterized by having an amino acid sequence shown as SEQ ID No. 1.

Further provided is a gene encoding the protein of claim 1. Preferably, the nucleotide sequence of the gene is shown as SEQ ID No. 2.

Further provides a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the nucleic acid molecule.

Also provides the application of the gene in preparing transgenic plants, wherein the gene is over-expressed in the plants by a transgenic method so as to prepare the plant varieties sensitive to rice blast germs. According to the invention, the expression vector of the MoERS1 gene in rice is constructed, and the vector is transferred into a rice susceptible variety TP309 by agrobacterium-mediated transformation, so that the transgenic rice over-expressing the MoERS1 gene is obtained. The invention finds that the Δ Mors 1 mutant can normally cause diseases on transgenic rice over-expressing the MoERS1 gene, and infected hyphae can normally expand. The plant variety sensitive to the rice blast germs prepared by the method can be used for evaluation or test of infection of the rice blast germs and can also be used as a susceptible variety contrast, so that the method has application significance in the rice blast resistance breeding process or identification of pathogenicity of the rice blast germs.

Preferably, the plant is a monocotyledonous plant, preferably the plant is rice.

Furthermore, the invention provides application of the effector MoErs1 protein in designing and/or screening a bactericide of rice blast fungus.

Specifically, the application of the effector MoErs1 protein as a target in designing and/or screening the bactericide of rice blast fungus is disclosed. Preferably, a small molecule compound is designed or predicted according to the crystal structure of the MoErs1 protein, the binding of the compound and MoErs1 is verified through a microcalorimetric electrophoresis experiment, the inhibition effect of the compound on the function of MoErs1 is preferably further determined, and the bacteriostatic effect of the compound is determined.

Experiments prove that the pathogenicity of the rice blast germs is obviously reduced due to the knockout of the coding gene of the effector protein derived from the rice blast germs, and the accumulation of active oxygen of the rice can not be inhibited. Therefore, the invention has theoretical guidance value for revealing the molecular mechanism of the interaction between the pathogenic bacteria and the host, analyzing the mechanism that the pathogenic bacteria breaks through the host defense and realizes the infection pathogenesis, and simultaneously, the invention is expected to design and screen the novel low-toxicity high-efficiency rice blast germ bactericide based on the expression and modification of the protein.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1: identification and cloning of the MoERS1 gene. Wherein A is predicted in a Magnaporthe grisea genome databaseMoERS1A schematic representation of the genes, and a modified schematic representation in Pyricularia oryzae; b is an agarose gel electrophoresis picture of the cloned MoERS1 gene in a rice blast fungus wild type strain Guy 11; c is a sequencing result of cloning a MoERS1 gene in a rice blast fungus wild strain Guy11 and a comparison result of the sequencing result and the MoERS1 gene predicted by a genome website.

FIG. 2: a schematic diagram of the MoERS1 gene knockout process and Southern hybridization verify the knockout of the MoERS1 gene.

FIG. 3: observation of the subcellular localization of MoErs1 in rice. Wherein A is a rice blast fungus strain with MoERS1 gene fusion expressing red fluorescent protein RFP (MoERS 1: RFP), rice sheath cells are infected, and MoErs1 can be gathered in the BIC structure; b is a rice blast strain with MoERS1 gene fusion expressing red fluorescent protein RFP and nuclear localization signal NLS (MoERS 1: RFP-NLS), which infects rice leaf sheath cells and can be secreted into rice cells and aggregated in rice cell nuclei.

FIG. 4: the morrs 1 gene deletion mutant has significantly reduced pathogenicity in mors 1. Wherein A is a pathogenic result of rice blast germ spore suspension liquid spray inoculation on rice leaves; b is the pathogenicity result of rice seedling inoculated by rice blast germ spore suspension; c and D are the statistical result of pathogenicity A; e is sporulation condition after lesion spots on the rice leaves in A are moisturized; f is the statistical result of sporulation and lesion on the rice leaves in A.

FIG. 5: the expansion capability of the infected hypha of the MoERS1 gene deletion mutant, Δ MoERS1, is obviously reduced. Wherein A and B are the expansion conditions of the infected hyphae 24 hours after the rice blast germ spore suspension is inoculated on the barley epidermis, and the hierarchical statistical analysis is carried out (grade I, only attachment cells penetrate through a host, grade II, a primary infected hyphae can be formed, grade III, secondary infected hyphae with 2 to 3 branches can be formed, grade IV, the infected hyphae expand and exceed 3 branches), and C is the expansion conditions of the infected hyphae 24 hours and 48 hours after the rice blast germ spore suspension is inoculated on the rice sheath.

FIG. 6: infection of the MoERS1 gene deletion mutant, Δ Mors 1, can not inhibit the accumulation of active oxygen of the host. Wherein, A and B are rice blast germ spore suspension liquid which is inoculated with rice leaf sheath for 24 hours, DAB is used for dyeing rice cells, and the number of brown rice cells is counted; c and D are the hypha expansion conditions of the rice blast fungi after the rice blast fungi spore suspension is inoculated on the rice leaf sheaths for 24 hours and 48 hours and treated by a rice NADPH oxidase inhibitor.

FIG. 7: after the effector coding gene MoERS1 is over-expressed in rice, the infection of rice blast germs can be promoted. Wherein A and B are pathogenicity results of rice blast germ spore suspension liquid inoculated rice leaves, and the statistical analysis of the disease incidence area is carried out; c and D are statistics of the hypha expansion conditions of the rice blast germs after the rice blast germs spore suspension is inoculated on the rice sheath for 24 hours and 48 hours.

