Rice sheath blight resistance related gene osbzip65 and application thereof

Abstract

The application discloses a rice disease resistance related gene OsbZIP65 and application thereof, and belongs to the technical field of plant genetic engineering.The application finds that OsbZIP65 negatively regulates the resistance of rice to sheath blight by constructing an overexpression rice strain and a gene editing rice strain of OsbZIP65 shown in SEQ ID NO.1.The application further finds that a 86bp base sequence in the 5'UTR region of the OsbZIP65 promoter is a core region responding to YWK196 induction of the sheath blight bacteria, the region is differentiated in different haplotypes, and the haplotype is related to the low pathogen-induced expression amount of OsbZIP65 and the enhanced resistance to the sheath blight.Therefore, the disease resistance of rice can be improved by genetic manipulation or haplotype hybridization of the OsbZIP65 gene, and the materials and products of the rice sheath blight resistance can be bred and created, and the disease resistance of rice can be further improved.

Description

Technical Field

[0001] The present 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 staple crops, crucial for supporting population growth and meeting a wide range of nutritional needs. As the staple food for more than half the world's population, the sustainability and stability of rice production are critical to global food security.

[0003] Throughout the growing season, rice is constantly threatened by pathogenic fungi, bacteria, nematodes, oomycetes, viruses, and herbivorous insects, resulting in annual yield losses of 10% to 30%. Over 100 rice diseases have been identified, and the three most destructive are rice blast, bacterial leaf blight, and sheath blight. These are caused by the semi-biotrophic fungus Magnaporthe oryzae, the semi-biotrophic bacterium Xanthomonas oryzae pv. oryzae, and the necrotrophic fungus Rhizoctonia solani, respectively. Rice sheath blight, primarily caused by Rhizoctonia solani, is one of the most widespread soil-borne fungal diseases worldwide. Rice sheath blight can occur from the seedling stage to the heading stage of rice, with the highest incidence rate around the heading stage. It mainly harms the rice leaf sheaths and leaves. In severe cases, it can invade the stem and spread to the ear. In the early stage of the disease, the leaf sheath appears as dark green water-soaked lesions, which later expand and merge into large irregular or cloud-like lesions. When conditions are suitable, the lesion edges are dark green and the center is gray-green. The lesion area expands rapidly and can develop to the entire rice plant including the ear. In severe cases, the entire plant will die. When the weather is dry, the lesion edges are brown and the center is straw yellow to grayish white. The diseased leaf sheaths wither and turn yellow due to tissue necrosis. The infected rice cannot head normally. Even if it heads, the spread of lesions to the ear will lead to an increase in the amount of barren grains and a decrease in yield.

[0004] To cope with environmental changes, plants have evolved complex signaling pathways typically composed of receptors, secondary signals, phytohormones, and signal transduction modules. Transcription factors are crucial components of biotic and abiotic stress signaling pathways, regulating downstream gene expression by binding to distinct cis-elements. A typical transcription factor is generally composed of four components: a DNA binding domain, a transcriptional regulatory domain, a nuclear localization signal peptide, and an oligomerization site. The combined action of these domains determines the temporal, spatial, and mode of transcriptional regulation. The bZIP family of transcription factors is widely involved in regulating plant defense responses to abiotic stresses. Studies have shown that the bZIP-type transcription factor APIP5 can form homodimers and interact with the rice blast effector AvrPiz-t in the cytoplasm, inhibiting its transcriptional activity and protein accumulation during the necrotrophic stage. Notably, APIP5 can undergo nucleocytoplasmic shuttling and, as an RNA-binding protein, regulates mRNA degradation of the cell death and defense-related genes OsLSD1 and OsRac1, acting at the posttranscriptional level. Furthermore, OsbZIP62 and OsbZIP1 regulate rice resistance to bacterial blight and 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 containing the complete coding region and 5'UTR of the corresponding related gene, and their application. The present invention confirmed through functional verification that the 86bp before the start codon of the 5'UTR region of the OsbZIP65 gene responds to induction by the sheath blight pathogen YWK196, thereby enhancing rice susceptibility to disease. Bioinformatics analysis revealed that there are 8 SNP sites with non-synonymous mutations in this segment, and a rare SNP site with an occurrence frequency of less than 5% was identified in 3024 rice materials. This haplotype reduces the expression level of OsbZIP65 after inoculation with the sheath blight pathogen, thereby enhancing rice resistance to sheath blight. Utilizing 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 solutions:

[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] In a second aspect, the present invention provides a 5'UTR of the rice gene OsbZIP65, which is the 86 bp region 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, the amino acid sequence of which 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 includes a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium.

[0011] In a fifth aspect, the present invention provides the use of the DNA fragment described in any one of a) to e) below 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, plant disease resistance is enhanced by down-regulating the transcription or expression level of any one of the DNA fragments a) to 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 in enhancing plant disease resistance.

