Rice sheath blight resistance related gene OsbZIP65 and application thereof

Abstract

The invention discloses a rice sheath blight resistance related gene OsbZIP65 and application thereof, and belongs to the technical field of plant genetic engineering. According to the invention, by constructing an overexpression rice strain and a gene editing rice strain of OsbZIP65 as shown in SEQ ID NO.1, the resistance of the OsbZIP65 to the sheath blight disease is negatively regulated and controlled by the OsbZIP65. Through promoter analysis and screening, it is also found that a section of base sequence with the length of 86 bp located in the 5 'UTR region of the OsbZIP65 promoter is a core region responding to rhizoctonia solani YWK196 induction, differentiation of different haplotypes exists in the region, and the haplotypes are related to the OsbZIP65 low pathogen induced expression quantity and sheath blight resistance enhancement. Therefore, the disease resistance character of rice can be improved through genetic manipulation or haplotype hybridization by utilizing the OsbZIP65 gene, and materials and production varieties capable of resisting the rice sheath blight disease are cultivated and created, so that the disease resistance of the rice is improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a rice OsbZIP65 transcription factor gene and an application thereof in improving rice resistance to sheath blight. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's three major food crops and is important in supporting population growth and meeting a wide range of nutritional needs. As the staple food for more than half of the world's population, the sustainability and stability of rice production is critical to global food security.

[0003] Throughout the growing season, rice is constantly threatened by pathogenic organisms such as pathogenic fungi, bacteria, nematodes, oomycetes, viruses and herbivorous insects, resulting in an annual yield loss of 10% to 30%. More than 100 rice diseases have been identified so far, and the three most destructive rice diseases are rice blast, bacterial leaf blight and sheath blight, which are caused by the semi-biotrophic fungus Magnaporthe oryzae, the semi-biotrophic bacteria Xanthomonas oryzae pv.oryzae and the necrotrophic fungus Rhizoctonia solani, respectively. Rice sheath blight, mainly caused by Rhizoctonia solani, is one of the most widespread soil-borne fungal diseases in the world. Rice sheath blight can occur from the seedling stage to the booting stage of rice, with the highest incidence rate around the heading stage. It mainly harms rice leaf sheaths and leaves. In severe cases, it can invade the stem and spread to the panicle. In the early stage of the disease, the leaf sheath appears as dark green water-soaked lesions, which then expand and merge into irregular or cloud-like large lesions. When conditions are suitable, the edge of the lesion is dark green and the center is gray-green. The lesion area expands rapidly and can develop to the entire rice plant including the panicle. In severe cases, the whole plant will die. When the weather is dry, the edge of the lesion is brown and the center is straw yellow to grayish white. The diseased leaf sheath withers and turns yellow due to tissue necrosis, and the infected rice cannot head normally. Even if it heads, the spread of lesions to the panicle will lead to an increase in the amount of barren grains and a decrease in yield. In 2024, the overall incidence of rice sheath blight in my country will be severe, with severe occurrences in the central and western parts of South China, the northern part of Southwest China, Jiangnan, and the middle and lower reaches of the Yangtze River, and moderate occurrences in other rice-growing areas. The total affected area nationwide is expected to exceed 240 million mu, which will rank first among all rice diseases. The development of rice breeding work for resistance to sheath blight is imminent.

[0004] In response to environmental changes, plants have evolved complex signaling pathways, which are usually composed of receptors, secondary signals, plant hormones, and signal transduction modules. Transcription factors are important components of biotic and abiotic stress signal transduction pathways, which regulate the expression of downstream genes by binding to different cis-elements. Typical transcription factors are generally composed of four parts: DNA binding domain, transcriptional regulatory domain, nuclear localization signal peptide, and oligomerization site. The combined action of these domains determines the time, space, and mode of action of transcription factor regulation. The bZIP family transcription factors are widely involved in regulating the defense response of plants to abiotic stresses. Studies have found that the bZIP-type transcription factor APIP5 can form homodimers and interact with the rice blast effector factor AvrPiz-t in the cytoplasm, inhibiting its transcriptional activity and protein accumulation during the necrotrophic stage. It is worth noting that APIP5 can undergo nucleocytoplasmic shuttling and, as an RNA-binding protein, regulates the mRNA degradation of cell death and defense-related genes OsLSD1 and OsRac1, acting at the post-transcriptional level. In addition, OsbZIP62 and OsbZIP1 can regulate rice resistance to bacterial blight and rice blast respectively through the salicylic acid pathway, while OsbZIP23 positively regulates resistance to sheath blight. Summary of the invention