Detailed Description

Example 1: isolation and cloning of the MoERS1 Gene

During the interaction process of the rice blast germs and the rice, a large number of effectors are secreted to inhibit the immune reaction of hosts and promote the infection of the rice blast germs. We identified a series of exosome-associated genes, including the MoERS1 gene, possibly as toxic effectors to suppress host immunity, through exosome proteomic analysis in the early days. To study the function of the gene, we obtained a predicted nucleic acid sequence of the gene with the accession number of MGG _13009 from the genomic database https:// fungidb.org/fungidb/app/record/gene of Pyricularia oryzae, and the predicted genomic nucleotide sequence of the website showed that the gene had two introns and three exons. In order to verify the prediction result, MoERS1 gene is respectively cloned from cDNA of a wild type strain Guy11 of rice blast fungus by using primers F1/R1 and F2/R2, and the results of experiments that F1/R1 cannot be cloned to the MoERS1 gene, F2/R2 is successfully cloned to the MoERS1 gene, the size is 645bp (A and B in figure 1) show that the website prediction result is wrong, and the MoERS1 gene only has one intron, so the cDNA sequence can be terminated early; meanwhile, the cloned cDNA sequence of the MoERS1 gene is connected to a pMD19-T (Takara Co, China) vector and sequenced (C in a figure 1), so that the cDNA sequence of the MoERS1 gene published by a website is wrong; the cDNA sequence of the MoERS1 gene is shown in SEQ ID No.2, and the amino acid sequence is shown in SEQ ID No. 1. The sequences of the primers used are detailed in Table 1.

Primers used in the description of Table 1

Example 2: knockout of the MoERS1 Gene

1、MoERS1Construction of Gene knockout vectors

Taking a rice blast fungus wild type strain Guy11 as a template, respectively amplifying an upstream flanking sequence of about 1kb and a downstream flanking sequence of about 1kb of a MoERS1 gene by using primers ERS1-p1-F/ERS1-P2-R and ERS1-p3-F/ERS1-p4-R, wherein the ERS1-p 2/ERS 1-p3 primers comprise an EcoR V enzyme cutting site. Two flanking sequences are connected by ERS1-p1-F/ERS1-p4-R, and the cloned 2 kb fragment is purified and constructed on a pMD19-T vector. The hygromycin HPH gene was amplified at about 1.4kb using FL1111/FL1112 and the fragment was inserted into pMD-MoERS1In a plasmid. The 3.4 kb fragment was amplified with ERS1-p1-F/ERS1-p4-R strain for protoplast transformation. The sequences of the primers used are detailed in Table 1.

The about 1kb upstream flanking sequence above is specifically (as shown in SEQ ID No. 3):

GTGTGCGTACGACAATGCTTGCCTGTTTGCCCATCTGTCTCGGGCTCCTCGTCATTTGTGTGAAAAGCGTCTGTTTGCAACGACGCAATGAGTCAACAGTACAAATTATAAGCTATAGTGAAGATGAAGCGAATGCCTGGTACGATAAACCTGTCGTGCCAAGGTAGAATTGACGAAGCCTTGCCTTGATCGATCGTCCAAGTTCAACATCATGTGAACAGAGCCCGTATGCAACCTGGTTGGCCCCCGATGCTTGTGCCATTCCTTGGCCCCAAGAACGTCAAGAGTAAAAAGCTAATCGGACACGACGGGTCCGATGAGACATGGTGAGAAGTTAGTGTTAGCTAAGACCAGGGGAGATCGAGAGCTGCAGCCTTGCCCCGCAGAACGGTAAATTCCCCAAACGAGCTAGACCAGTCCCTTCGCTCAAATACGCCCCTGGGCTGACGAGAAGGACCACACATGCCACTATGTACAGGACCCGGTATAATGTAATTGAGAGAAGGCTGCCTAAGCGCTAAACAAAATGGTCCAGTCGGGGCGAATTCAAGGTAAACGAACATACGGAATAATGGAAGAACTGGCTACCAGGAAGGATCATTAACCTCCACCTGCACCCCCTGAAAGACGAGCGCTTTTTTTTTTTTTTTTTTTGCCAGCGGCACAGTCGCCAAGGGTGGTGATTTTCCCCATTCCATCGTTGGCTCTTTCCTGCACGAGCTGATCCACCACTGTTCGAGATTTGATTCCCCTGCCTGGTCGGGCATGGAACTTCTGGTAATGATGTACCGTAGACCGAATGGCCCTCAATGTGGTTCCGCAAATCTGTAAATTGCCATCAGGCAACAGGCGGGCAAGCCGGGTGTACCGATGGCGAACAGGATAAATACCCCTCGACCGTCCTCGACTTGAATCCCAGGTAGAAGGAAGAGACAGCACTCGCCACAGCTCCTCTGACATTCGCTTTCCCATCGCCCACAACATTTGGCCCTGCTCTCATTCCTTGGGTTGATTTTTTTTCTTTTCTTTCGCAAGCACCGAAAACTTTAGTCAAA

the about 1kb downstream flanking sequence above is specifically (as shown in SEQ ID No. 4):

GCCTGACATAACATTTTGCTGGGTAATCGGTTCAACATGTGAGCATTGCGTGATGTTTACCCTGTATTTTTACAGGTATACAGGGTGGACGTGGAAGCAAACACCCAGCTCCCGTCGGTCTGAGAGCATGAATGCCAATGTTGTGCAGCATGATGGATCGCTGCCTGGTTCTAGAATATATAGCAAGTACCGTACGGAAGACATGCTTCAGATATGGGTACTTCGAATAGATAGCCTGGGTCAGGTAGCACAGATGGGCAGGGTTGGTTCATGAGCACGAGTCAATTTAATGTAAATGATTTGACGATACCCCGGATTACTGCGCCGTTTGGGTTTATGCATGTCGCCTAAACGCAACATGTAGTCCGCAAACGCTCCGTAACCCATGAGAGGCTGCTGCACTTTTGCTGCTTTGTTTGCCAACATCTGAGACCCCCCGACCAGTCATGTCTGCTCGGTTGCATGTGCGTTGCAAGGAATGTCAGTCAACAAAGTCCATCAAACGTGAACCGAATCCAACGTCAACAAGCAATGTTATCTCGCTGCCATCTGCCATCTGCCATCTGGGTATCTGCGCGTCAACCCTGAGCTACTAGCCCCACCGCCTTTGGCCCACGGCTTTTCTCGGTTGGTGGGCGCTAAGTATAATGAATTCGGTCAGCTTTGGCCCCAACCAGGTTGTTTGTTAACAAGCTAGATCCATGTACCAAATGTAAACAAATACTACTCTGAGCCATCACACCGTCAACCGGAACCAGGCTTGCTAGGCACGAAGTAATCATCTCTCGGCAAGGAAGAAGAAAAACAAAAAAAAAAAAACCCTGGCGCGGGCACCGGCGGGGTGATGATGGAATTTTGCGGGAACAAAGATACAGTAGGCAGTCGGACACAAGGCAAGCGAAATGGGCAGGCGGGGCAGCTGTAGCAGTACCATCGATGACGCTTCTTGATAAGGTACTGTGATCAAGCTACCTAGGTACGGTATGGCTTACCGTAGGACGAACGGTG

the about 1.4kb hygromycin gene sequence above is specifically as follows (as shown in SEQ ID No. 5):

GGAGGTCAACACATCAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAG。

2. acquisition of MoERS1 Gene knockout mutant

1) Preparation of culture Medium

The CM medium configuration method comprises the following steps: 50ml of 20 xnitrate (120g of sodium nitrate, 10.4g of potassium chloride, 10.4g of magnesium sulfate heptahydrate, 30.4g of potassium dihydrogen phosphate dissolved in distilled water to 1L), 50ml of 1000 xtrace elements (2.2g of zinc sulfate heptahydrate, 1.1g of boric acid, 0.5g of manganese chloride tetrahydrate, 0.5g of iron sulfate heptahydrate, 0.17g of cobalt chloride hexahydrate, 0.16g of copper sulfate pentahydrate, 0.15g of sodium manganate dihydrate, 5g of tetrasodium EDTA dissolved in distilled water to 100ml), 1ml of a vitamin solution (0.01g of biotin, 0.01g of vitamin B6, 0.01g of vitamin B1, 0.01 of riboflavin, 0.01 of p-formic acid, 0.01 of nicotinic acid, 100ml of aminobenzene dissolved in distilled water), 1ml of glucose (10 g of glucose, 2g of peptone, 1g of yeast extract, 1g of casamino acid, 15g of agar powder, quantitative determination of distilled water to 1L, cooling in a flask at 20 ℃ for 20 minutes.

The configuration method of the spore production culture medium comprises the following steps: the corn flour and the rice straw are prepared, 100g of the rice straw is weighed, 1L of water is added to the rice straw to be boiled for 30 molecules, 40g of the corn flour and 15g of agar powder are added to be boiled for 20 minutes, finally, distilled water is used for fixing the volume to 1L, the materials are separately packed in triangular bottles, the materials are sterilized for 20 minutes at the temperature of 121 ℃, and the materials are cooled for standby application.

1 × STC configuration method: weighing 20% of sucrose by mass volume ratio, weighing 50 mM Tris.Cl PH 8.0, 50 mM calcium chloride, adding distilled water to quantify to 1L, subpackaging in triangular flasks, sterilizing at 121 ℃ for 20 minutes, and cooling for later use.

TB3 medium configuration method: weighing 3g yeast extract, 3g Casamino Acids and 20% sucrose, adding distilled water to a fixed amount of 1L, subpackaging in triangular flasks, sterilizing at 121 ℃ for 20 minutes, and cooling for later use.

The configuration method of the PTC comprises the following steps: 60% PEG4000 was weighed out and dissolved in 1 × STC, and filtered through a bacterial filter for use.

Preparation of enzyme solution: the enzyme (lysing Enzymes from Trichoderma) was dissolved in 0.7M NaCl solution

Harzianum, sigma), the concentration of the enzyme solution is 7.5-10 mg/ml, and the enzyme solution is filtered and sterilized to be used.

2) Preparation of protoplasts (Protoplasting)

a. Activating the strain: the inoculated mycelium pellet was grown on CM plates for 3-4 d, with a colony diameter of about 3 CM. Note that the strain should not be too old

b. Colonies with a diameter of about 3 CM were excised, minced as much as possible, placed in about 100ml of CM or 5 XYEG liquid medium (containing 50. mu.g/ml Amp), and cultured at 28 ℃ for 2 d (36-48 h) with shaking at 150 rpm.

c. 1-2 layers of Miracloth are filtered to collect hyphae, the hyphae are washed twice by sterile water, and the water is slightly sucked dry by absorbent paper.

d. Placing the hyphae in a 50ml centrifuge tube containing 10-20 ml enzyme solution (containing 50 mug/ml Amp) for enzymolysis. The centrifuge tube is laid flat, and the enzymolysis is carried out at 30 ℃ and 60 rpm for 1.5-2 h. In the enzymolysis process, the bacterial liquid is required to be absorbed for microscopic examination, and the release condition of the protoplast is checked.

e. The following steps are carried out at 4 ℃ with 0.7M NaCl solution, 1 × STC first at 4 ℃ for pre-cooling.

f. Taking out the enzymolysis solution, adding a little 0.7M NaCl solution, shaking gently, pouring into three-layer sterilized mirror paper for filtering, washing gently with 0.7M NaCl solution for 1-2 times, removing residues, and collecting filtrate in a 50ml centrifuge tube.

g. Centrifuging at 3000 rpm at 4 deg.C for 10 min.

h. The supernatant was carefully discarded, suspended with 10-20 ml of 1 × STC, gently blown with a tip-cut tip, and then centrifuged at 3000 rpm at 4 ℃ for 10 min.

i. Repeat step h twice.

j. Adding a proper amount (about 300 mu l) of 1 × STC for suspension, counting to ensure that the final concentration of protoplasts is 108/ml, and subpackaging into 150 mu l/tube. Transformation was immediately or stored at-70 deg.C (normally no storage, 7% DMSO if required).

3) Transformation of rice blast bacteria

a. 2 mu g of DNA (5-10 mu l) is added into 150 mu l of protoplast, the protoplast is gently mixed, and the DNA concentration needs to be sufficiently large and clean after standing at room temperature for 25 min.

b. Adding 1ml PTC 2-3 times, mixing, and standing at room temperature for 25 min. The standing time is not suitable to be too long, and PTC has toxicity to protoplasts. After addition, TB3 solid medium was thawed.

c. The protoplast is added into about 10 ml of TB3 solid culture medium (containing 50 mug/ml Amp) which is melted and cooled to 45-50 ℃, and the mixture is poured into a culture dish after being mixed evenly. Culturing at 28 deg.C in the dark for 24 hr

Optinal or protoplasts were added to 5-10 ml TB3 (containing 50. mu.g/ml Amp) solution and cultured overnight at 28 ℃ with gentle shaking.

d. An additional 10 ml of TB3 solid medium (containing 50. mu.g/ml Amp) containing 300. mu.g/ml Hygromycin B/Bleomycin was poured into the culture dish.

e. Culturing at 28 deg.C in dark for 7-10 days, and determining whether the transformation is successful after 5 days.

f. When the colony diameter of the transformant is about 5 mm, selecting a small lump mycelium block from the colony, and transferring the small lump mycelium block into a CM solid culture medium containing 150 mu g/ml of Hygromycin B/Bleomycin to screen the transformant.

g. Further, for the transformant verification by PCR and Southern verification, the internal probe used was amplified with primers ERS1-p5-F/ERS1-p6-R and the HPH probe was amplified with FL1111/FL 1112.

h. The mutants were analyzed for phenotype.