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

[0027] An eighth aspect of the present invention provides use of a regulator capable of downregulating the expression level and / or activity of any one of the proteins described in 1) to 3) in 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 to screen for 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 to screen for rice plants with improved disease resistance;

[0031] or hybridizing with 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 may be used to create an A at the 32nd SNP site in SEQ ID NO. 3, and rice plants with improved disease resistance may be screened.

[0033] In the above method, the method for downregulating 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] In a tenth aspect of the present invention, a rice SNP site or a molecular marker containing the SNP site in the rice genome is provided for use in screening, identifying, or improving rice resistant to sheath blight. The SNP site is rs16372715 (Os-Nipponbare-Reference-IRGSP-1.0 / MSU, https: / / riceome.hzau.edu.cn / ), which is position 32 in the sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3, and the site is G or A. Rice with the SNP site A is sheath blight-resistant rice. The application of screening or identifying sheath blight-resistant rice can be achieved by detecting the SNP site or molecular marker; the application of improving sheath blight-resistant rice can be achieved by mutating the site in rice to A.

[0036] Beneficial effects of the present invention: The present invention provides, for the first time, the use of the rice OsbZIP65 transcription factor gene for improving rice resistance to sheath blight. Reverse genetics methods were used to investigate the function of rice OsbZIP65, as shown in SEQ ID NO. 1, and it was found that OsbZIP65 negatively regulates rice resistance to sheath blight. Transgenic technology was used to transform the rice variety Zhonghua 11 with an overexpression vector for the OsbZIP65 gene. The resulting OsbZIP65-overexpressing rice exhibited significantly reduced resistance to sheath blight. Specifically, promoter analysis identified an 86-bp base sequence located in the 5'UTR region of the OsbZIP65 promoter as a core region responsive to induction by the sheath blight pathogen YWK196, with differentiation of different haplotypes within this region. Therefore, the OsbZIP65 gene can be used to improve rice disease resistance, cultivate and create materials and varieties resistant to sheath blight, and thereby enhance rice disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Results of the OsbZIP65 rice resistance test to sheath blight in Example 1 are shown. (A) PCR identified 21 OsbZIP65 overexpressing transgenic T0-generation positive plants; (B) Three different osbzip65 gene-edited T1-generation homozygous lines were identified; (C) qRT-PCR analysis of the 21 OsbZIP65 overexpressing transgenic T0-generation lines in Figure A showed significantly increased transcription levels; (D) ZH11, OsbZIP65-OE, and osbzip65 materials were inoculated with the sheath blight pathogen YWK196 using the artificial embedding method, and lesion lengths were photographed and recorded; (E) Lesion lengths in Figure D were statistically analyzed; (F) Genomic DNA was extracted from the diseased stems in Figure D, and the sheath blight biomass in plant tissues was determined using real-time fluorescence quantification.

[0038] Figure 2The OsbZIP65 gene in Example 2 responds to induction by Rhizoctonia solani YWK196. (A) OsbZIP65 is induced by Rhizoctonia solani; (B) The OsbZIP65 promoter can respond to induction by Rhizoctonia solani and activate the expression of downstream reporter genes GFP and LUC (P 2H16 -GFP as a 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 induction by the sheath blight pathogen YWK196. (A) A series of truncated vectors at the 5' end of the OsbZIP65 promoter were constructed, using GFP as a reporter gene. (B) Using PDA medium without sheath blight inoculation as a negative control, no significant change in GFP fluorescence intensity was observed in the four truncated vectors. (C) Further truncations of the segments from +229 bp to +711 bp were performed into five mutually exclusive segments. After inoculation with sheath blight, only the segments from +625 bp to +711 bp responded to 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 accessions. rs16372715, a rare SNP site (Os-Nipponbare-Reference-IRGSP-1.0 / MSU), only occurs in a few accessions. (B) In ZH11, the relative expression of OsbZIP65 increases with the time of inoculation with R. solani. However, in W029, the relative expression of OsbZIP65 loses its ability to increase with the time of inoculation with R. solani. (C) After mutation of the rare SNP site in (A), the ability of the OsbZIP65 promoter to respond to R. solani induction was significantly reduced. (D) The lesion length of different haplotypes after inoculation with R. solani YWK196 was measured and statistically analyzed using the artificial embedding method. (E) The lesion length in D was photographed and statistically analyzed. DETAILED DESCRIPTION

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

[0042] The present invention encompasses DNA fragments homologous to the OsbZIP65 gene, as long as they encode proteins that are functionally equivalent to the protein set forth in SEQ ID NO. 4. As used herein, "functionally equivalent to the protein set forth in SEQ ID NO. 4" means that the protein encoded by the target DNA fragment is identical or similar to the protein set forth in SEQ ID NO. 4 in terms of biological function, physiological and biochemical characteristics, and other characteristics. A typical biological function of the protein set forth in SEQ ID NO. 4 is regulating disease resistance in rice. By downregulating the expression and / or activity of the protein set forth 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 derivative genes corresponding to the nucleotide sequence of the present invention (SEQ ID NO. 1); the proteins they encode are similar to the protein shown in SEQ ID NO. 4 of the present invention, or have one, several, or dozens of amino acid substitutions, deletions, or insertions, all of which fall within the scope of the present invention.