[0005] The purpose of the present invention is to provide a rice disease resistance-related gene OsbZIP65 and a DNA fragment of a complete coding region and 5'UTR of a corresponding related gene and its application. The present invention confirms through functional verification that the 86bp before the start codon of the 5'UTR region of the OsbZIP65 gene responds to the induction of the sheath blight pathogen YWK196, thereby enhancing the susceptibility of rice to disease. Bioinformatics analysis found that there were 8 SNP sites with non-synonymous mutations in the segment, and a rare SNP site with an occurrence frequency of less than 5% was identified in 3024 rice materials. The expression of OsbZIP65 in this haplotype decreased after inoculation with the sheath blight pathogen, thereby enhancing the resistance of rice to sheath blight. Using the present invention, the OsbZIP65 gene or its 5'UTR region or the SNP site can be applied to rice disease resistance breeding and the cultivation of germplasm materials.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a rice gene OsbZIP65 for enhancing disease susceptibility is provided. The sequence of the rice gene OsbZIP65 is shown in SEQ ID NO.1.

[0008] The second aspect of the present invention provides a 5'UTR of the rice gene OsbZIP65, which is the region of 86 bp before the start codon of OsbZIP65, and the sequence is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0009] The third aspect of the present invention provides a protein encoded by the rice gene OsbZIP65, wherein the amino acid sequence of the protein is shown in SEQ ID NO.4.

[0010] A fourth aspect of the present invention provides a biological material carrying the rice gene OsbZIP65, wherein the biological material comprises a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium.

[0011] In a fifth aspect of the present invention, a DNA fragment as described in any one of a) to e) below is provided as a gene regulating disease resistance in regulating plant disease resistance;

[0012] a) the DNA fragment shown in SEQ ID NO.1;

[0013] b) the DNA fragment shown in SEQ ID NO.2;

[0014] c) the DNA fragment shown in SEQ ID NO.3;

[0015] d) a DNA fragment encoding the amino acid sequence shown in SEQ ID NO.4;

[0016] e) a DNA fragment having 70% or more identity with the DNA fragment defined in a) or b) or c) or d), and encoding a protein that is functionally equivalent to the protein shown in SEQ ID NO.4.

[0017] In the above application, the plant disease resistance is enhanced by down-regulating the transcription or expression level of any one of the DNA fragments in a)-e).

[0018] Preferably, the plant is rice, and the disease resistance is resistance to sheath blight.

[0019] The sixth aspect of the present invention provides the use of the protein described in any one of 1) to 3) below in regulating plant disease resistance;

[0020] 1) A protein having an amino acid sequence as shown in SEQ ID NO.4;

[0021] 2) A protein having the same function as the protein shown in SEQ ID NO.4 obtained by replacing, deleting or inserting one, several or dozens of amino acids in the amino acid sequence shown in SEQ ID NO.4;

[0022] 3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.4.

[0023] In the above application, the disease resistance of the plant is improved by down-regulating the expression level and / or activity of the protein described in any one of 1) to 3).

[0024] Preferably, the plant is rice, and the disease resistance is resistance to sheath blight.

[0025] The seventh aspect of the present invention provides the use of a gene expression regulator capable of down-regulating the expression of any one of the DNA fragments described in a) to e) in plants for enhancing plant disease resistance.

[0026] Preferably, the gene expression regulator is selected from T-DNA or interfering RNA.