A knockout mutant of the MoERS1 gene, designated as Δ Moers1, was successfully obtained in the wild-type strain Guy11 by the above method (FIG. 2). The Δ Mors 1 mutant phenotypes such as virulence and suppression of host immunity are described in example 5 through example 8.

Example 3: construction of complementary vectors

Amplification with primers ERS1-p7-F/ERS1-p8-R, containing an upstream about 1.5kb self promoter, insertion of the fragment into the pYF11 vector by yeast transformation (bleomycin resistance) resulted in pYF11-MoERS1And (3) a carrier. And the vector was introduced into the Δ Mors 1 mutant by protoplast transformation to obtain a complementary strain of Δ Mors 1/MoERS 1. Transformants are first screened for phenotype, then verified by PCR, and finally verified for complementation by pathogenicity, growth, and other phenotypic determinations.

The about 1.5kb promoter sequence above is specifically as follows (as shown in SEQ ID No. 6):

CATATAGGTAGACATATCTTGGGGTATTCGACCCGAAGCTGATCTTTTCTCCCCTCCAAAAACAGTGTGCGGATGTGCCGTCGGTGTAGCGACCGCCGAAGTATCGGGGTTAATAGCGAGACCAGGTCCGACCGGAACTGCCTCCGTCTCGGCGTCATATTCTTACATGAGCGGCAGCGGCTTCTCCGTGGTCTCATCCTTGACGGTCATCAACAAAGCGGCCTCCGGTACCGCAACGCCAAGCTTCATGCCCCAGGTAGACCAACCGAGCCAAATGGTGCCAATCCACAAGAGAGACGTCGAGGAAGACCACGAACCGCCAAAGAGCTTCGAACCGGCTAACAAGGCCGCAGTACAGGCCAGCTCCGGGACGTCGTCTAGTCATTACGGAAATATGCGTATTTCGGGTTGGGTCATATCGGTTGTACCTACATTGTTGGTTGGTCTGATTGTATAGAAAGAAAAGAAGAGAAAGAGGAAAAAGAAAGAAAAAAAAGGGTGAAATACGGGGATCAAGGAAAGTGTGCGTACGACAATGCTTGCCTGTTTGCCCATCTGTCTCGGGCTCCTCGTCATTTGTGTGAAAAGCGTCTGTTTGCAACGACGCAATGAGTCAACAGTACAAATTATAAGCTATAGTGAAGATGAAGCGAATGCCTGGTACGATAAACCTGTCGTGCCAAGGTAGAATTGACGAAGCCTTGCCTTGATCGATCGTCCAAGTTCAACATCATGTGAACAGAGCCCGTATGCAACCTGGTTGGCCCCCGATGCTTGTGCCATTCCTTGGCCCCAAGAACGTCAAGAGTAAAAAGCTAATCGGACACGACGGGTCCGATGAGACATGGTGAGAAGTTAGTGTTAGCTAAGACCAGGGGAGATCGAGAGCTGCAGCCTTGCCCCGCAGAACGGTAAATTCCCCAAACGAGCTAGACCAGTCCCTTCGCTCAAATACGCCCCTGGGCTGACGAGAAGGACCACACATGCCACTATGTACAGGACCCGGTATAATGTAATTGAGAGAAGGCTGCCTAAGCGCTAAACAAAATGGTCCAGTCGGGGCGAATTCAAGGTAAACGAACATACGGAATAATGGAAGAACTGGCTACCAGGAAGGATCATTAACCTCCACCTGCACCCCCTGAAAGACGAGCGCTTTTTTTTTTTTTTTTTTTGCCAGCGGCACAGTCGCCAAGGGTGGTGATTTTCCCCATTCCATCGTTGGCTCTTTCCTGCACGAGCTGATCCACCACTGTTCGAGATTTGATTCCCCTGCCTGGTCGGGCATGGAACTTCTGGTAATGATGTACCGTAGACCGAATGGCCCTCAATGTGGTTCCGCAAATCTGTAAATTGCCATCAGGCAACAGGCGGGCAAGCCGGGTGTACCGATGGCGAACAGGATAAATACCCCTCGACCGTCCTCGACTTGAATCCCAGGTAGAAGGAAGAGACAGCACTCGCCACAGCTCCTCTGACATTCGCTTTCCCATCGCCCACAACATTTGGCCCTGCTCTCATTCCTTGGGTTGATTTTTTTTCTTTTCTTTCGCAAGCACCGAAAACTTTAGTCAAA。

example 4: observation of subcellular localization of effector MoErs1 in Rice

1. Construction of fluorescent expression vectors

The full-length gene of MoERS1 is cloned from the genome of a rice blast fungus wild type strain Guy11, comprises a 1.5kb self promoter, fusion expression fluorescent protein coding gene RFP or RFP-NLS (nuclear localization sequence), and is introduced into a pYF11 vector through yeast homologous recombination to form a vector of pYF11-MoERS1-RFP and pYF11-MoERS 1-RFP-NLS. The vector was introduced into the Δ Mors 1 mutant by transformation of Magnaporthe grisea protoplasts, and a fluorescent expression strain was obtained by the Magnaporthe grisea transformation method in example 2.

2. Observation of subcellular localization of effector MoErs1 in Rice

The preparation method of the spore-forming culture medium is detailed in example 2. A method for inoculating rice leaves by injecting rice blast fungus spore liquid comprises the following specific steps:

1) firstly, inducing rice blast germs to generate conidia, inoculating rice blast germs Guy11 mycelium blocks on a CM culture medium to an SDC culture medium, culturing for 4 days in the dark at 28 ℃, scraping surface hyphae, and inducing for 3 days under a black light to obtain the conidia.

