Use of fsd2 in modulating resistance to rice sheath blight and white leaf blast disease

Abstract

This invention provides the application of FSD2 in regulating resistance to rice sheath blight and bacterial leaf blight, belonging to the field of gene breeding technology. This invention provides... FSD2 The application of genes and / or encoded proteins in regulating resistance in rice to infectious diseases caused by pathogenic microorganisms, the aforementioned FSD2 Genes are involved in regulating rice resistance to sheath blight and bacterial leaf blight. This invention obtains these genes through genetic transformation. FSD2 Overexpressing plants, compared with wild-type plants, showed that overexpressing plants... FSD2 The gene expression level was significantly higher than that of the wild type; after inoculation with *Rhizoctonia solani* and *Bacterium oxysporum*, the overexpressing plants showed significantly enhanced resistance to *Rhizoctonia solani* and *Bacterium oxysporum*. This invention provides a theoretical basis for elucidating the disease-resistant properties of plants, and can also be applied to the screening and creation of disease-resistant rice materials, laying a germplasm foundation for future variety improvement.

Description

Technical Field

[0001] This invention belongs to the field of gene breeding technology, specifically involving the use of FSD2 in regulating resistance to rice sheath blight and bacterial blight. Background Technology

[0002] Rice disease resistance breeding is a breeding technology that aims to improve rice's resistance to target diseases. It combines multiple disciplines such as genetics, molecular biology, and crop breeding to screen or create disease-resistant germplasm resources and cultivate rice varieties with high disease resistance, high quality, high yield, and wide adaptability. It is the most economical, environmentally friendly, and efficient core means of controlling rice diseases. It can reduce pesticide use and yield loss from the source, and ensure the safety and quality improvement of rice production.

[0003] Rice is one of the world's most important food crops, but it is threatened by a variety of diseases during its growth. For example, sheath blight and bacterial blight cause a lot of yield loss every year. Therefore, disease prevention and control is crucial to ensuring food production.

[0004] Currently, there is a lack of rice varieties with high resistance to sheath blight in production. Furthermore, since the vast majority of sheath blight pathogens in the field have binucleate or multinucleate genomes, it is difficult to carry out genetic transformation, resulting in a relatively slow research on the pathogenesis of sheath blight. This brings great difficulties to the prevention and control of the disease.

[0005] Although some rice bacterial blight resistant varieties exist in production, the increased selection pressure caused by large-scale field planting leads to easy mutation of the pathogen, thus overcoming existing resistance genes. Multi-resistance gene aggregation breeding is an important solution to this problem, and discovering resistance genes is a crucial prerequisite for resistance breeding. Summary of the Invention

[0006] This invention provides the use of FSD2 in regulating resistance to rice sheath blight and bacterial blight, wherein FSD2 is directly related to rice resistance to major diseases, especially overexpression of FSD2. FSD2 At that time, the resistance of rice to major diseases was significantly improved.

[0007] This invention provides FSD2 The application of genes and / or encoded proteins in regulating the resistance of rice to infectious diseases caused by pathogenic microorganisms, wherein the amino acid sequence of the encoded protein is shown in SEQ ID No. 1.

[0008] In one specific embodiment of the present invention, the infectious diseases caused by the pathogenic microorganisms include infectious diseases caused by fungi and infectious diseases caused by bacteria.

[0009] In one specific embodiment of the present invention, the infectious diseases caused by the pathogenic microorganisms include sheath blight caused by fungi and bacterial blight caused by bacteria.

[0010] In one specific embodiment of the present invention, the... FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

[0011] In one specific embodiment of the present invention, the... FSD2 The CDS sequence of the gene is shown in SEQ ID No. 3.

[0012] In one specific embodiment of the present invention, the overexpression of the... FSD2 Genes, Enhancement FSD2 Increasing the content and / or activity of gene-encoded proteins enhances rice's resistance to infectious diseases caused by pathogenic microorganisms.

