Magnaporthe oryzae MoPCP1 Gene and Its Application

Abstract

The present invention belongs to the field of biological genetic engineering, and specifically relates to a Magnaporthe oryzae MoPCP1 gene and its application. The full-length sequence of the Magnaporthe oryzae MoPCP1 gene is shown in SEQ ID NO.1, the cDNA sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3. Experiments show that after replacing the Magnaporthe oryzae MoPCP1 gene with the hygromycin resistance gene, the obtained Magnaporthe oryzae knockout mutant has obvious changes in vegetative growth ability, appressorium formation ability, and conidia morphology and size compared with the wild-type Magnaporthe oryzae, and there are obvious differences in the response to external environmental factors; the pathogenicity experiment shows that MoPCP1 the deletion of the gene significantly reduces the pathogenicity of Magnaporthe oryzae, indicating that MoPCP1 the gene plays a significant role in regulating the pathogenicity of Magnaporthe oryzae. The MoPCP1 gene and its application provided by the present invention play an important role in the pathogenicity of Magnaporthe oryzae, providing a new direction for further using this gene to control Magnaporthe oryzae.

Description

Technical Field

[0001] The present invention belongs to the field of biological genetic engineering, and specifically relates to a pathogenicity gene MoPCP1 derived from Magnaporthe oryzae, and also relates to the use of this gene. Background Art

[0002] The rice blast caused by Magnaporthe oryzae will cause a serious reduction in rice production, and its destructive power will seriously threaten the production of rice and international food security.

[0003] Mitochondria provide a large amount of ATP for the life activities of cells and participate in various physiological processes. Serine protease is a class of proteolytic enzymes with a serine side chain. In yeast, mitochondrial serine protease Pcp1 is a member of the rhomboid protease family in the membrane. Mitochondrial serine protease plays a crucial role in maintaining mitochondrial homeostasis. It is necessary for processing various mitochondrial proteins and maintaining mitochondrial DNA and morphology; however, the function of mitochondrial serine protease Pcp1 in Magnaporthe oryzae and even plant pathogenic fungi has not been reported. At present, there are few studies on how to regulate mitochondrial homeostasis in Magnaporthe oryzae and the impact of mitochondrial homeostasis on the pathogenic ability of Magnaporthe oryzae. Therefore, using Magnaporthe oryzae to study the function of mitochondrial serine protease in the pathogenic process lays a foundation for further revealing the pathogenic mechanism of mitochondrial homeostasis on the infection of rice by Magnaporthe oryzae, and has important theoretical significance and application value for the comprehensive control of Magnaporthe oryzae. Summary of the Invention

[0004] Aiming at the current problems of serious harm of rice blast and lack of effective control methods, the purpose of the present invention is to provide a Magnaporthe oryzae MoPCP1 gene and its application. The MoPCP1 gene plays an important role in the vegetative growth of mycelia, conidia production, appressorium formation and pathogenicity of Magnaporthe oryzae.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A Magnaporthe oryzae MoPCP1 gene, the full-length sequence of the Magnaporthe oryzae MoPCP1 gene is shown in SEQ ID NO.1, the cDNA sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0007] The application of the above-mentioned Magnaporthe oryzae MoPCP1 gene in inhibiting the vegetative growth of Magnaporthe oryzae.

[0008] The application of the above-mentioned Magnaporthe oryzae MoPCP1 gene in reducing the tolerance of Magnaporthe oryzae to oxidative stress.

[0009] Use of the above-mentioned Magnaporthe oryzae MoPCP1 gene in altering the morphology and size of Magnaporthe oryzae conidia.

[0010] Use of the above-mentioned Magnaporthe oryzae MoPCP1 gene in delaying the formation of appressoria of Magnaporthe oryzae.

[0011] Use of the above-mentioned Magnaporthe oryzae MoPCP1 gene in reducing the pathogenicity of Magnaporthe oryzae, wherein reducing the pathogenicity of Magnaporthe oryzae is reducing the pathogenicity of Magnaporthe oryzae to rice.

