Application of rice OsPBL8 gene in enhancing resistance of rice to magnaporthe oryzae

Abstract

The invention relates to the technical field of biology, in particular to application of a rice OsPBL8 gene in enhancing resistance of rice to magnaporthe oryzae. The rice OsPBL8 gene is a rice endogenous gene participating in the processes of plant growth and development, abiotic stress response, hormone signal channel regulation and control and the like. The invention provides application of the OsPBL8 gene in the field of disease resistance, the OsPBL8 gene is overexpressed through gene editing, and the expression quantity of receptor-like cytoplasm kinase OsPBL8 in plants is increased, so that a plurality of signal channels are regulated and controlled, and the germ resistance of the plants is synergistically improved. By adjusting key hub nodes of plant targeted immune signal transduction, more lasting broad-spectrum disease resistance can be provided, good universality is achieved, disease resistance and growth can be balanced, good practicability is achieved, and improvement of disease resistance of various rice varieties is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae. BACKGROUND

[0002] Magnaporthe oryzae is a disease that occurs on rice caused by Magnaporthe oryzae, which can occur throughout the growth period of rice, and can harm seedlings, leaves, ears, and nodes, respectively known as seedling blight, leaf blight, ear blight, and node blight. Magnaporthe oryzae is widely distributed and has a great impact on rice yield. In order to prevent and control rice blast, current methods mainly include cultivating high-quality seedlings, managing water and fertilizer, strengthening field management, implementing control measures, and using chemical agents.

[0003] In order to reduce the adverse effects of rice blast on rice yield, researchers in the field use biotechnology to improve the varieties of rice. For example, Chinese patent CN112779271A provides rice gene OsFd2 and its application in rice resistance to Magnaporthe oryzae. However, the current transgenic rice still has low resistance to Magnaporthe oryzae, so further research on genes affecting the resistance of rice to Magnaporthe oryzae is still needed. SUMMARY

[0004] The purpose of the present application is to provide a research direction for improving the resistance of rice to Magnaporthe oryzae.

[0005] To achieve the above purpose, the present application provides the following technical solutions: Application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae.

[0006] Optionally, the nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, its coding sequence is SEQ ID No: 2, and the amino acid sequence of the encoded receptor-like cytoplasmic kinase is SEQ ID No: 3.

[0007] Optionally, the OsPBL8 gene sequence is transferred into the initial rice plant to form the transgenic rice overexpressing the OsPBL8 gene.

[0008] Optionally, the transgenic rice is formed by Agrobacterium-mediated transformation method using Agrobacterium strain EHA105 loaded with the OsPBL8 gene to transfect the initial rice plant.

[0009] Optionally, the variety of the initial rice is Nipponbare.

[0010] Optionally, the ability of the transgenic rice to burst reactive oxygen under the induction of chitin is stronger than the ability of the initial rice to burst reactive oxygen under the induction of chitin.

[0011] Optionally, the stem base of the transgenic rice or the initial rice is soaked in a treatment liquid containing the chitin.

[0012] Optionally, the concentration of the chitin in the treatment liquid is any value ranging from 3 μg / mL to 5 μg / mL.

[0013] Optionally, the overexpression of the OsPBL8 gene promotes the expression of resistance genes phenylalanine ammonia lyase (PAL) and WRKY transcription factor 45 (WRKY45).

[0014] The present application has the beneficial effect that by regulating the key pivotal nodes of targeted immune signaling of plants through transgenic technology, more persistent broad-spectrum disease resistance can be provided, and the disease resistance and growth are balanced, and the practicality is good, which is helpful to improve the disease resistance of various rice varieties.

[0015] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, and the content of the specification can be implemented. The following will be described in detail with the preferred embodiments of the present application and with the help of the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is an agarose gel electrophoresis diagram of DNA of multiple rice plants in the first embodiment of the present application; Figure 2 Figure 2 is an expression amount analysis diagram of the OsPBL8 gene of the multiple rice plants in the first embodiment of the present application; Figure 3 Figure 3 is a phenotype of the multiple rice plants after inoculating the rice blast spores in the first embodiment of the present application; Figure 4 Figure 4 is a relative biomass analysis diagram of the multiple rice plants after inoculating the rice blast spores in the first embodiment of the present application; Figure 5 Figure 5 is an active oxygen release amount analysis diagram of the multiple rice plants after treating the base of the plants with chitin in the first embodiment of the present application; Figure 6 Figure 6 is an expression situation analysis diagram of the resistance genes in the leaves of the multiple rice plants in the first embodiment of the present application. DETAILED DESCRIPTION

[0017] The technical solutions of the present application will be described in detail below with the help of the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0018] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0019] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0020] In addition, the technical features involved in the different embodiments of the application described below can be combined with each other as long as there is no conflict between them.