[0044] Those skilled in the art can readily mutate the nucleotide sequence of the OsbZIP65 gene of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 70% or greater identity with the nucleotide sequence of the OsbZIP65 gene of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as the encoded protein has the same function as the protein set forth in SEQ ID NO. 4.

[0045] From an 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, a DNA fragment that can activate or increase the transcription level, translation level, or protein activity of the rice OsbZIP65 gene (SEQ ID NO.1) or its homologous gene is mined. For another example, for the rice OsbZIP65 gene (SEQ ID NO.1) or its homologous gene, there are microRNA molecules (microRNA, miRNA), small interfering RNA (small interfering RNA, siRNA), or artificial miRNA (artificial microRNA, amiRNA) that interact with it; 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, thereby obtaining 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 with reference to 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 will be able to ascertain the essential features of the present invention and, without departing from the spirit and scope of the present invention, will be able to make various changes and modifications to the present invention to adapt it to various uses and conditions. Unless otherwise noted, the present invention utilizes prior art in the art.

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

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

[0050] Using OMEGA's EZNA RNA from rice Zhonghua 11 was extracted using an RNA Kit and reverse transcribed using the HiFiScript cDNA Synthesis Kit from Kangwei Century to generate cDNA. Using this cDNA as a template, PCR was used to amplify the DNA fragment of the OsbZIP65 gene using primers (5′-ATGACCCTGTCAGGTGGGACC-3′) and (5′-CTAGCACTGGTACAGAAACTG-3′). The PCR reaction procedure included 35 cycles of initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. The resulting gene was named OsbZIP65, and its nucleotide sequence is shown in SEQ NO. 1. It encodes a 180-amino acid protein, the amino acid sequence of which 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 line is pCXUN-Myc, 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 a forward primer (5′-ccccctttcgccaggggtaccATGACCCTGTCAGGTGGGACC-3′) and a reverse primer (5′-tacgaattcgagctcggtaccCTAGCACTGGTACAGAAACTGAT-3′). The PCR reaction protocol was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 40 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, and post-extension at 72°C for 5 min. The OsbZIP65 gene fragment was obtained. The genetic transformation vector pCXUN-Myc, carrying the maize ubiquitin promoter, was digested with Kpn I and the digested product was purified. Homologous recombination and transformation were performed using the OsbZIP65 gene fragment and the digested vector. 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 using Agrobacterium-mediated genetic transformation, commissioned by Wuhan Boyuan Biotechnology Co., Ltd. The resulting transgenic material was named OsbZIP65-OE. A total of 21 independently transformed plants were obtained in this example.

[0055] The gene-edited rice of OsbZIP65 was commissioned to be prepared by Baige Biotechnology Co., Ltd.

[0056] 3. Verification of the disease resistance function of the 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 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 generation rice was inoculated with the sheath blight pathogen YWK196, and the lesion length 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), overexpressing plants with significantly elevated expression levels showed significantly reduced resistance to sheath blight, indicating that the product encoded by the OsbZIP65 gene plays a negative regulatory role in rice's resistance to sheath blight. This suggests that inhibiting OsbZIP65 expression can confer resistance to Rhizoctonia solani, thereby improving rice's resistance to fungal diseases.

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

[0060] 1. OsbZIP65 expression pattern induced by Rhizoctonia solani

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

[0062] Punch a hole through the stored culture medium of the YWK196 strain. Use an inoculating needle to select the punched bacterial cake and place it upside down, with the mycelium attached, in the center of the PDA culture medium. Seal with parafilm. Incubate in a 28°C incubator for two days, until the mycelium completely covers a 90 mm Petri dish. Use a clean 0.7 cm hole puncher to punch holes in the outermost circle of the culture medium covered with mycelium for inoculation. Place two pieces of filter paper in a square Petri dish and moisten the filter paper with sterile water. Use clean scissors to cut leaves from 8-week-old rice plants, ensuring uniform inoculation. Place the bacterial cake with mycelium attached on the cut leaves. Incubate in a 28°C incubator. Inoculate leaves at 0, 4, 8, 12, 24, and 36 hours after inoculation.