[0012] A method for constructing a Magnaporthe oryzae mutant with the MoPCP1 gene knocked out, comprising the following steps: using the genomic DNA of Magnaporthe oryzae as a template, amplifying the upper arm fragment of the MoPCP1 gene with MoPCP1-F1 and MoPCP1-R1 as primers, amplifying the lower arm fragment of the MoPCP1 gene with MoPCP1-F2 and MoPCP1-R2 as primers, then fusing the upper arm fragment of the MoPCP1 gene, the hygromycin resistance gene fragment and the lower arm fragment of the MoPCP1 gene, amplifying the fused product in large quantities with NEXT-F and NEXT-R as primers to obtain a large amount of DNA fused product, performing genetic transformation to obtain transformants, and screening and identification;

[0013] The full-length sequence of the Magnaporthe oryzae MoPCP1 gene is shown in SEQ ID NO.1, the cDNA sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3;

[0014] The sequences of MoPCP1-F1 and MoPCP1-R1 are shown in SEQ ID NOs.4-5;

[0015] The sequences of MoPCP1-F2 and MoPCP1-R2 are shown in SEQ ID NOs.6-7;

[0016] The sequences of NEXT-F and NEXT-R are shown in SEQ ID NOs.8-9;

[0017] The sequence of the hygromycin resistance gene fragment is shown in SEQ ID NO.13.

[0018] Use of the Magnaporthe oryzae mutant constructed by the above-mentioned construction method, including one or more of the following:

[0019] 1) Use in inhibiting the vegetative growth of Magnaporthe oryzae;

[0020] 2) Use in reducing the tolerance of Magnaporthe oryzae to oxidative stress;

[0021] 3) Use in altering the morphology and size of Magnaporthe oryzae conidia;

[0022] 4) Application in delaying the formation of appressoria of Magnaporthe oryzae;

[0023] 5) Application in reducing the pathogenicity of Magnaporthe oryzae to rice.

[0024] The remarkable advantages of the present invention are as follows:

[0025] The present invention discovers for the first time the gene MoPCP1 encoding the serine protease of Magnaporthe oryzae, which can affect the growth, development and pathogenic process of Magnaporthe oryzae. There is no similar research at home and abroad at present, so the present invention has a certain novelty. The present invention uses molecular biology experimental methods to verify that the deletion of the MoPCP1 gene can significantly affect the growth, development and pathogenic process of Magnaporthe oryzae, which has a certain creativity. The MoPCP1 gene and its application provided by the present invention play an important role in the pathogenicity of Magnaporthe oryzae, can provide important drug targets for the design and screening of targeted drugs against Magnaporthe oryzae, and provide a new direction for further using this gene to control Magnaporthe oryzae, which has a certain practicality.

[0026] In summary, the present invention verifies the physiological functions such as vegetative growth, conidia germination, appressorium determination, and pathogenicity of the MoPCP1 gene of Magnaporthe oryzae, and proves that replacing the MoPCP1 gene of Magnaporthe oryzae with the hygromycin resistance gene significantly inhibits the growth and development of Magnaporthe oryzae and leads to the reduction of the pathogenicity of Magnaporthe oryzae, thus indicating that the MoPCP1 gene is an important gene in the pathogenic process of Magnaporthe oryzae. The MoPCP1 gene and its application provided by the present invention provide a new direction for the prevention and control of Magnaporthe oryzae. Brief Description of the Drawings

[0027] Figure 1 : Schematic diagram of the construction process of the MoPCP1 gene knockout mutant.

[0028] Figure 2 : Southern hybridization was used to analyze the single-copy integration in the mutant. The genomic DNAs of Guy11 and the Mopcp1 gene knockout mutant were digested with EcoR I, and then hybridized with the probe to verify the single-copy integration of the HPH gene in the mutant genome.

[0029] Figure 3 : Photographs of the colony and aerial mycelium morphology of the MoPCP1 gene knockout mutant on the CMⅡ, MM, SDC, and RBM medium plates.

[0030] Figure 4 : Photographs of the colony morphology of the MoPCP1 gene knockout mutant under oxidative stress.

[0031] Figure 5 : Comparison of the conidia morphology between the MoPCP1 gene knockout mutant and the wild type.

[0032] Figure 6 : Germination of appressoria of the MoPCP1 gene knockout mutant and the wild type at different times.

[0033] Figure 7 : Pathogenicity map of the MoPCP1 gene knockout mutant and the wild type on rice leaves. Specific implementation manners

[0034] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0035] The test materials involved in the present invention are as follows:

[0036] Test strains and plants:

[0037] The rice blast fungus strain is the wild type strain Guy11 of the rice blast fungus, and the test rice is the rice susceptible to rice blast, variety Co39.

[0038] Preparation of relevant culture media and solutions:

[0039] Complete medium (CMⅡ): 20× nitrate 50 mL / L, 1000× trace elements 1 mL / L, 1000× vitamin solution 1 mL / L, glucose 10 g / L, peptone 2 g / L, acid-hydrolyzed casein 1 g / L, yeast extract 1 g / L, agar powder 20 g / L added to the solid medium;

[0040] Trace elements: zinc sulfate heptahydrate 22 g / L, boric acid 11 g / L, manganese chloride tetrahydrate 5 g / L, ferrous sulfate heptahydrate 5 g / L, cobalt chloride hexahydrate 1.7 g / L, copper sulfate pentahydrate 1.6 g / L, sodium molybdate pentahydrate 1.5 g / L, disodium ethylenediaminetetraacetate 22 g / L.