[0013] This invention provides a method for improving the resistance of rice to infectious diseases caused by pathogenic microorganisms, comprising overexpressing in the target rice genome FSD2 Gene; The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

[0014] In one specific embodiment of the present invention, when the genetic transformation method is used to perform the above... FSD2 When a gene is overexpressed, including by using the aforementioned... FSD2 Transformation with an overexpression vector for the gene, wherein the base vector of the overexpression vector includes pBWA(V)HU.

[0015] The present invention also provides FSD2 The application of genes and / or encoded proteins in rice resistance breeding, wherein the rice resistance breeding includes resistance to infectious diseases caused by pathogenic microorganisms; The amino acid sequence of the encoded protein is shown in SEQ ID No. 1.

[0016] This invention also provides a rice breeding method, including the analysis of the target rice genome. FSD2 By controlling gene expression levels, rice varieties and / or lines with different resistance to infectious diseases caused by pathogenic microorganisms can be obtained. The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

[0017] Beneficial effects: This invention provides a rice SOD gene FSD2 The novel function of (Fe-dependent superoxide dismutase 2), the FSD2 Genes are involved in regulating rice resistance to sheath blight and bacterial leaf blight. In this embodiment of the invention, the genes are obtained through genetic transformation. FSD2Overexpressing plants, compared with wild-type plants, showed that overexpressing plants... FSD2 The gene expression level was significantly higher than that of the wild type; after inoculation with *Rhizoctonia solani* and *Bacillus thuringiensis*, the overexpressing plants showed significantly enhanced resistance to *Rhizoctonia solani* and *Bacillus thuringiensis*; disease resistance tests also proved that the overexpressing plants showed significantly enhanced resistance to *Rhizoctonia solani* and *Bacillus thuringiensis* compared to the wild type, providing a theoretical basis for elucidating the disease resistance of plants, and also applicable to screening and creating disease-resistant rice materials, laying the germplasm foundation for future variety improvement. Attached Figure Description

[0018] Figure 1 for FSD2 Schematic diagram of the overexpression vector; Figure 2 for FSD2 Overexpression in rice and wild-type rice FSD2 The relative expression level of genes; Figure 3 Wild-type rice plants and FSD2 Statistics on the length of lesions after rice inoculation with sheath blight, where ZH11 represents wild-type rice Zhonghua 11; Figure 4 Wild-type rice plants and FSD2 Statistics on the length of lesions after overexpression of rice inoculated with bacterial blight, where ZH11 represents wild-type rice Zhonghua 11. Detailed Implementation

[0019] This invention provides FSD2 The application of genes and / or encoded proteins in regulating the resistance of rice to infectious diseases caused by pathogenic microorganisms, wherein the amino acid sequence of the encoded protein is shown in SEQ ID No. 1.

[0020] The present invention FSD2 The gene is a member of the SOD gene family. Higher plants possess active oxygen scavenging systems, such as superoxide dismutase (SOD), one of the most important scavenging enzymes. SOD catalyzes the dismutation of superoxide free radicals into the reactive oxygen species hydrogen peroxide (H2O2), playing a crucial role in plant resistance to biotic and abiotic stresses. As confirmed by the embodiments of this invention, the... FSD2 The (Fe-dependent superoxide dismutase 2) gene is involved in regulating resistance to major rice diseases, which are infectious diseases caused by pathogenic microorganisms, specifically including infectious diseases caused by fungi and infectious diseases caused by bacteria. In one embodiment, the infectious diseases caused by pathogenic microorganisms include sheath blight caused by fungi and bacterial blight caused by bacteria.