[0021] Application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae.

[0022] The rice OsPBL8 gene is a rice endogenous gene involved in plant growth and development, non-biological stress response, and regulation of hormone signal pathways and the like. The application provides the application of the OsPBL8 gene in the field of rice blast resistance. By gene editing to overexpress the OsPBL8 gene, the expression amount of the plant cytoplasmic receptor-like kinase OsPBL8 is increased, thereby regulating multiple signal pathways and synergistically realizing the disease resistance of the plant.

[0023] By regulating the key hub node of targeted immune signal transduction of the plant through transgenic technology, more persistent broad-spectrum disease resistance can be provided, and the method has good universality, can balance disease resistance and growth, has good practicability, and is helpful to improve the disease resistance of various rice varieties.

[0024] The optimization of the traits of agricultural products by transgenic technology is usually based on gene introduction or replacement, and usually relies on the specific recognition mechanism of "gene to gene", and the vertical resistance of the plant is improved based on a single gene. In this application, the function of the endogenous gene is optimized, the improvement direction is more accurate and sustainable, the network regulation of the targeted immune signal transduction in the plant is realized, the horizontal resistance is realized through signal integration, thereby providing more persistent and broad-spectrum disease resistance, breaking through the thinking limitation of traditional disease-resistant genes, and providing a new direction for crop disease-resistant breeding.

[0025] In some embodiments, the nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, the coding sequence thereof is SEQ ID No: 2, and the amino acid sequence of the receptor-like cytoplasmic kinase encoded thereby is SEQ ID No: 3.

[0026] In some embodiments, the OsPBL8 gene sequence is introduced into an initial rice plant to form a transgenic rice with overexpression of the OsPBL8 gene.

[0027] In some embodiments, the transgenic rice is formed by a method of Agrobacterium-mediated transformation of an initial rice plant by an Agrobacterium strain EHA105 loaded with the OsPBL8 gene.

[0028] In some embodiments, the transgenic rice has a stronger ability to burst reactive oxygen under the induction of chitin than the initial rice.

[0029] In some embodiments, the stem base of the transgenic rice or the initial rice is soaked in a treatment liquid containing chitin.

[0030] In some embodiments, the concentration of chitin in the treatment liquid is any value ranging from 3 μg / mL to 5 μg / mL, for example, any value of 3 μg / mL, 4 μg / mL and 5 μg / mL.

[0031] In some embodiments, the overexpression of the OsPBL8 gene promotes the expression of the resistance genes phenylalanine ammonia lyase (PAL) gene and WRKY transcription factor 45 (WRKY45).

[0032] For details, please refer to the following examples.

[0033] Example 1: The rice OsPBL8 gene was inserted into the initial rice genome to obtain transgenic rice, and the OsPBL8 gene was overexpressed. The initial rice used in this embodiment is japonica rice, and the variety is Nipponbare. The nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, the coding sequence (CDS) is SEQ ID No: 2, and the amino acid sequence of the encoded receptor-like cytoplasmic kinase is SEQ ID No: 3. The specific operation method for preparing transgenic rice is as follows: According to the sequence of the rice OsPBL8 gene, primers OsPBL8-F and OsPBL8-R were designed to amplify the complete open reading frame (ORF) thereof, wherein the nucleotide sequence of OsPBL8-F is SEQ ID No: 4, and the nucleotide sequence of OsPBL8-R is SEQ ID No: 5. OsPBL8-F and OsPBL8-R were both prepared into a solution with a concentration in the range of 10 pmol / L to 15 pmol / L.

[0034] The total RNA of the Nipponbare variety rice plant was used as a template to reverse transcribe the first strand cDNA, and the first strand cDNA was used as an amplification template, and OsPBL8-F and OsPBL8-R were used as amplification primers for amplification. The polymerase required for amplification was a premixed DNA polymerase kit of the PrimeSTAR brand of Takara, which includes 5×PrimeSTAR GXL Buffer, dNTP Mixture, and PrimeSTAR GXL DNA Polymerase. The specific amplification system is shown in Table 1 below.