2) The greenhouse-cultured 20-day rice seedlings were used for inoculation experiments, and conidia on the SDC plates were collected at a concentration of 1X 10⁵And (4) each/ml, injecting the mixture into a rice leaf sheath, and carrying out dark moisturizing culture for 30 hours.

3) And tearing off the inner epidermis by using medical forceps, carrying out microscopic observation, and observing BIC positioning of the infected hyphae and positioning on rice cell nuclei.

The results of the experiment show that the fusion of MoErs1 with the fluorescent strain expressing RFP and RFP-NLS concentrated fluorescence in the BICs after infecting the leaf sheath (A in FIG. 3). Meanwhile, fluorescence can be accumulated in the rice nucleus after infecting the leaf sheath with a fluorescent strain of MoERS1: RFP-NLS (B in FIG. 3). The above demonstrates that MoErs1 are a class of cytoplasmic effector molecules and are secreted into rice cells during the infection phase.

Example 5: pathogenicity determination of MoERS1 gene deletion mutant

1. A method for inoculating rice leaves by spraying rice blast germ spore liquid comprises the following specific steps:

1) first, the rice blast fungus is induced to produce conidia, and the specific method is shown in item 2 of example 4.

2) Rice seedlings cultured in the greenhouse for 14 days were used for the spray inoculation experiment, and 4ml of conidia were collected from the SDC plates at a concentration of 5X 10⁴Each/ml, and containing gelatin at a total concentration of 0.2% (w/v), sprayed onto rice leaves, and cultured in the dark for 24 hours, followed by culturing alternately in the dark for 5-7 days.

3) And (5) counting the disease area and the number of the disease spots.

4) Each treatment was repeated three times.

2. A method for inoculating rice seedlings by injecting rice blast fungus spore liquid comprises the following specific steps:

1) first, the rice blast fungus is induced to produce conidia, and the specific method is shown in item 2 of example 4.

2) The rice seedlings cultured in the greenhouse for 20 days are used for inoculation experiments, and conidia on the SDC plate are collected at the concentration of1×10⁵Injecting the cells/ml into rice seedling stalks, carrying out dark moisture-preserving culture for 24 hours, and then carrying out light-dark alternate culture for 5-7 days.

3) And (5) counting the disease area and the number of the disease spots.

4) Each treatment was repeated three times.

The results of the rice spray inoculation experiments show that the lesions on the patients' own Moers1 mutant are significantly reduced compared with the complementary strains obtained from the wild type Guy11 and example 3, and the lesions do not normally spread (A, C, D, F in FIG. 4); the result of the leaf sheath injection shows that little lesions appear only near the injection point for the patients with the Mors 1 mutant, while the lesions of the wild type and the complementary strain spread normally (B in FIG. 4). The hyphal growth on the lesions of the inoculated leaves was further observed, and it was found that after induction of moisture retention, the lesions of the wild type and the complemented strain (80%, n = 100) produced a large number of conidia, while the lesions of the rice leaves inoculated with the mutant (Morers 1) did not produce spores (< 20%, n = 100) (E in FIG. 4). This result indicates that MoErs1 regulates the virulence of Magnaporthe grisea.

Example 6: determination of invasion hypha expansion capacity of MoERS1 gene deletion mutant

The experimental method for rice blast fungus infection is shown in example 4. To explain the cause of the reduced pathogenicity of the Δ Mors 1 mutant, i.e., the reduction of typical lesion number and the failure to induce the production of spores at lesions normally. Infection experiments were performed in barley, onion epidermis and rice sheath, respectively. After 30 h of infection on barley, 100 attachment cell infection sites are observed and graded statistics is carried out (grade I, only attachment cells do not penetrate through hosts; grade II, a primary infection hypha can be formed; grade III, a secondary infection hypha with 2 to 3 branches can be formed; grade IV, the infection hypha expands and exceeds 3 branches). In the wild-type and complementary strains, 80% of the sites of infection formed grade III and IV hyphae, while in the mutants, less than 30% of the sites of infection formed grade III and IV hyphae (A and B in FIG. 5). In rice leaf sheaths, the mutant invasion rate was significantly lower than that of the wild type (10% of mutant, 80% of wild type) at 24 hours of infection, and hyphae of the mutant did not yet spread to neighboring cells at 48 hours after infection, while the wild type spread to neighboring cells (C in fig. 5). The above results indicate that MoErs1 is essential for the growth of the infecting hyphae, and that normal extension of the infecting hyphae is critical for the formation of typical lesions of the rice blast fungus.

Example 7: the MoERS1 gene deletion mutant can not inhibit the accumulation of host active oxygen

DAB (3, 3' -diamino-benzidine) staining method: the method for infecting rice sheath with rice blast fungus is shown in example 4. After 24 hours of infection, the rice leaf sheaths were stained with 1 mg/ml DAB (pH 3.5) for 8 hours at room temperature in the dark, then decolorized with ethanol/acetic acid (94: 4) for 1 hour, and the inner skin of the leaf sheaths was torn with forceps for microscopic observation.

Reactive Oxygen Species (ROS) staining observation is carried out on infected rice cells by DAB, and the experimental result shows that no ROS is generated in the rice leaf sheath infected by the wild type strain Guy11, but in ΔMoers1Mutant-infected rice leaf sheath cells accumulated large amounts of ROS (A and B in FIG. 6). Treating rice leaf sheaths with DPI (diphenyleneiodonium, NADPH oxidase inhibitor), inhibiting the generation of ROS, observing the growth condition of the infected hyphae in the leaf sheath cells, counting 100 infected sites, and carrying out graded statistics (I grade, only attachment cells can not form primary infected hyphae; II grade, primary infected hyphae; III grade, secondary infected hyphae can not be expanded to adjacent cells; IV grade, the infected hyphae is expanded to adjacent cells). The results show that the mutant invasion hyphal extension defect can be complemented back after DPI treatment (C and D in fig. 6). The result shows that MoErs1 can inhibit the generation of host active oxygen and promote the extension of infected hyphae.