[0027] The eighth aspect of the present invention provides the use of a regulator capable of down-regulating the expression level and / or activity of any one of the proteins described in 1) to 3) for enhancing plant disease resistance.

[0028] A ninth aspect of the present invention provides a method for cultivating rice with improved disease resistance, the method comprising:

[0029] Down-regulating the expression of the rice gene OsbZIP65 shown in SEQ ID NO.1, and screening rice plants with improved disease resistance;

[0030] or down-regulating the expression of the protein encoded by the rice gene OsbZIP65 shown in SEQ ID NO.4, and screening out rice plants with improved disease resistance;

[0031] or hybridizing a variety carrying the SNP site A at position 32 in SEQ ID NO.3 to obtain a rice plant with improved disease resistance;

[0032] Alternatively, mutation or gene editing is used to create the SNP site at position 32 in SEQ ID NO.3 as A, and rice plants with improved disease resistance are screened.

[0033] In the above method, the method for down-regulating the expression of the rice gene OsbZIP65 includes: mutating or knocking out the entire or partial sequence of the gene shown in SEQ ID NO.1 in rice; or using interfering RNA to interfere with the expression of the gene shown in SEQ ID NO.1; or using a gene silencing system to silence the gene shown in SEQ ID NO.1.

[0034] Preferably, the mutation is induced by CRISPR / Cas9 or TELLEN technology or T-DNA insertion or EMS mutagenesis.

[0035] The tenth aspect of the present invention provides an application of a rice SNP site or a molecular marker containing the SNP site in a rice genome in screening, identifying or improving rice resistant to sheath blight, wherein the SNP site is rs16372715 (Os-Nipponbare-Reference-IRGSP-1.0 / MSU, https: / / riceome.hzau.edu.cn / ), which is the 32nd position in the sequence shown in SEQ ID NO.2 or SEQ ID NO.3, and the site is G or A. The rice with the SNP site A is rice resistant to sheath blight. The application of screening or identifying rice resistant to sheath blight can be achieved by detecting the SNP site or molecular marker; the application of improving rice resistant to sheath blight can be achieved by mutating the site of rice to A.

[0036] Beneficial effects of the present invention: The present invention provides for the first time the application of rice OsbZIP65 transcription factor gene in improving rice resistance to sheath blight. The function of rice OsbZIP65 as shown in SEQ ID NO.1 was studied by reverse genetics method, and it was found that OsbZIP65 negatively regulates rice resistance to sheath blight. The overexpression vector of OsbZIP65 gene was transformed into rice variety Zhonghua 11 by transgenic technology, and the resistance of OsbZIP65 overexpressing rice to sheath blight was significantly weakened. Specifically, by promoter analysis, a 86 bp base sequence located in the 5'UTR region of OsbZIP65 promoter was screened as the core region responsive to the induction of sheath blight pathogen YWK196, and there was differentiation of different haplotypes in this region. Therefore, the OsbZIP65 gene can be used to improve the disease resistance of rice, cultivate and create materials and production varieties resistant to rice sheath blight, and thus improve the disease resistance of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of the OsbZIP65 gene rice resistance to sheath blight in Example 1 are verified. (A) PCR identified 21 OsbZIP65 over-transgenic T0 positive plants; (B) Three different osbzip65 gene-edited T1 homozygous lines were identified; (C) The transcription level of the 21 OsbZIP65 over-transgenic T0 lines in Figure A was significantly increased by qRT-PCR detection; (D) The ZH11, OsbZIP65-OE, and osbzip65 materials were inoculated with sheath blight pathogen YWK196 by artificial embedding method, and the length of the lesions was recorded by taking photos; (E) The length of the lesions in Figure D was counted; (F) The genomic DNA of the stems of the diseased parts in Figure D was extracted, and the biomass of sheath blight pathogens in plant tissues was determined by real-time fluorescence quantitative method.