[0041] Vitamins: biotin 0.1 g / L, vitamin B 0.1 g / L, thiamine 0.1 g / L, riboflavin 0.1 g / L, p-aminobenzoic acid 0.1 g / L, nicotinic acid 0.1 g / L.

[0042] Protoplast regeneration medium (TB3): yeast extract 3 g / L, acid-hydrolyzed casein 3 g / L, sucrose 200 g / L, agar powder 12 g / L added to the solid medium.

[0043] Rice straw medium (SDC): corn flour 25 g / L, dry rice straw 150 g / L, agar powder 20 g / L. Used to induce sporulation of the rice blast fungus.

[0044] Oat medium (OMA): oats 50 g / L, agar powder 20 g / L added to prepare the solid medium.

[0045] Minimal medium (MM): Sodium nitrate 6 g / L, potassium chloride 0.52 g / L, magnesium sulfate heptahydrate 0.312 g / L, potassium dihydrogen phosphate 1.52 g / L, thiamine 0.01 g / L, trace elements 1 mL / L, glucose 10 g / L. For solid medium, add agar powder 20 g / L.

[0046] Example 1: Method for knocking out MoPCP1 gene

[0047] The full-length sequence of the MoPCP1 gene of Magnaporthe oryzae in the present invention is shown as SEQ ID NO.1, its cDNA sequence is shown as SEQ ID NO.2, and the amino acid sequence of the pathogenicity protein MoPcp1 of Magnaporthe oryzae encoded by it is shown as SEQ ID NO.3.

[0048] (1) Using the wild-type Guy11 genome of Magnaporthe oryzae as a template, the upper arm fragment of the MoPcp1 gene was amplified with primers MoPCP1-F1 and MoPCP1-R1, and the lower arm fragment of the MoPcp1 gene was amplified with primers MoPCP1-F2 and MoPCP1-R2 ( Figure 1 ). Then, the upper arm fragment of the MoPcp1 gene, the hygromycin resistance gene fragment (SEQ ID NO.13), and the lower arm fragment of the MoPcp1 gene were fused. The fusion product was amplified in large amounts with NEXT-F and NEXT-R to obtain a large amount of fusion product. The fusion product was transformed into protoplasts to replace the MoPcp1 gene of Magnaporthe oryzae.

[0049] (2) Process of protoplast transformation:

[0050] ① Add about 10 μL of the fusion product to 150 μL of the well-divided protoplasts of the wild-type strain Guy11 of Magnaporthe oryzae. Gently flick the bottom of the test tube to mix and then let it stand on ice for 20 - 25 minutes (to allow the protoplasts to fully contact with the DNA);

[0051] ② Add 1 mL of PTC solution (sterilized by suction filtration) to the above-mentioned standing protoplasts. Gently flick the bottom of the tube to mix well and then let it stand at room temperature for 10 - 20 minutes;

[0052] ③ Add liquid TB3 medium to the standing protoplast tube and make up the volume to 10 mL. Seal the tube mouth and incubate in the dark overnight at 90 rpm and 28 °C in an incubator for recovery;

[0053] ④Dissolve 200 mL of TB3 solid medium by microwave heating. After allowing it to cool slightly, pour 10 mL of the cells that have completed overnight resuscitation into a 50 mL centrifuge tube. Add the non-solidified TB3 solid medium to make the volume up to 50 mL. Add 300 μg / mL hygromycin required for the first layer of screening and 0.1 g / mL ampicillin / carbenicillin antibiotics to prevent bacterial contamination. Mix well by inverting the tube up and down. After mixing, pour it onto a plate and wait for it to solidify.

[0054] ⑤Pour the remaining non-solidified TB3 solid medium into a 50 mL centrifuge tube (the purpose is to completely cover the first layer of solidified medium). Add 600 μg / mL hygromycin required for the second layer of screening. After shaking and mixing well, pour it onto the first layer of solidified medium. After the second layer of medium has also dried, cooled, and solidified, seal it with a sealing film and place it upside down in a 28 °C constant temperature incubator for dark culture for about 5 - 8 days.

[0055] ⑥When transformants grow on the second layer of medium, carefully pick a single colony with a toothpick and continue to culture it on CMII solid medium for subsequent PCR verification.