[0021] The amino acid sequence of the FSD2 protein described in this invention is as shown in SEQ ID. Shown in No.1: MAFATLVGVGGLSPALFSPSRPLSCSSSTSVSAPFILRAGGGGDARRHGLRRLVTPLRGSACRGESTNSRVLQCANEANVVTEDDIVNDGIDDETASDAEMDEDAEANGDESSGTDEDASVSWIEQQPLPYPSDALEPYISKETVEQHWGVHQNIHVERLNGMIGGSEWEGMSLGQMMLSSFNEGREAPHPP FFHAAQIWNHDFYWRSMQPGGGGKPPERLLKFINRDFGSYDGMIRQFMDAASTQFGSGWVWLCYKTSKLPHVKSRSPIPSDNYGRLVISKSPNAINPLVW GHSPLLAIDLWEHAYYLDYEDRRSDYVSTFLEKLVSWETVESRLKKAVQRAVERDEYVSTKHIRKQLLARAKSQIRAMPQQVNGDAREQTSGQEKSLGV; The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2: The FSD2 The CDS sequence of the gene is shown in SEQ ID No. 3:

[0022] In one embodiment of the present invention, the gene represented by SEQ ID No. 3 is overexpressed in rice. FSD2 The gene can significantly improve the resistance of rice to sheath blight and bacterial blight.

[0023] This invention provides a method for improving the resistance of rice to infectious diseases caused by pathogenic microorganisms, comprising overexpressing in the target rice genome FSD2 Gene; The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

[0024] This invention does not specifically limit the overexpression method; any method in the art that can significantly increase gene expression levels can be used, such as EMS chemical mutagenesis, radiation mutagenesis, T-DNA insertion, CRISPR gene editing technology, or genetic transformation. In the examples, an overexpression vector was constructed, and Agrobacterium-mediated rice callus was used to obtain... FSD2 Highly expressed transgenic rice, but this should not be considered the entire scope of protection of this invention. In one embodiment of this invention, FSD2 The strong promoter of pBWA(V)HU (vector digested with Bsa I / Eco31I) was inserted via homologous recombination. Ubi Subsequently, the pBWA(V)HU-FSD2 overexpression vector was constructed, and overexpression plants were obtained through Agrobacterium-mediated genetic transformation.

[0025] After obtaining the overexpressing plant, the present invention also includes verifying the... FSD2 Regarding the gene expression level, this invention does not specifically limit the detection method; methods such as qRT-PCR, Western blotting, or ELISA can be used for detection. In one embodiment, the expression level is verified using qRT-PCR. FSD2 The expression level of the gene, but this should not be considered the entire scope of protection of this invention. When performing the qRT-PCR described in this invention, the expression level is... Actin The primer sequences used are as follows: (This is for internal reference only) FSD2 -F (SEQ ID No.4): CCAATGAGGCTAATGTGGTGACC; FSD2 -R (SEQ ID No.5): TGATGAACTCCCCAGTGCTGTC; Actin-F (SEQ ID No.6):GAGTATGATGAGTCGGGTCCAG; Actin -R (SEQ ID No. 7): ACACCAACAATCCCAAACAGAG.

[0026] This invention preferably collects leaves from wild-type and mutant organisms, extracts RNA, and reverse transcribes it into cDNA. The cDNA is used as a template to prepare the reaction system. In the examples, qRT-PCR was performed using the ChamQ universal SYBR qPCR Master Mix (Vazyme, China) kit. The operation steps are as follows: Step 1: 95℃, 30s; Step 2: 95℃, 5s; 60℃, 30s; Step 2 was repeated 40 times; Melting curve program: 95℃, 15s; 60℃, 30s; 95℃, 15s.

[0027] The present invention also provides FSD2 The application of genes and / or encoded proteins in rice resistance breeding, wherein the rice resistance breeding includes resistance to infectious diseases caused by pathogenic microorganisms; The amino acid sequence of the encoded protein is shown in SEQ ID No. 1.

[0028] This invention can be controlled by adjusting the above FSD2 The expression level of genes can be controlled to regulate the resistance of rice to sheath blight and bacterial blight, thereby regulating the expression levels of these genes. FSD2 The expression levels of the gene were used to obtain rice varieties and / or lines with different resistances to sheath blight and bacterial blight, for example, let the gene expression levels be as described. FSD2 Gene overexpression can significantly improve resistance to sheath blight and bacterial blight, and can be used to breed highly resistant rice varieties and / or lines.