[0035]

[0036] The reaction conditions of the amplification system are shown in Table 2 below.

[0037]

[0038] After amplification, agarose gel electrophoresis was performed and the gel was cut to recover the amplification product with an agarose gel recovery kit purchased from Omega Bio-Tek, obtaining an OsPBL8 gene fragment with relatively high purity. The OsPBL8 gene fragment was connected with the special vector for blunt-end cloning provided in the blunt-end cloning kit purchased from TransGen Biotech Co., Ltd. of Beijing, and the connection product was transferred into the E. coli DH5α competent cells purchased from Beijing Tianren Biotechnology Co., Ltd., to obtain a modified bacterial solution.

[0039] The modified bacteria solution was coated on Luria-Bertani (LB) agar plate medium containing 100 mg / mL ampicillin (Amp), and after 12 h culture at 37°C, colonies were picked and the length of the inserted fragment in the bacteria was confirmed by colony polymerase chain reaction (PCR) to be consistent with the expected length. The bacteria were sent to Nanjing Kingsway Biotech Co., Ltd. for sequencing, and the sequence determination results showed that the bacteria were nucleotide sequences including the OsPBL8 gene in the gene sequence, and could encode the protein encoded by the OsPBL8 gene. The plasmid in the correct sequencing bacteria was extracted, and the obtained plasmid was named pEASY-Blunt-OsPBL8.

[0040] The purchased pCAMBIA1300.1 vector was single-cut with self-restriction endonuclease Sac I purchased from Thermo Fisher Scientific, and the enzyme cutting buffer for pCAMBIA1300.1 was 10 times enzyme cutting buffer (10×Buffer), which was purchased together with pCAMBIA1300.1. The enzyme cutting system is shown in Table 3 below.

[0041]

[0042] After 1 h of enzyme cutting at 37°C, agarose gel electrophoresis was performed and the gel was cut. The agarose gel recovery kit of Omega Biological Technology Co., Ltd. was used to recover the enzyme-cut vector and reserve it for later use. Nanjing Kingsway Biotech Co., Ltd. was commissioned to synthesize the DNA sequence of double copies of the Cauliflower mosaic virus (CAMV) 35S promoter, and the DNA fragment with the nucleotide sequence of SEQ ID No: 6 was added at the 5' end, and the DNA fragment with the nucleotide sequence of SEQ ID No: 7 was added at the 3' end. The artificially synthesized DNA fragment was named 2×35s. The In-Fusion brand recombinant kit of Takara Co., Ltd. was used to homologously recombine the 2×35s fragment into the vector recovered after enzyme cutting of the pCAMBIA1300.1 vector, and the recombinant product was transformed into the E. coli DH5α competent cells purchased from Beijing Tiangen Biotech Co., Ltd. by heat shock method. All the transformed cells were uniformly coated on Luria-Bertani solid medium containing 50 mg / L kanamycin, and after 16 h culture at 37°C, colonies were picked and sent to Nanjing Kingsway Biotech Co., Ltd. for sequencing. The plasmid extracted from the correctly sequenced colonies was named pCAMBIA1300-2×35s.

[0043] The prepared pCAMBIA1300-2x35s plasmid was single-enzyme cut by purchasing self-limiting restriction enzyme BamHI of ThermoFisher Science Company, and the enzyme cutting system is shown in Table 4 below.

[0044]

[0045] After 37°C enzyme cutting for 1h, agarose gel electrophoresis was performed and the gel was cut, and the enzyme cut vector was recovered by using the agarose gel recovery kit of Omega Bio-tek Company and was used as needed.