Example 8: after the effector coding gene MoERS1 is over-expressed in rice, the infection of rice blast germs can be promoted

The rice spray test method is described in example 5. The coding region of a target gene MoERS1 is constructed into a pCAM2300 vector by using an enzyme digestion connection method, XbaI and PstI enzyme digestion sites are respectively added to an upstream primer and a downstream primer of MoERS1 during primer design, the MoERS1 gene is amplified, the target gene and a vector are subjected to enzyme digestion linearization by using restriction enzymes XbaI and PstI, and the vector and a fragment are connected by using T4 ligase to form the vector of the pCAM 2300-MoERS 1. Then the gene expression of the MoERS1 gene is started by an actin promoter through the vector, and the gene expression is provided with a Flag label for later verification; constructing the vector, and then entrusting Wuhan Boehfar biological company to transform to obtain a plant with the MoERS1 gene over-expression;

the experiment result shows that both the wild strain Guy11 and the mutant strain Mors 1 can attack on over-expression plants of MoERS1-OX (A and B in figure 7), which indicates that MoErs1 can inhibit the immune response of a host and promote the infection of rice blast bacteria after entering the host cells. Meanwhile, leaf sheath infection observation is carried out, and the overexpression and the strain of the MoERS1-OX are found to be the same as TP309, after the rice blast germs are infected for 48 hours, the rice blast germs are infected with diseases very much, and the infected hyphae are very severely expanded (C and D in figure 7). These results indicate that, in the rice blast pathogen infection process, the MoErs1 can inhibit the disease-resistant reaction of rice and promote the infection of rice blast pathogen.

Example 9: design and screening of bactericide by using expression of effector MoErs1 protein as target

A fragment (21-214 aa) of MoErs1 signal-removed peptide was cloned from cDNA of a wild type strain Guy11 of Pyricularia oryzae, and constructed on a pET15b vector (Novagen, Madison, Wis., USA). The above vector plasmid was expressed in E.coli BL21 and shaken at 37 ℃ in liquid LB medium (containing 100. mu.g/ml ampicillin) to an OD600 of between 0.4 and 0.8. The culture temperature was lowered to 16 ℃ and the expression of the MoErs1 protein was induced by the addition of 0.4 mM IPTG (isoproyl. beta. -D-1-thiogalactopyranoside). After 12 hours, 5300g was centrifuged for 15 minutes to collect the bacterial suspension. The pellet was lysed by suspension in 1 mM lysis buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10mM Imidazole) followed by sonication and the supernatant was collected as protein in MoErs 1. Protein expression can be detected by western. The protein crystal structure of MoErs1 was further obtained by the sink method.

Small molecule compounds are designed or predicted according to the crystal structure of the MoErs1 protein, the combination of the compounds and the MoErs1 is verified through a micro-scale thermophoresis (MST) experiment, the inhibition effect of the compounds on the functions of the MoErs1 is further determined through the methods provided by the different implementation cases, and the bacteriostatic effect of the compounds is determined.

The crystal structure of the MoErs1 protein is obtained by adopting a dropping method and a hanging dropping method, and the specific method comprises the following steps:

1. the concentration of the purified MoErs1 protein was adjusted to 10 mg/ml, and the protein was dissolved in 20 mM Tris-HCl (pH 8.0), 800 mM NaCl, 5 mM DTT solution;

2. mixing 0.4 mu l of protein in 1 with a liquid storage agent (0.2M magnesium chloride hexahydrate, 0.1M Tris pH 8.5, 3.4M 1, 6-glycol), placing the mixture in a drip plate, and culturing crystals at 4 ℃;

3. after 3 days of culture, MoErs1 can generate crystals, and the culture conditions of the crystals are further optimized by using a pendant drop method;

4. mixing 1.5 mul of purified MoErs1 protein with an equal amount of storage solution, wherein the storage solution comprises 0.05M zinc acetate and 20% PEG3350, and well-grown crystals are used for data analysis;

5. before data collection, the crystals were transferred to a stock solution containing 25% glycerol for cryoprotection and then snap frozen into liquid nitrogen;

6. the crystal structure of MoErs1 was analyzed by single wavelength anomalous diffraction (SAD).

After the MoErs1 crystal is obtained by the method, a potential small molecular compound combined with MoErs1 is designed around key interaction sites Loop2, Loop4, Loop8 and beta 11 of MoErs1 and rice cysteine protease through a ClusPro 2.0 tool. One specific compound designed is FY21001, and the structural formula is as follows:

the binding ability of small molecule compounds to the above sites was verified using the MST method in example 1. The experimental result shows that the MoErs1 protein and FY21001 have strong binding capacity (Kd = 0.28), and further experiments show that the compound has strong inhibitory activity on pathogenicity of rice blast bacteria and EC₅₀The value was 231.07 [ mu ] M (80.445 [ mu ] g/mL), and EC for tricyclazole₅₀The use concentration of 224.08 mu M (42.405 mu g/mL) is similar,therefore, the bactericidal composition has excellent control effect on rice blast and can be used for preparing bactericides.

Finally, it is also noted that the above list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

<110> Nanjing university of agriculture

<120> effector protein MoErs1 derived from Magnaporthe grisea, and coding gene and application thereof

<160>30

<170>Patent-In 3.3

<210> 1

<211>214

<212>PRT

<213>Magnaportheoryzae

<220>

<223> sequence description: amino acid sequence of MoErs1 protein

<400> 1

MRTQFSLLGVAALASTVVNAMPSTLEARALPQVSAVAKPRACSSYPTFDPATGEATEFIFYADSTEEPVAPFAGSVVGKLANPNLAIARIGIAVRGDLAKVVTKCFPDGGEEGLRTRTHGDWRRLTLAGGEDENIILIGQGPVAHRPLTPHDHFFANGTQQPGVFMGDNGSTTWAFSRKDASASEPFDQYEIRLLKSADSPLRNGEFRGFVRAA 214