[0029] This invention also provides a rice breeding method, including the analysis of the target rice genome. FSD2 By controlling gene expression levels, rice varieties and / or lines with different resistance to infectious diseases caused by pathogenic microorganisms can be obtained. The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

[0030] The present invention relates to the above. FSD2 There are no specific limitations on the methods for controlling gene expression levels; conventional methods in the field can be used for regulation, such as EMS chemical mutagenesis, radiation mutagenesis, T-DNA insertion, CRISPR gene editing technology, or genetic transformation.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, illustrates the use of FSD2 provided by the present invention in regulating resistance to rice sheath blight and bacterial blight, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 according to FSD2 Full-length primers were designed based on the sequence (SEQ ID No. 2) to obtain the target gene from cDNA containing the target gene via PCR. FSD2 The CDS fragment is ultimately connected to the entire fragment. Ubi Promoter-driven plant overexpression vectors ( Figure 1 The specific experimental method is as follows: Using bioinformatics software such as Snapgene, design FSD2 The amplification primers for the gene were SEQ ID No. 8 and SEQ ID No. 9. The amplification was performed using TOYOBO high-fidelity PCR polymerase KOD-Plus. FSD2 Gene cloning experiments.

[0033] FSD2 - OX -F (SEQ ID No.8):ttgtttggtgttacttctgttgcaacATGGCGTTCGCCACACTG; FSD2 - OX -R (SEQ ID No.9): tagtctccgtcgtggtctttgtaatcCACCCCTAGGGACTTCTCTTGAC; After gel electrophoresis, the PCR products were recovered using the Kangwei Century Gel Extraction Kit.

[0034] The purified PCR product was mixed with the pBWA(V)HU vector at a molar ratio of 3:1. 10 μL of 2×Multif Seamless Assembly Mix recombinase was added, followed by ddH2O to a final volume of 20 μL. The mixture was incubated at 50°C for 15 minutes. The ligation system was then transformed into *E. coli* DH5α, and positive clones were screened and sent to a biotechnology company for sequencing analysis. Ensure the insertion... OsFSD2 The full-length CDS sequence is shown in SEQ ID No. 3.

[0035] Plasmids were extracted and introduced into Agrobacterium tumefaciens GV3101 to transform callus tissue of rice variety Zhonghua 11. T0 generation rice with hygromycin resistance was obtained. Individual plants were harvested and sown to produce T1 generation rice. RNA was extracted from rice leaves, reverse transcribed into cDNA, and overexpressing plants were identified by qRT-PCR.

[0036] by Actin The primer sequences used are as follows: (This is for internal reference only) FSD2 -F (SEQ ID No.4): CCAATGAGGCTAATGTGGTGACC; FSD2 -R (SEQ ID No.5): TGATGAACTCCCCAGTGCTGTC; Actin -F (SEQ ID No.6):GAGTATGATGAGTCGGGTCCAG; Actin -R (SEQ ID No. 7): ACACCAACAATCCCAAACAGAG.

[0037] Leaves from wild-type and mutant strains were collected, RNA was extracted, and cDNA was reverse transcribed. The cDNA was used as a template to prepare the reaction system, and the ChamQ universal SYBR qPCR Master Mix (Vazyme, China) kit was used. The procedure was followed according to the kit instructions. Reaction program: Step 1: 95℃, 30s; Step 2: 95℃, 5s; 60℃, 30s; repeat 40 times; Melting curve program: 95℃, 15s; 60℃, 30s; 95℃, 15s.

[0038] RNA was extracted from leaves and reverse transcribed into cDNA. The expression levels in overexpressing plants and wild-type Zhonghua 11 were detected by qRT-PCR. FSD2 Gene expression levels, results as follows Figure 2 As shown, compared to the wild type, the mutant has... FSD2 The expression level was significantly higher in the wild type than in the genotype.