[0046] The pEASY-Blunt-OsPBL8 plasmid prepared in the above experiment was used as a template, and the corresponding primers OsPBL8-BamHI-F and OsPBL8-BamHI-R were used for amplification. The nucleotide sequence of OsPBL8-BamHI-F is SEQ ID No: 8, and the nucleotide sequence of OsPBL8-BamHI-R is SEQ ID No: 9. After the amplification product was recovered and purified, the purified amplification product was cloned into the product recovered after BamHI enzyme cutting of pCAMBIA1300-2x35s by using the recombination kit of In-Fusion brand of Takara Company, and the recombined product was transformed into the E. coli DH5a competent cells purchased from Beijing Tiangene Biological Technology Co., Ltd. by heat shock method. The whole bacterial liquid obtained by transformation was uniformly coated on Luria-Bertani solid medium containing 50 mg / L kanamycin. After 37°C culture for 16h, colonies were picked and sent to Nanjing Kingsriver Biological Technology Co., Ltd. for sequencing. The plasmid extracted from the colony with correct sequencing was named as pCAMBIA1300-2x35s-OsPBL8.

[0047] In this embodiment, pCAMBIA1300-2x35s-OsPBL8 was first transformed into Agrobacterium, and then OsPBL8 gene sequence was transformed into rice plants by Agrobacterium-mediated transformation. The Agrobacterium used in this embodiment is Agrobacterium strain EHA105 purchased from Shanghai Weidi Biological Technology Co., Ltd. pCAMBIA1300-2x35s-OsPBL8 was transformed into the strain by liquid nitrogen freeze-thaw method, thereby obtaining the transformed strain. The specific method includes: The EHA105 competent cells were thawed in ice bath, at least 100 ng of plasmid was added, and then mixed gently. After 5 min of ice bath, the mixture was quickly frozen in liquid nitrogen for 5 min, and then heat shocked at 37℃ for 5 min. After completion, the mixture was quickly placed on ice for 1-2 min. 800 μL of Luria-Bertani medium without antibiotics was added to the mixture, and then the mixture was recovered in a 28℃ shaker at 200 rpm for 3.5 h. The mixture was centrifuged at 4000 rpm for 3 min, and then the supernatant was removed by pipetting. The remaining bacterial solution was mixed, and then spread on solid Luria-Bertani medium containing 100 mg / mL of carbenicillin and 100 mg / mL of rifampicin. The medium was inverted and cultured at 28℃ for 30-48 h. The colonies were picked for colony polymerase chain reaction. The positive clones were named as EHA105:pCAMBIA1300-2×35s-OsPBL8, and then stored at 4℃ for later use.

[0048] The EHA105:pCAMBIA1300-2×35s-OsPBL8 was used to transform rice plants of Nipponbare variety by conventional Agrobacterium-mediated method. A total of 7 EHA105:pCAMBIA1300-2×35s-OsPBL8 transgenic plants were obtained in T0 generation.

[0049] The genomic DNA of the above 7 single plants was used as a template, and the primer hyg283-F and hyg283-R were used to amplify the specific fragment of the transgenic genetic transformation marker gene, i.e., the hygromycin phosphotransferase gene, for detection. The nucleotide sequence of hyg283-F was SEQ ID No: 10, and the nucleotide sequence of hyg283-R was SEQ ID No: 11. The 2×Taq DNA Polymerase Mix purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. was used for amplification. The specific amplification system is shown in Table 5 below.

[0050]

[0051] The genomic DNA of EHA105:pCAMBIA1300-2×35s-OsPBL8 was used as a template to form a positive control group, and water was used as a template to form a negative control group. The DNA molecular weight marker (DNA Marker) was labeled as M, the positive control group was labeled as +, the negative control group was labeled as -, and the experimental systems for detecting the genomic DNA of the 7 single plants were labeled with numbers 1 to 7. The reaction conditions of the amplification system are shown in Table 6 below.

[0052]

[0053] The amplification products were separated by agarose gel electrophoresis, and the results were recorded by taking pictures with a gel imaging instrument.

[0054] See Figure 1 It can be seen that the electrophoretic pattern of the DNA of the seven strains of transgenic plants of EHA105:pCAMBIA1300-2x35s-OsPBL8 is similar to that of the positive control template, indicating that the target product is amplified.

[0055] The plant corresponding to the DNA corresponding to the number 1 which amplifies the target product is named OsPBL8-OE1, and the plant corresponding to the DNA corresponding to the number 6 which amplifies the target product is named OsPBL8-OE6, and the Nip variety rice plant without transgenic treatment is named NIP.