[0056] (3) Use the internal primers KO-F and KO-R of the MoPCP1 gene for PCR knockout verification. Use distilled water as the negative control and the DNA of the wild-type strain Guy11 of Magnaporthe oryzae as the positive control. Select the transformants without bands for the next verification. Use the primers MoPCP1-F1 and HYG-R for BY verification. Use distilled water as the negative control and select the transformants with bands for southern blot verification.

[0057] (4) Southern hybridization identification: Select the restriction enzyme EcoR I to digest the genomic DNA of the wild-type strain Guy11 of Magnaporthe oryzae and the candidate positive transformants after MoPCP1 knockout ( Figure 2 ). The results show that when using the coding region of the MoPCP1 gene as a probe, a clear band of the preset size of 5.2 kb appears at the corresponding position of the wild-type Guy11, while there is no band in the corresponding lanes of the obtained knockout transformants ΔMopcp1-8, ΔMopcp1-14, and ΔMopcp1-40, indicating that the three candidate transformants have all been successfully targeted for knockout. At the same time, when using the HPH gene as a probe, all three candidate transformants show clear and single target bands of 4.7 kb in size, while there is no band at the corresponding lane position of the wild-type strain Guy11, indicating that these three candidate transformants are all single-copy insertions.

[0058] (5) Primer sequences used:

[0059] MoPCP1-F1: 5'-TGGTAGTGTGTCCATGTGGA-3' (SEQ ID NO.4)

[0060] MoPCP1-R1:

[0061] 5'-CATTCATTGTTGACCTCCACTAGCTCCAGGCGTTCGAGACGGGAGCCG-3' (SEQ ID NO.5)

[0062] MoPCP1-F2:

[0063] 5'-GCAAAGGAATAGAGTAGATGCCGACCGGCGGGGTCGAAATTTGATCC-3' (SEQ ID NO.6)

[0064] MoPCP1-R2: 5'-GGAGTCACGAGTACGGCCAC-3' (SEQ ID NO.7)

[0065] NEXT-F: 5'-AGAGTCCACTGAACCGATCA-3' (SEQ ID NO.8)

[0066] NEXT-R: 5'-TGATATCCGGCTGCCGCAAG-3' (SEQ ID NO.9)

[0067] KO-F: 5'-GGCGAACAAGCTGCTGCGCA-3' (SEQ ID NO.10)

[0068] KO-R: 5'-CCGATGTCGCACGCTTGGTA-3' (SEQ ID NO.11)

[0069] HYG-R: 5'-CGGTGGTGCAGATGAACTTC-3' (SEQ ID NO.12)

[0070] (6) PCR reaction system used in the experiment:

[0071] Amplification PCR reaction system (50 μL): template 0.5 μL, 2x reaction mix 25 μL, upstream primer 2 μL, downstream primer 2 μL, ddH2O 25 μL. The PCR reaction procedure is: 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C at 1 kb / min, 30 cycles, 72°C for 10 min, 16°C for 10 min.

[0072] Fusion PCR reaction system (25 μL): The upper arm fragment of the MoPcp1 gene, the hygromycin resistance gene fragment, and the lower arm fragment of the MoPcp1 gene were added at a molar ratio of 1:3:1, 2.5 μL of 10x PCR buffer, 0.5 μL of dNTP Mixture, 0.25 μL of LATaq enzyme, and 21.75 μL of ddH2O. The PCR reaction program was: 94°C for 3 min, 94°C for 35 s, 58°C for 5 min, 72°C for 5 min, 10 cycles, and 16°C for 10 min.

[0073] Example 2: MoPcp1 regulates the vegetative growth of Magnaporthe oryzae

[0074] The wild-type Guy11 and the MoPcp1 knockout mutant strains were respectively inoculated in the center of different medium plates with a diameter of 70 mm, and cultured in an incubator at 28°C for 7 days. The colonies to be measured were observed and photographed, and the colony area diameter was measured and photographed for preservation. The experimental results showed that in the four media, the colony growth diameter of the MoPcp1 knockout mutant was smaller than that of the wild-type ( Figure 3 ), and at the same time, it could be observed that the aerial hyphae also decreased in the SDC and RBM sporulation media, indicating that MoPcp1 regulates the vegetative growth of Magnaporthe oryzae.

[0075] Example 3: MoPcp1 is involved in regulating the response of Magnaporthe oryzae to oxidative environmental stress

[0076] The wild-type Guy11 and the MoPcp1 knockout mutant strains were inoculated in a complete medium supplemented with 2.5 mM hydrogen peroxide (H2O2) and 5 mM hydrogen peroxide (H2O2), and cultured in the dark at 28°C for 7 d. The results showed that the MoPcp1 knockout mutant became more sensitive to 5 mM hydrogen peroxide (H2O2), and its tolerance to oxidative stress decreased significantly compared with the wild-type ( Figure 4 ). The results indicate that MoPcp1 is involved in regulating the response of Magnaporthe oryzae to oxidative stress environmental stress.