<210>2

<211>645

<212> DNA

<213>Magnaportheoryzae

<220>

<223> sequence description: nucleotide sequence of MoErs1 protein coding gene

<400>2

ATGCGCACCCAGTTCTCTCTCCTCGGAGTCGCGGCTCTCGCCAGCACCGTCGTCAACGCCATGCCCTCCACGCTCGAGGCCAGGGCCCTTCCCCAGGTTTCGGCCGTCGCCAAGCCGAGGGCGTGCTCCTCGTACCCGACCTTTGATCCCGCCACCGGCGAGGCTACCGAATTCATATTCTATGCCGACTCGACCGAGGAGCCTGTCGCTCCGTTCGCCGGTAGCGTGGTGGGGAAGTTGGCCAACCCCAACCTGGCCATTGCACGGATCGGAATCGCCGTCCGCGGAGATCTCGCGAAGGTCGTGACCAAGTGCTTCCCCGACGGCGGCGAAGAGGGACTCCGCACCCGCACGCACGGCGACTGGAGACGTCTCACCCTTGCCGGAGGCGAGGACGAAAACATCATCTTGATCGGCCAAGGTCCAGTGGCCCACCGACCCTTGACCCCCCACGATCACTTCTTCGCCAACGGCACGCAGCAGCCCGGCGTCTTTATGGGCGACAACGGATCGACCACCTGGGCCTTCTCGAGGAAGGACGCCAGCGCCAGTGAGCCGTTCGACCAGTACGAGATCCGTCTTCTGAAGAGCGCAGACTCGCCTCTGAGGAATGGAGAGTTCAGGGGCTTTGTGCGTGCTGCTTGA 645

<210>3

<211> 1055

<212> DNA

<213>Magnaportheoryzae

<220>

<223> sequence description: upstream flanking nucleotide sequence of MoErs1 protein coding gene

<400>3

GTGTGCGTACGACAATGCTTGCCTGTTTGCCCATCTGTCTCGGGCTCCTCGTCATTTGTGTGAAAAGCGTCTGTTTGCAACGACGCAATGAGTCAACAGTACAAATTATAAGCTATAGTGAAGATGAAGCGAATGCCTGGTACGATAAACCTGTCGTGCCAAGGTAGAATTGACGAAGCCTTGCCTTGATCGATCGTCCAAGTTCAACATCATGTGAACAGAGCCCGTATGCAACCTGGTTGGCCCCCGATGCTTGTGCCATTCCTTGGCCCCAAGAACGTCAAGAGTAAAAAGCTAATCGGACACGACGGGTCCGATGAGACATGGTGAGAAGTTAGTGTTAGCTAAGACCAGGGGAGATCGAGAGCTGCAGCCTTGCCCCGCAGAACGGTAAATTCCCCAAACGAGCTAGACCAGTCCCTTCGCTCAAATACGCCCCTGGGCTGACGAGAAGGACCACACATGCCACTATGTACAGGACCCGGTATAATGTAATTGAGAGAAGGCTGCCTAAGCGCTAAACAAAATGGTCCAGTCGGGGCGAATTCAAGGTAAACGAACATACGGAATAATGGAAGAACTGGCTACCAGGAAGGATCATTAACCTCCACCTGCACCCCCTGAAAGACGAGCGCTTTTTTTTTTTTTTTTTTTGCCAGCGGCACAGTCGCCAAGGGTGGTGATTTTCCCCATTCCATCGTTGGCTCTTTCCTGCACGAGCTGATCCACCACTGTTCGAGATTTGATTCCCCTGCCTGGTCGGGCATGGAACTTCTGGTAATGATGTACCGTAGACCGAATGGCCCTCAATGTGGTTCCGCAAATCTGTAAATTGCCATCAGGCAACAGGCGGGCAAGCCGGGTGTACCGATGGCGAACAGGATAAATACCCCTCGACCGTCCTCGACTTGAATCCCAGGTAGAAGGAAGAGACAGCACTCGCCACAGCTCCTCTGACATTCGCTTTCCCATCGCCCACAACATTTGGCCCTGCTCTCATTCCTTGGGTTGATTTTTTTTCTTTTCTTTCGCAAGCACCGAAAACTTTAGTCAAA 1055

<210>4

<211> 1008

<212> DNA

<213>Magnaportheoryzae

<220>

<223> sequence description: downstream flanking nucleotide sequence of MoErs1 protein coding gene

<400>4

GCCTGACATAACATTTTGCTGGGTAATCGGTTCAACATGTGAGCATTGCGTGATGTTTACCCTGTATTTTTACAGGTATACAGGGTGGACGTGGAAGCAAACACCCAGCTCCCGTCGGTCTGAGAGCATGAATGCCAATGTTGTGCAGCATGATGGATCGCTGCCTGGTTCTAGAATATATAGCAAGTACCGTACGGAAGACATGCTTCAGATATGGGTACTTCGAATAGATAGCCTGGGTCAGGTAGCACAGATGGGCAGGGTTGGTTCATGAGCACGAGTCAATTTAATGTAAATGATTTGACGATACCCCGGATTACTGCGCCGTTTGGGTTTATGCATGTCGCCTAAACGCAACATGTAGTCCGCAAACGCTCCGTAACCCATGAGAGGCTGCTGCACTTTTGCTGCTTTGTTTGCCAACATCTGAGACCCCCCGACCAGTCATGTCTGCTCGGTTGCATGTGCGTTGCAAGGAATGTCAGTCAACAAAGTCCATCAAACGTGAACCGAATCCAACGTCAACAAGCAATGTTATCTCGCTGCCATCTGCCATCTGCCATCTGGGTATCTGCGCGTCAACCCTGAGCTACTAGCCCCACCGCCTTTGGCCCACGGCTTTTCTCGGTTGGTGGGCGCTAAGTATAATGAATTCGGTCAGCTTTGGCCCCAACCAGGTTGTTTGTTAACAAGCTAGATCCATGTACCAAATGTAAACAAATACTACTCTGAGCCATCACACCGTCAACCGGAACCAGGCTTGCTAGGCACGAAGTAATCATCTCTCGGCAAGGAAGAAGAAAAACAAAAAAAAAAAAACCCTGGCGCGGGCACCGGCGGGGTGATGATGGAATTTTGCGGGAACAAAGATACAGTAGGCAGTCGGACACAAGGCAAGCGAAATGGGCAGGCGGGGCAGCTGTAGCAGTACCATCGATGACGCTTCTTGATAAGGTACTGTGATCAAGCTACCTAGGTACGGTATGGCTTACCGTAGGACGAACGGTG 1008

<210>5

<211> 1349

<212> DNA

<213>

<220>

<223> sequence description: nucleotide sequence of hygromycin encoding gene

<400>5

GGAGGTCAACACATCAATGCCTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCATCCAAATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTCCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCATGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCCTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGCCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAG 1349