[0056] Take the whole plant of OsPBL8-OE1, OsPBL8-OE6 and NIP, and extract total RNA, and detect the expression of OsPBL8 gene in the tissue sample by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). In the real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription are RT-OsPBL8-F and RT-OsPBL8-R, wherein the nucleotide sequence of RT-OsPBL8-F is SEQ ID No: 12, and the nucleotide sequence of RT-OsPBL8-R is SEQ ID No: 13. See Figure 2 It can be seen that the expression amount of OsPBL8 gene in OsPBL8-OE1 and OsPBL8-OE6 is significantly up-regulated compared with NIP, indicating that the OsPBL8 gene in OsPBL8-OE1 and OsPBL8-OE6 is overexpressed.

[0057] OsPBL8-OE1, OsPBL8-OE6 and NIP are inoculated with rice blast, and the inoculation method comprises: The strain of Magnaporthe oryzae is inoculated on complete (CM) medium for activation, and cultured at 28°C for 12 days, and treated with 12h light and 12h dark every 24h. The culture medium is washed with 0.05% Tween 20 sterilized water, and the spore solution is washed off with a glass rod, filtered with a 40μm filter membrane, and the spore concentration is detected under a microscope, and finally adjusted to 1x10 5 The spore suspension is uniformly sprayed on the surface of rice leaves, and placed in a 28°C incubator, and after 24h dark treatment, treated with 12h light and 12h dark cycle, and observed after 7 days of culture.

[0058] See Figure 3It can be seen that the lesion length of OsPBL8-OE6 after inoculation of rice blast spores is about 0.3 cm, and the lesion length of OsPBL8-OE1 after inoculation of rice blast spores is about 0.2 cm, and the lesion length of NIP after inoculation of rice blast spores is significantly larger, indicating that the insertion of OsPBL8 gene helps to improve the ability of rice to resist rice blast.

[0059] Seven days after spraying rice blast spores, the leaf samples of OsPBL8-OE1, OsPBL8-OE6 and NIP were taken to extract DNA, and the contents of rice reference gene UBQ and rice blast reference gene MoPot in the DNA were detected by real-time fluorescent quantitative polymerase chain reaction, so as to obtain the biomass of rice blast in OsPBL8-OE1, OsPBL8-OE6 and NIP relative to the leaf tissue, that is, the relative biomass. When detecting the content of UBQ gene by real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription are UBQ-RT-F and UBQ-RT-R, and the nucleotide sequence of UBQ-RT-F is SEQ ID No: 14, and the nucleotide sequence of UBQ-RT-R is SEQ ID No: 15. When detecting the content of MoPot gene by real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription are MoPot-RT-F and MoPot-RT-R, and the nucleotide sequence of MoPot-RT-F is SEQ ID No: 16, and the nucleotide sequence of MoPot-RT-R is SEQ ID No: 17. The experimental results are shown in Table 2. Figure 4 It can be seen that the relative biomass of rice blast in OsPBL8-OE1 and OsPBL8-OE6, which overexpress OsPBL8 gene, is significantly lower than that of the control group NIP, indicating that the development of rice blast is inhibited.

[0060] The rice seeds obtained from culturing OsPBL8-OE1, OsPBL8-OE6 and NIP were peeled and sterilized, and then were placed in Hoggland nutrient solution in a glass tube, and were cultured aseptically for 7 days to obtain multiple rice seedlings. 150 μL of double distilled water was added to each well of a black 96-well enzyme-coated plate, and the rice seedlings with uniform size and stem thickness were selected, and the stem base was cut to obtain a small piece with a length of about 1 mm, and the small piece samples obtained by cutting were placed in the small holes of the black enzyme-coated plate, 5 small pieces per hole. When cutting, the white stem with a length of about 2 mm near the seed was removed, and then the stem base tissue was cut. The black enzyme-coated plate was placed at room temperature in the dark overnight, and the room temperature was detected to be 25°C. On the second day, a chitin mother liquor with a concentration of 2 mg / mL was prepared, and 10 mL of reaction solution was prepared therefrom. The preparation method of the reaction solution includes: adding 20 μL of horseradish peroxidase (HRP) solution, 20 μL of chemiluminescent probe L-012 solution and 20 μL of chitin mother liquor into a test tube, and adding double distilled water to make up to 10 mL. The horseradish peroxidase solution is a laboratory-prepared solution, which is prepared by adding 15 mg of horseradish peroxidase powder to 1 mL of sterile water, and is stored in a -20°C refrigerator. The chemiluminescent probe L-012 solution is a laboratory-prepared solution, which is prepared by adding 15 mg of chemiluminescent probe L-012 powder to 1 mL of sterile water, and is stored in a -20°C refrigerator. The black enzyme-coated plate placed in the dark overnight was taken out, the liquid in each hole was sucked out with a pipette and discarded, and 100 μL of reaction solution or double distilled water was added, respectively, to obtain six groups of samples treated with reaction solution or water, wherein the sample obtained by treating NIP with chitin is labeled as group one, the sample obtained by treating OsPBL8-OE1 with chitin is labeled as group two, the sample obtained by treating OsPBL8-OE6 with chitin is labeled as group three, the sample obtained by treating NIP with water is labeled as group four, the sample obtained by treating OsPBL8-OE1 with water is labeled as group five, and the sample obtained by treating OsPBL8-OE6 with water is labeled as group six. The enzyme-coated plate without cover was placed in an enzyme marker, and each hole was detected for about 30 ms. The enzyme marker setting parameters are shown in Table 7.