[0038] Figure 2The OsbZIP65 gene in Example 2 responds to the induction of Rhizoctonia solani YWK196. (A) OsbZIP65 is induced by Rhizoctonia solani; (B) The OsbZIP65 promoter can respond to the induction of Rhizoctonia solani and activate the expression of downstream reporter genes GFP and LUC (P 2H16 -GFP as positive control).

[0039] Figure 3 The 86 bp before the start codon of the 5'UTR region of the OsbZIP65 gene in Example 3 is the core region that responds to the induction of the sheath blight pathogen YWK196. (A) A series of truncated vectors at the 5' end of the OsbZIP65 promoter were constructed, with GFP as the reporter gene; (B) PDA medium without inoculation of sheath blight pathogen was used as a negative control, and it was found that the GFP fluorescence intensity of the four truncated vectors had no obvious change; (C) +229bp to +711bp was further truncated into 5 mutually exclusive segments, and after inoculation of sheath blight pathogen, it was found that only +625bp to +711bp could respond to its induction.

[0040] Figure 4 A rare SNP site in the 5'UTR region of the OsbZIP65 gene in Example 4 is involved in the resistance of rice to sheath blight. (A) SNP site variation in the 5'UTR region of the OsbZIP65 gene and its frequency of occurrence in 3024 rice materials, among which rs16372715 only appeared in individual materials and was a rare SNP site (Os-Nipponbare-Reference-IRGSP-1.0 / MSU); (B) In ZH11 material, the relative expression level of OsbZIP65 was upregulated with the increase of the time of inoculation with Rhizoctonia solani, while in W029 material, the relative expression level of OsbZIP65 lost the ability to upregulate with the time of inoculation with Rhizoctonia solani; (C) After the rare SNP site in (A) was mutated, it was found that the ability of the OsbZIP65 promoter to respond to the induction of Rhizoctonia solani was significantly reduced; (D) The artificial embedding method was used to measure and count the lesion length of different haplotype materials after inoculation with Rhizoctonia solani YWK196; (E) The lesion length in D was photographed and counted. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0042] The present invention includes DNA fragments homologous to the OsbZIP65 gene, as long as the proteins they encode are functionally equivalent to the protein shown in SEQ ID NO.4. "Functionally equivalent to the protein shown in SEQ ID NO.4" as referred to herein means that the protein encoded by the target DNA fragment is the same or similar to the protein shown in SEQ ID NO.4 in the present invention in terms of biological function and physiological and biochemical characteristics. The typical biological function of the protein shown in SEQ ID NO.4 is to regulate rice disease resistance. By downregulating the expression amount and / or activity of the protein shown in SEQ ID NO.4, rice disease resistance can be enhanced.

[0043] These DNA fragments homologous to the OsbZIP65 gene include alleles, homologous genes, mutant genes and derived genes corresponding to the nucleotide sequence (SEQ ID NO.1) of the present invention; the proteins encoded by them are similar to the protein shown in SEQ ID NO.4 of the present invention, or there are one, several or dozens of amino acid substitutions, deletions or insertions, all of which belong to the content of the present invention.

[0044] Those skilled in the art can easily mutate the nucleotide sequence of the OsbZIP65 gene of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 70% or higher identity with the nucleotide sequence of the OsbZIP65 gene of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the same function as the protein shown in SEQ ID NO.4.

[0045] From the application perspective, by regulating the expression of the rice OsbZIP65 gene (SEQ ID NO.1) of the present invention, it is very likely to create a phenotype with enhanced disease resistance. The "regulated expression" referred to here includes three levels: DNA transcription level, cDNA translation level and protein product activity, including upregulation and downregulation. For example, according to the present invention, the DNA fragment that can activate or improve the transcription level or translation level or protein activity of the gene is mined from the rice OsbZIP65 gene (SEQ ID NO.1) or its homologous gene. For another example, for the rice OsbZIP65 gene (SEQ ID NO.1) or its homologous gene, there are microRNA molecules (microRNA, miRNA), interfering small RNA (small interfering RNA, siRNA) or artificial miRNA (artificial microRNA, amiRNA) that act thereon; by rationally controlling the expression of specific small RNA molecules (amiRNA, miRNA and siRNA), the accumulation of the OsbZIP65 gene or its homologous gene mRNA is downregulated to obtain a phenotype with enhanced disease resistance.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0047] The present invention is further defined in the following examples. Based on the above description and these examples, those skilled in the art can determine the essential features of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions. Unless otherwise specified, the present invention adopts the prior art in the art.