<210>6

<211> 1576

<212> DNA

<213>Magnaportheoryzae

<220>

<223> sequence description: promoter nucleotide sequence

<400>6

CATATAGGTAGACATATCTTGGGGTATTCGACCCGAAGCTGATCTTTTCTCCCCTCCAAAAACAGTGTGCGGATGTGCCGTCGGTGTAGCGACCGCCGAAGTATCGGGGTTAATAGCGAGACCAGGTCCGACCGGAACTGCCTCCGTCTCGGCGTCATATTCTTACATGAGCGGCAGCGGCTTCTCCGTGGTCTCATCCTTGACGGTCATCAACAAAGCGGCCTCCGGTACCGCAACGCCAAGCTTCATGCCCCAGGTAGACCAACCGAGCCAAATGGTGCCAATCCACAAGAGAGACGTCGAGGAAGACCACGAACCGCCAAAGAGCTTCGAACCGGCTAACAAGGCCGCAGTACAGGCCAGCTCCGGGACGTCGTCTAGTCATTACGGAAATATGCGTATTTCGGGTTGGGTCATATCGGTTGTACCTACATTGTTGGTTGGTCTGATTGTATAGAAAGAAAAGAAGAGAAAGAGGAAAAAGAAAGAAAAAAAAGGGTGAAATACGGGGATCAAGGAAAGTGTGCGTACGACAATGCTTGCCTGTTTGCCCATCTGTCTCGGGCTCCTCGTCATTTGTGTGAAAAGCGTCTGTTTGCAACGACGCAATGAGTCAACAGTACAAATTATAAGCTATAGTGAAGATGAAGCGAATGCCTGGTACGATAAACCTGTCGTGCCAAGGTAGAATTGACGAAGCCTTGCCTTGATCGATCGTCCAAGTTCAACATCATGTGAACAGAGCCCGTATGCAACCTGGTTGGCCCCCGATGCTTGTGCCATTCCTTGGCCCCAAGAACGTCAAGAGTAAAAAGCTAATCGGACACGACGGGTCCGATGAGACATGGTGAGAAGTTAGTGTTAGCTAAGACCAGGGGAGATCGAGAGCTGCAGCCTTGCCCCGCAGAACGGTAAATTCCCCAAACGAGCTAGACCAGTCCCTTCGCTCAAATACGCCCCTGGGCTGACGAGAAGGACCACACATGCCACTATGTACAGGACCCGGTATAATGTAATTGAGAGAAGGCTGCCTAAGCGCTAAACAAAATGGTCCAGTCGGGGCGAATTCAAGGTAAACGAACATACGGAATAATGGAAGAACTGGCTACCAGGAAGGATCATTAACCTCCACCTGCACCCCCTGAAAGACGAGCGCTTTTTTTTTTTTTTTTTTTGCCAGCGGCACAGTCGCCAAGGGTGGTGATTTTCCCCATTCCATCGTTGGCTCTTTCCTGCACGAGCTGATCCACCACTGTTCGAGATTTGATTCCCCTGCCTGGTCGGGCATGGAACTTCTGGTAATGATGTACCGTAGACCGAATGGCCCTCAATGTGGTTCCGCAAATCTGTAAATTGCCATCAGGCAACAGGCGGGCAAGCCGGGTGTACCGATGGCGAACAGGATAAATACCCCTCGACCGTCCTCGACTTGAATCCCAGGTAGAAGGAAGAGACAGCACTCGCCACAGCTCCTCTGACATTCGCTTTCCCATCGCCCACAACATTTGGCCCTGCTCTCATTCCTTGGGTTGATTTTTTTTCTTTTCTTTCGCAAGCACCGAAAACTTTAGTCAAA 1576

<210>7

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>7

ATGCGCACCCAGTTCTC 17

<210>8

<211>20

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>8

TCAGACCGACGGGAGCTGGG 20

<210>9

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>9

ATGCGCACCCAGTTCTC 17

<210>10

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>10

TCAAGCAGCACGCACAAAG 19

<210>11

<211>21

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>11

GTGTGCGTACGACAATGCTTG 21

<210>12

<211>48

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>12

CAGCAAAATGTTATGTCAGGCGATATCTTTGACTAAAGTTTTCGGTGC 48

<210>13

<211>48

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>13

GCACCGAAAACTTTAGTCAAAGATATCGCCTGACATAACATTTTGCTG 48

<210>14

<211>21

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>14

CACCGTTCGTCCTACGGTAAG 21

<210>15

<211>21

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>15

GAGGCTACCGAATTCATATTC 21

<210>16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>16

CAGTACGAGATCCGTCTTCTG 21

<210>17

<211>58

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>17

ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTCATATAGGTAGACATATCTTGG 58

<210>18

<211>54

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>18

CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACAGCAGCACGCACAAAGCC 54

<210>19

<211>58

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>19

ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTCATATAGGTAGACATATCTTGG 58

<210>20

<211>37

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>20

GTCCTCGGAGGAGGCCATGACCGACGGGAGCTGGGTG 37

<210>21

<211>37

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>21

CACCCAGCTCCCGTCGGTCATGGCCTCCTCCGAGGAC 37

<210>22

<211>52

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>22

CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACGGCGCCGGTGGAGTGG 52

<210>23

<211>58

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>23

ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTCATATAGGTAGACATATCTTGG 58

<210>24

<211>37

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>24

GTCCTCGGAGGAGGCCATGACCGACGGGAGCTGGGTG 37

<210>25

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>25

CACCCAGCTCCCGTCGGTCATGGCCTCCTCCGAGGAC 37

<210>26

<211> 118

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>26

CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACTTAAACCTTTCTCTTCTTCTTAGGAACCTTTCTCTTCTTCTTAGGAACCTTTCTCTTCTTCTTAGGGGCGCCGGTGGAGTGG 118

<210>27

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>27

GGAGGTCAACACATCAATG 19

<210>28

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>28

CTCTATTCCTTTGCCCTCG 19

<210>29

<211>30

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>29

ACGCGTCGACATGCCCTCCACGCTCGAGGC 30

<210>30

<211>26

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>30

AACTGCAGAGCAGCACGCACAAAGCC 26