[0061]

[0062] The detection results are shown in Table 8. Figure 5 It can be seen that chitin can trigger the active oxygen burst of the plant, and the insertion of the OsPBL8 gene does not affect this active oxygen burst. In addition, after chitin treatment, the active oxygen production level of the OsPBL8 gene overexpression plant is about 2 times higher than that of NIP.

[0063] The whole plants of OsPBL8-OE1, OsPBL8-OE6 and NIP were taken, and total RNA was extracted, and the expression of phenylalanine ammonia-lyase (PAL) gene and WRKY transcription factor 45 (WRKY45) in the tissue samples was detected by real-time fluorescent quantitative polymerase chain reaction. In the detection of the expression of phenylalanine ammonia-lyase (PAL), the primers used in reverse transcription in the real-time fluorescent quantitative polymerase chain reaction were RT-OsPAL-F and RT-OsPAL-R, and the nucleotide sequence of RT-OsPAL-F was SEQ ID No: 18, and the nucleotide sequence of RT-OsPAL-R was SEQ ID No: 19. In the detection of the expression of WRKY transcription factor 45 (WRKY45), the primers used in reverse transcription in the real-time fluorescent quantitative polymerase chain reaction were RT-OsWRKY-F and RT-OsWRKY-R, and the nucleotide sequence of RT-OsWRKY-F was SEQ ID No: 20, and the nucleotide sequence of RT-OsPAL-R was SEQ ID No: 21. The detection results are shown in Table 1. Figure 6 It can be seen that the plants overexpressing OsPBL8 gene have a significantly stronger ability to resist rice blast than the plants without inserting OsPBL8 gene.

[0064] In summary, it can be seen that overexpression of rice OsPBL8 gene can enhance the resistance of rice to Magnaporthe oryzae.

[0065] The technical features of the above-described embodiments can be combined arbitrarily, and to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, it should be considered that it is within the scope of the present disclosure.

[0066] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Application of the rice OsPBL8 gene in enhancing rice resistance to rice blast fungus.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, its coding sequence is SEQ ID No: 2, and the amino acid sequence of the receptor-like cytokinase it encodes is SEQ ID No:

3.

3. The application as described in claim 1, characterized in that, The OsPBL8 gene sequence was transferred into the initial rice plants to form transgenic rice with OsPBL8 gene overexpression.

4. The application as described in claim 3, characterized in that, The transgenic rice was formed by transfecting the initial rice plants with Agrobacterium strain EHA105 carrying the OsPBL8 gene via Agrobacterium-mediated transformation.

5. The application as described in claim 3, characterized in that, The initial rice variety was Nipponbare.

6. The application as described in claim 3, characterized in that, The transgenic rice exhibits a stronger ability to generate reactive oxygen species (ROS) under chitin-induced conditions than the original rice.

7. The application as described in claim 6, characterized in that, The stem base of the genetically modified rice or the initial rice is immersed in a treatment solution containing chitin.

8. The application as described in claim 7, characterized in that, The concentration of chitin in the treatment solution is any value between 3 μg / mL and 5 μg / mL.

9. The application as described in claim 1, characterized in that, Overexpression of the OsPBL8 gene promotes the expression of the resistance gene phenylalanine ammonia-lyase and WRKY transcription factor 45.

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