[0048] Example 1: OsbZIP65 negatively regulates rice resistance to sheath blight

[0049] 1. Isolation and cloning of OsbZIP65 gene from rice varieties

[0050] Adopting OMEGA's EZNA RNA from rice Zhonghua 11 was extracted using RNA Kit, and cDNA was obtained by reverse transcription using HiFiScript cDNA Synthesis Kit of Kangwei Century Company. Using the cDNA as a template, the DNA fragment of OsbZIP65 gene was amplified by PCR technology using primers (5′-ATGACCCTGTCAGGTGGGACC-3′) and primers (5′-CTAGCACTGGTACAGAAACTG-3′). The PCR reaction program was pre-denaturation at 94℃ for 3min, denaturation at 94℃ for 30s, annealing at 56℃ for 30s, extension at 72℃ for 30s, reaction for 35 cycles, and post-extension at 72℃ for 5min. The obtained gene was named OsbZIP65 gene, and its nucleotide sequence is shown in SEQ NO.1. The gene encodes a protein composed of 180 amino acids, and its amino acid sequence is shown in SEQ NO.4.

[0051] 2. Construction of OsbZIP65 overexpression and gene-edited rice lines

[0052] The vector used to construct the OsbZIP65 overexpression rice strain is pCXUN-Myc, which is an Agrobacterium-mediated genetic transformation vector carrying the maize ubiquitin constitutive overexpression promoter.

[0053] Using rice Zhonghua 11 cDNA as a template, the gene fragment was amplified using forward primer (5′-ccccctttcgccaggggtaccATGACCCTGTCAGGTGGGACC-3′) and reverse primer (5′-tacgaattcgagctcggtaccCTAGCACTGGTACAGAAACTGAT-3′). The PCR reaction procedure was as follows: pre-denaturation at 94℃ for 5min, denaturation at 94℃ for 40s, annealing at 56℃ for 30s, extension at 72℃ for 1min, reaction for 35 cycles, and post-extension at 72℃ for 5min to obtain the OsbZIP65 gene fragment. The genetic transformation vector pCXUN-Myc carrying the maize ubiquitin promoter was digested with Kpn I, and the digestion product was purified. The OsbZIP65 gene fragment and the digested vector were used for homologous recombination and transformation, and positive clones were screened by PCR; the constructed overexpression vector was named 35S::OsbZIP65-Myc.

[0054] The overexpression vector was introduced into the susceptible rice variety Zhonghua 11 by Agrobacterium-mediated genetic transformation, and Wuhan Boyuan Biotechnology Co., Ltd. was commissioned to obtain the transgenic material named OsbZIP65-OE. A total of 21 independent transformed plants were obtained in this example.

[0055] The preparation of the gene-edited rice of OsbZIP65 was commissioned by Biogen Biotech Co., Ltd.

[0056] 3. Verification of the disease resistance function of OsbZIP65 gene

[0057] A total of 21 OsbZIP65 overexpressing transgenic T0 lines were obtained by positive identification at the DNA and RNA levels using pCXUN-Myc vector fragment and gene fragment specific primers. Figure 1 A, C). Through sequencing and identification, a total of three homozygous OsbZIP65 gene-edited T0 rice lines were obtained. Figure 1 As shown in B, the osbzip65-1 strain lacked 6 bases in the target region and terminated translation prematurely at the 537th base; the osbzip65-2 strain lacked 14 bases in the target region and terminated translation prematurely at the 69th base; the osbzip65-3 strain had 2 bases inserted in the target region, resulting in premature termination of translation at the 39th base.