[0077] Example 4: MoPcp1 is involved in regulating the asexual reproduction process of Magnaporthe oryzae

[0078] The wild-type Guy11 and the MoPcp1 knockout mutant strains were respectively inoculated in the center of SDC medium plates. After dark incubation at 28 °C for 5 days, the aerial hyphae on the surface of the medium were scraped off, and then placed in a 28 °C greenhouse and induced to sporulate for 3 days under the regulation of black light. The conidia yields of the corresponding strains in the SDC medium were quantitatively counted. The experimental results showed that some spores produced by the MoPcp1 knockout mutant were abnormally shaped. The conidia of different strains were stained with CFW to further observe their morphology: It was found from microscopic observation that the conidia of the wild-type and complementary strains were basically three-celled with two septa, while there were three spore morphologies in the mutant: no septum, one septum, and two septa. Among them, the proportion of spores with a single septum was 70%, and the size of the conidia was also reduced compared to the wild-type ( Figure 5 ). The above results indicate that MoPcp1 is crucial for the asexual reproduction process of Magnaporthe oryzae.

[0079] Example 5: Deletion of MoPCP1 delays the maturation of appressoria

[0080] The conidia of the wild-type Guy11 and the MoPcp1 knockout mutant were collected separately, and their appressorial germination was induced under hydrophobic conditions. The germination and formation of appressoria of different strains were observed under a microscope at 2, 4, 6, 8, and 12 hours respectively. The experiment found that during the germination stage, the conidia of the wild-type Guy11 and the MoPcp1 knockout mutant basically germinated, but in the later appressorium formation stage, the proportion of conidia of the MoPcp1 knockout mutant forming appressoria was significantly lower than that of the wild-type ( Figure 6 ). The above results show that the deletion of MoPCP1 delays the formation of appressoria of Magnaporthe oryzae.

[0081] Example 6: MoPcp1 is involved in regulating the pathogenic process of Magnaporthe oryzae

[0082] The conidial suspensions (1×10 5 per mL) of the wild-type Guy11 and the MoPcp1 knockout mutant were collected and respectively inoculated on three-week-old rice leaves for a rice spraying experiment. The leaf lesions were observed after 6 days. The results showed that on the rice leaves, the wild-type Guy11 could produce typical rice blast lesions, while the MoPcp1 knockout mutant failed to produce lesions ( Figure 7 ). In summary, the above results indicate that the deletion of the MoPCP1 gene significantly reduces the pathogenicity of Magnaporthe oryzae, indicating that the MoPCP1 gene plays an important role in the pathogenic process of Magnaporthe oryzae.

[0083] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. Knockout of Rice Blast Fungus MoPCP1 The invention relates to an application of a rice blast fungus mutant containing a gene, characterized in that: Selected from: 1) Application in inhibiting the vegetative growth of rice blast fungus; 2) Application in reducing the tolerance of rice blast fungus to oxidative stress; 3) Application in changing the morphology and size of conidia of rice blast fungus; 4) Application in delaying the formation of appressorium of rice blast fungus; 5) Application in reducing the pathogenicity of rice blast fungus to rice; Magnaporthe grisea MoPCP1 The full-length sequence of the gene is shown in SEQ ID NO.1, its cDNA sequence is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.3; The method for constructing the rice blast fungus mutant is as follows: using the rice blast fungus genomic DNA as a template, using MoPCP1-F1 and MoPCP1-R1 as primers to amplify MoPCP1 The upper arm fragment of the gene was amplified using MoPCP1-F2 and MoPCP1-R2 as primers MoPCP1 The lower arm fragment of the gene will be MoPCP1 Gene upper arm fragment, hygromycin resistance gene fragment and MoPCP1 The lower arm fragments of the gene are fused, and the fusion products are amplified in large quantities using NEXT-F and NEXT-R as primers to obtain a large number of DNA fusion products, genetic transformation, transformants are obtained, and screening and identification are performed; the sequences of MoPCP1-F1 and MoPCP1-R1 are shown in SEQ ID NO.4~5; the sequences of MoPCP1-F2 and MoPCP1-R2 are shown in SEQ ID NO.6~7; the sequences of NEXT-F and NEXT-R are shown in SEQ ID NO.8~9; the sequence of the hygromycin resistance gene fragment is shown in SEQ ID NO.13.

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