[0039] The obtained overexpression lines were inoculated with *Rhizoctonia solani* and *Bacterium oxysporum* AG-IA (Li, D., Wu, X., Huang, C., Lin, Q., Wang, Y., Yang, X., Wang, C., Xuan, Y., Wei, S., and Mei, Q. (2023). This enhanced rice resistance to sheath blight through NitrateTransporter 1.1B mutation without yield loss under NH4+. +Fertilization. Journal of Agricultural and Food Chemistry 71, 19958-19969.), measuring the length of lesions after disease onset. For identification of sheath blight, a leaf sheath method is used. Activated sheath blight fungus mycelium is collected using a punch and placed in the center of a new PDA culture dish. Wood bark is placed radially around the mycelium. After the mycelium has covered the entire culture dish, the wood bark at the same distance from the mycelium is removed and placed in a cultured rice leaf sheath. The dish is then sprayed twice with distilled water and wrapped with plastic wrap to maintain moisture. Ten overexpressing plants and ten wild-type plants are inoculated. The lesion length is measured one week later. The results are as follows: Figure 3 As shown.

[0040] The bacterial blight pathogen PXO86 (Mei, Q., Fu, YW, Li, TM, and Xuan, YH (2022). Ac / Ds-Induced Receptor-like Kinase Genes Deletion Provides Broad-Spectrum Resistance to Bacterial Blight in Rice. International Journal of Molecular Sciences 23, 4561.) was identified using the leaf-cutting method. Frozen bacterial blight pathogens were streaked onto plates, and the resulting single colonies were cultured on a shaker at 28°C and 220 rpm. Once the OD value reached 0.8-1.0, 1 cm of the rice leaf tip was cut off using scissors dipped in the bacterial solution. Ten overexpressing plants and ten wild-type plants were inoculated. The length of lesions was counted two weeks later. The results are as follows: Figure 4 As shown.

[0041] The results showed that the lesion length of the overexpressing plants was significantly shorter than that of the wild-type control, indicating that the overexpressing plants had significantly improved resistance to sheath blight and bacterial blight.

[0042] Table 1. Statistics on lesion length after inoculation with *Rhizoctonia solani*.

[0043] Table 2. Statistics on lesion length after inoculation with bacterial blight pathogen.

[0044] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. FSD2 The application of genes and / or encoded proteins in regulating resistance in rice to infectious diseases caused by pathogenic microorganisms, characterized in that, The amino acid sequence of the encoded protein is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The infectious diseases caused by the pathogenic microorganisms include infectious diseases caused by fungi and infectious diseases caused by bacteria.

3. The application according to claim 1 or 2, characterized in that, The infectious diseases caused by the pathogenic microorganisms include sheath blight caused by fungi and bacterial leaf blight caused by bacteria.

4. The application according to claim 1, characterized in that, The FSD2 The genome sequence of the gene is shown in SEQ ID No.

2.

5. The application according to claim 1 or 4, characterized in that, The FSD2 The CDS sequence of the gene is shown in SEQ ID No.

3.

6. The application according to claim 1, characterized in that, Overexpression FSD2 Genes, Enhancement FSD2 Increasing the content and / or activity of gene-encoded proteins enhances rice's resistance to infectious diseases caused by pathogenic microorganisms.

7. A method for improving the resistance of rice to infectious diseases caused by pathogenic microorganisms, characterized in that, Including overexpression in the target rice genome FSD2 Gene; The FSD2 The genome sequence of the gene is shown in SEQ ID No.

2.

8. The method according to claim 7, characterized in that, When using genetic transformation methods to perform the above FSD2 When a gene is overexpressed, including by using the aforementioned... FSD2 Transformation with an overexpression vector for the gene, wherein the base vector of the overexpression vector includes pBWA(V)HU.

9. FSD2 The application of genes and / or encoded proteins in rice resistance breeding is characterized by, The rice resistance breeding includes resistance to infectious diseases caused by pathogenic microorganisms; The amino acid sequence of the encoded protein is shown in SEQ ID No.

1.

10. A method for rice breeding, characterized in that, Including the target rice genome FSD2 By controlling gene expression levels, rice varieties and / or lines with different resistance to infectious diseases caused by pathogenic microorganisms can be obtained. The FSD2 The genome sequence of the gene is shown in SEQ ID No. 2.

Images