[0058] The T1 rice plants were inoculated with the sheath blight pathogen YWK196, and the length of the lesions was measured and counted. The results showed that the gene-edited rice with the OsbZIP65 gene had significantly enhanced resistance to sheath blight ( Figure 1 DF), the overexpression plants with significantly increased expression levels showed significantly reduced resistance to sheath blight, indicating that the encoded product of the OsbZIP65 gene plays a negative regulatory role in the disease resistance of rice to sheath blight. It can be seen that inhibiting the expression of the OsbZIP65 gene can give plants a disease resistance response to diseases caused by Rhizoctonia solani, thereby improving the resistance of rice to fungal diseases.

[0059] Example 2: OsbZIP65 is induced to express by Rhizoctonia solani

[0060] 1. Expression pattern of OsbZIP65 induced by Rhizoctonia solani

[0061] In order to confirm that the OsbZIP65 gene is involved in the regulation of disease resistance, this example uses quantitative RT-PCR technology to analyze the expression pattern of the OsbZIP65 gene in Zhonghua 11 after induction by Rhizoctonia solani YWK196.

[0062] Punch the preserved YWK196 strain culture medium, pick out the punched bacterial cake with an inoculation needle, and place the side with hyphae upside down in the center of the PDA culture medium and seal it with a sealing film. Culture in a constant temperature incubator at 28°C for 2 days until the hyphae cover the 90mm culture dish. Use a clean 0.7cm puncher to punch holes in the outermost circle of the culture medium covered with hyphae and use it for the inoculation test. Then take two filter papers and spread them in a square culture dish, and moisten the filter papers with an appropriate amount of sterilized water. Cut the leaves of 8-week-old rice with clean scissors to ensure the uniformity of the inoculation position. Place the bacterial cake with hyphae on the cut leaves. Culture in a 28°C culture room. Take the inoculated leaves at 0h, 4h, 8h, 12h, 24h and 36h after inoculation.

[0063] The expression difference of OsbZIP65 gene at different time points after inoculation was analyzed by quantitative RT-PCR using Bio-Rad quantitative PCR instrument and SYBR Green fluorescent embedding dye (Kangwei Century). The specific PCR primers of OsbZIP65 gene were: 5′-GACCTTACCTCACAGGTGAAC-3′, 5′-ATCTCACGGAGCGCTGATAG-3′. Parallel qRT-PCR of rice actin was used as the normalization internal reference control.

[0064] The analysis results showed that after inoculation with Rhizoctonia solani, the OsbZIP65 gene in the susceptible material Zhonghua 11 was induced to express ( Figure 2 A), indicating that the OsbZIP65 gene is involved in the response to sheath blight.

[0065] 2. OsbZIP65 promoter responds to induction by Rhizoctonia solani

[0066] The inventor team previously developed a transient expression reporter gene system of Nicotiana benthamiana induced by sheath blight inoculation to study the analysis technology of sheath blight-induced promoter (Li N, Wei S, Chen J, Yang F, Kong L, Chen C, Ding X*, Chu Z*. OsASR2 regulates the expression of a defense-related gene Os2H16 by targeting the GT-1 cis-element. Plant Biotechnology Journal, 2018, 16 (3): 771-783.). Referring to the above analysis technology, in this study, the 1988bp promoter sequence (SEQ ID NO.5) of the 5'UTR of the OsbZIP65 gene was cloned into the GFP reporter system vector pCXGFP to obtain the pCXGFP D0 plasmid, which was transformed into Agrobacterium to infect Nicotiana benthamiana leaves for 48 hours and then inoculated with sheath blight YWK196. After 24 hours of inoculation, the sheath blight was able to activate the expression of the downstream GFP reporter gene, causing the tobacco cells to excite green fluorescence at 488nm ( Figure 2 B). The promoter sequence was connected to the luciferase reporter vector pGREEN-0800-LUC, and similar transcriptional activation effects were obtained. The above experiments prove that the promoter sequence containing the 5'UTR of the OsbZIP65 gene can respond to the induction of Rhizoctonia solani and activate the expression of downstream reporter genes ( Figure 2 B).