[0063] Quantitative RT-PCR using a Bio-Rad quantitative PCR instrument and SYBR Green fluorescent intercalating dye (Congwei Century) was used to analyze differential expression of the OsbZIP65 gene at different time points after inoculation. The OsbZIP65 gene-specific PCR primers were: 5′-GACCTTACCTCACAGGTGAAC-3′, 5′-ATCTCACGGAGCGCTGATAG-3′. A parallel qRT-PCR assay for rice actin was used as an internal control for normalization.

[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 inventors' team previously developed a transient expression reporter gene system in Nicotiana benthamiana induced by sheath blight inoculation to study the analysis technology of sheath blight-induced promoters (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. The plasmid was transformed into Agrobacterium and infected with Nicotiana benthamiana leaves for 48 hours. After inoculation with sheath blight YWK196, 24 hours after inoculation, the sheath blight bacteria were able to activate the expression of the downstream GFP reporter gene, causing the tobacco cells to emit green fluorescence at 488nm ( Figure 2 B). Similar transcriptional activation effects were obtained by ligating the promoter sequence into the luciferase reporter vector pGREEN-0800-LUC. The above experiments demonstrated 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 the downstream reporter gene ( 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 sheath blight induction, 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 with Agrobacterium to infect Nicotiana benthamiana leaves 48 hours after inoculation with sheath blight pathogen YWK196. The uninoculated strain was used as a control. After 24 hours, the tobacco cells were observed under 488 nm excitation light to see whether they emitted green fluorescence. The results showed that the GFP fluorescence observed in leaves infected with vectors containing 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 pCXGFP D1. The leaves infected on D0 were almost identical with no significant changes, indicating that the +229 to +711 segment has complete pathogen-induced promoter function ( Figure 3 AB).

[0069] To identify the core cis-acting element, the +229 to +711 segment was further truncated into five parts, labeled R1 (+229 to +369), R2 (+350 to +446), R3 (+427 to +551), R4 (+529 to +648), and R5 (+626 to +711), respectively. These parts were ligated into the pGREEN-0800-LUC vector to construct pGREEN R1, R2, R3, R4, and R5, respectively, and then transformed into Nicotiana benthamiana leaves. The pGREEN-0800-LUC vector was used as a negative control for transient expression in Nicotiana benthamiana leaves. Only Nicotiana benthamiana leaves transformed with pGREEN R5 induced reporter gene expression 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 perform haplotype division on the 150bp (+561 to +711) sequence upstream of the ATG of the OsbZIP65 gene. A total of 8 SNP sites were detected, including ZH11, Heibiao (CH1004), Gaoliqi The 150bp upstream ATG sequence of the OsbZIP65 gene in 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 is a rare mutation ( Figure 4 A). Further testing of the transcriptional level of OsbZIP65 in Zhonghua 11 and W029 after inoculation with YWK196 revealed that in Zhonghua 11, OsbZIP65 was upregulated by YWK196; whereas in W029, YWK196 could not induce upregulation of OsbZIP65 expression, suggesting that this site variation 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. Transient expression in Nicotiana benthamiana leaves without inoculation with YWK196 was used as a negative control. Compared with R5, R5GAmutant did not induce reporter gene expression after inoculation with the 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). ZH11, CH1004, CH1032, C051, C052, and W029 were further inoculated with YWK196 using the artificial embedding method at the heading stage. The lesion length was counted 5 days after inoculation. The results showed that the average lesion length of ZH11 was 6.06 cm, while the average lesion lengths of C052 and W029 were 4.77 cm and 4.53 cm, respectively. These were significantly shorter than those of ZH11, indicating enhanced disease resistance. This suggests that the mutation at this site is associated with the loss of pathogen-induced expression ability of the OsbZIP65 gene and rice resistance to sheath blight ( Figure 4 DE).

Claims

1. A rice gene OsbZIP65 The DNA fragment of 5'UTR is characterized by The sequence of the DNA fragment of the 5'UTR is shown as SEQ ID NO.2 or SEQ ID NO.

3.

2. Use of the DNA fragment according to claim 1 in regulating resistance to rice sheath blight.

3. The use according to claim 2, characterized in that By down-regulating the transcription or expression level of the DNA fragment according to claim 1, the resistance of rice to sheath blight is enhanced.

4. A method for cultivating rice with improved resistance to sheath blight, characterized in that: The method comprises: A rice plant with improved resistance to sheath blight is obtained by hybridizing a variety carrying the SNP site A at position 32 in SEQ ID NO. 3; Alternatively, mutation or gene editing may be used to create an SNP site A at position 32 in SEQ ID NO. 3, and rice plants with improved resistance to sheath blight may be screened.

5. A method for screening, identifying or improving rice resistant to sheath blight disease using a rice SNP site or a molecular marker containing the SNP site in a rice genome, wherein: The SNP site is position 32 in the sequence shown in SEQ ID NO. 3, and the site is G or A; the rice with the SNP site being A is resistant to sheath blight.

Images