[0067] Example 3: The 86 bp before the start codon of the 5'UTR region of the OsbZIP65 gene is the core region that responds to the induction of the sheath blight pathogen YWK196

[0068] In order to identify the core cis-acting elements of the OsbZIP65 promoter and 5'UTR region that respond to the induction of sheath blight, this example continued to construct a series of truncated vectors (pCXGFP D1, pCXGFP D2, pCXGFP D3, and pCXGFP D4) at the 5' end of the OsbZIP65 promoter. The constructed truncated vectors were transformed into Agrobacterium to infect Nicotiana benthamiana leaves, and then inoculated with sheath blight YWK196 for 48 hours. The uninoculated strain was used as a control. After 24 hours, the tobacco cells were observed under 488nm excitation light to see whether they emitted green fluorescence. The results showed that the GFP fluorescence observed in the leaves infected with the vectors containing the deletion structures of the regions from -864 to +711 (pCXGFP D1, the transcription start site is marked as +1), -270 to +711 (pCXGFP D2), +34 to +711 (pCXGFP D3), and +229 to +711 (pCXGFP D4) was similar to that of the pCXGFP D1. The leaves infected on D0 were almost identical with no significant changes, indicating that the segment from +229 to +711 has complete pathogen-induced promoter function ( Figure 3 AB).

[0069] In order to identify the core cis-acting element, the +229 to +711 segment was further truncated into five parts, marked as R1 (+229 to +369), R2 (+350 to +446), R3 (+427 to +551), R4 (+529 to +648) and R5 (+626 to +711), respectively, and connected to the pGREEN-0800-LUC vector to construct pGREEN R1, R2, R3, R4, and R5, respectively, and then transferred into Nicotiana benthamiana leaves. The pGREEN-0800-LUC vector was used as a negative control for transient expression in Nicotiana benthamiana leaves. Only the Nicotiana benthamiana leaves transferred with pGREEN R5 induced the expression of the reporter gene after inoculation with the sheath blight pathogen YWK196, indicating that the +626 to +711 interval (SEQ ID NO.2) is the core region of the OsbZIP65 promoter in response to YWK196 induction ( Figure 3 C).

[0070] Example 4: Haplotype analysis and key SNP identification in the OsbZIP65 promoter region

[0071] The above promoter deletion experiment proved that the 5'UTR region of OsbZIP65 gene +626 to +711 is the core region of OsbZIP65 promoter in response to YWK196 induction. Therefore, in this example, 3024 Asian rice materials were used as a population to divide the haplotype of the 150bp (+561 to +711) sequence upstream of the ATG of the OsbZIP65 gene. A total of 8 SNP sites were detected, and ZH11, Heibiao (CH1004), Gaoliqi The 150 bp sequence upstream of the ATG of the OsbZIP65 gene in the u (CH1032), PeiC122 (C051), Guihuahuang (C052) and Ai_Chueh_Ta_Pai_Ku (W029) was sequenced and compared, and it was found that one SNP variation GA was shared by C052 and W029 and did not appear in other varieties. At the same time, the frequency of this SNP variation in 3024 materials was only 2.74%, which was a rare mutation ( Figure 4 A). Further detection of the transcription level of OsbZIP65 in Zhonghua11 and W029 after inoculation with YWK196 revealed that in Zhonghua11, OsbZIP65 was upregulated by YWK196; whereas in W029, YWK196 could not induce OsbZIP65 upregulation, suggesting that the mutation of this site plays a key role in the regulation of OsbZIP65 resistance to rice sheath blight ( Figure 4 B).

[0072] The DNA sequence of the mutation (SEQ ID NO.3) at this site was connected to the pGREEN-0800-LUC vector to construct pGREENR5 GAmutant. The mutant was transiently expressed in leaves of Nicotiana benthamiana without inoculation of YWK196 as a negative control. Compared with R5, R5GAmutant did not induce reporter gene expression after inoculation of sheath blight pathogen YWK196, indicating that this site plays a key role in the regulation of OsbZIP65 in rice resistance to sheath blight ( Figure 4 C). The ZH11, CH1004, CH1032, C051, C052 and W029 materials at the heading stage were further inoculated with YWK196 by artificial embedding method. The lesion length was counted 5 days after inoculation. The results showed that the average lesion length of ZH11 was 6.06 cm, and the average lesion length of C052 and W029 was 4.77 cm and 4.53 cm, respectively, which were significantly shorter than that of ZH11, showing enhanced disease resistance, indicating that the variation of this site is related to the loss of pathogen-induced expression ability of OsbZIP65 gene and rice resistance to sheath blight ( Figure 4 DE).

Claims

1. A rice gene OsbZIP65 that enhances disease susceptibility, characterized in that: The sequence of the rice gene OsbZIP65 is shown as SEQ ID NO.

1.

2. A 5'UTR of the rice gene OsbZIP65 according to claim 1, characterized in that: The sequence of the 5'UTR is shown as SEQ ID NO.2 or SEQ ID NO.

3.

3. A protein encoded by the rice gene OsbZIP65 according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

4.

4. A biological material carrying the rice gene OsbZIP65 according to claim 1, characterized in that: The biological materials include recombinant expression vectors, transgenic cell lines, and genetically engineered bacteria.

5. Use of the DNA fragment described in any one of a) to e) below or the protein described in any one of 1) to 3) below in regulating plant disease resistance; a) the DNA fragment shown in SEQ ID NO.1; b) the DNA fragment shown in SEQ ID NO.2; c) the DNA fragment shown in SEQ ID NO.3; d) a DNA fragment encoding the amino acid sequence shown in SEQ ID NO.4; e) a DNA fragment having 70% or more identity with the DNA fragment defined in a) or b) or c) or d), and encoding a protein that is functionally equivalent to the protein shown in SEQ ID NO.4; 1) A protein having an amino acid sequence as shown in SEQ ID NO.4; 2) A protein having the same function as the protein shown in SEQ ID NO.4 obtained by replacing, deleting or inserting one, several or dozens of amino acids in the amino acid sequence shown in SEQ ID NO.4; 3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.

4.

6. The use according to claim 5, characterized in that: The plant disease resistance is enhanced by down-regulating the transcription or expression level of any one of the DNA fragments in a)-e); or by down-regulating the expression amount and / or activity of any one of the proteins in 1)-3).

7. Use of a gene expression regulator capable of down-regulating the expression of the DNA fragment described in any one of a) to e) of claim 5 in a plant or a regulator capable of down-regulating the expression level and / or activity of the protein described in any one of 1) to 3) of claim 6 in improving plant disease resistance.

8. A method for cultivating rice with improved disease resistance, characterized in that: The method comprises: Down-regulating the expression of the rice gene OsbZIP65 shown in SEQ ID NO.1, and screening rice plants with improved disease resistance; or down-regulating the expression level and / or activity of the protein shown in SEQ ID NO.4 to screen out rice plants with improved disease resistance; or hybridizing a variety carrying the SNP site A at position 32 in SEQ ID NO.3 to obtain a rice plant with improved disease resistance; Alternatively, mutation or gene editing is used to create the SNP site at position 32 in SEQ ID NO.3 as A, and rice plants with improved disease resistance are screened.

9. The method according to claim 8, characterized in that The method for down-regulating the expression of rice gene OsbZIP65 includes: mutating or knocking out the entire or partial sequence of the gene shown in SEQ ID NO.1 in rice; or using interfering RNA to interfere with the expression of the gene shown in SEQ ID NO.1; or using a gene silencing system to silence the gene shown in SEQ ID NO.

1.

10. Use of a rice SNP site or a molecular marker containing the SNP site in a rice genome in screening, identifying or improving rice resistant to sheath blight, characterized in that: The SNP site is rs16372715, which is G or A; the rice with the SNP site being A is rice resistant to sheath blight.

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