Application of OsCBM1 gene in improving disease resistance of rice

Abstract

The invention discloses application of an OsCBM1 gene in improvement of rice disease resistance, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the OsCBM1 gene is as shown in SEQ ID NO: 1. The invention proves that the inhibition or overexpression of the OsCBM1 gene in the rice enables the rice to show resistance change on different bacterial blight germs. Through systematic research, it is found that the OsCBM1 gene has an important regulation and control function in the aspect of bacterial blight resistance of rice.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and more particularly to the application of OsCBM1 gene in improving the disease resistance of rice. Background Art

[0002] Rice is an important food crop in the world, and its stable yield plays a significant role in world food security. Rice yield is affected by many factors, among which disease is one of the important factors affecting rice yield. Traditional disease prevention and control mainly involves spraying chemical agents. From the perspective of sustainable agricultural development, long-term spraying of chemical agents is prone to pollute the environment on the one hand, and on the other hand, chemical agents will accelerate the mutation of pathogens and cause rice to become diseased. Therefore, discovering rice's own disease-resistant genes and improving rice's own defense system are of great significance for cultivating high-quality disease-resistant varieties.

[0003] The disease resistance of plants is a complex process regulated by multiple genes. The genes involved in plant disease resistance are divided into two categories: major disease resistance genes and disease resistance-related genes. At present, more than 30 major disease resistance genes have been cloned from rice, such as rice bacterial blight resistance genes Xa21, Xa1, Xa26, xa5, Xa27, xa13, etc., and multiple rice blast resistance genes such as Pib, Pi-ta, Pi9, Pi2, etc. However, the resources of major disease resistance genes are limited. Most of the major disease resistance genes are only resistant to one or a few pathogenic species, and the range of disease resistance is limited. In addition, the cloning progress of major disease resistance genes is always slower than the changes in the pathogenicity of pathogens, which greatly limits the application of major disease resistance genes in agricultural production.

[0004] All genes involved in disease resistance in plants except the main disease resistance genes are called disease resistance-related genes. Their encoded products participate in the synthesis of disease resistance signal molecules, signal transduction or defense reactions in plants. At present, many disease resistance-related genes have been cloned. More than a dozen disease resistance-related genes have been identified from rice, such as OsWRKY45, OsGH3-8, OsEDR1, OsMAPK6, OsYLP4 and other genes. These genes encode different types of proteins and participate in the disease resistance of rice to different pathogens by regulating different reaction pathways in rice. Most of these disease resistance-related genes have broad-spectrum disease resistance to different types of pathogens.

[0005] Sugar-binding lectin domain or lectin-like domain proteins (CBMs) are a subfamily of lectin proteins in the biological world. CBMs in bacteria and fungi encode the non-catalytic domain of glycoside hydrolases and function in glycoside hydrolases that degrade plant cell wall polysaccharides. CBMs in plants are malectin-like receptor kinases, a new subfamily of receptor-like kinases (RLKs); they are also commonly known as Catharanthus roseus receptor-like kinase-like proteins (CrRLK1Ls), which are involved in plant immunity, cell wall integrity and other biological functions. The CrRLK1L protein family in plants has a malectin-like domain, a transmembrane domain and an intracellular Ser and Thr kinase domain. FERONIA (FER) is the most functional protein in the CrRLK1Ls family. FER can interact with ROS and Ca2+ to mediate pollen tube signal reception and mechanical signal transduction in Arabidopsis. CBM1 in rice was found to contain only one malectin-like domain. OsCBM1 physically interacts with OsRbohA and OsRacGEF1 to jointly regulate the production of ROS. Overexpression of the OsCBM1 gene can significantly respond to abiotic stresses such as oxidative stress and drought.

[0006] Although the OsCBM1 gene has been reported to be involved in the regulation of abiotic stress, its function in biotic stress is still unknown. On this basis, the present invention found that plants that inhibit the expression of the OsCBM1 gene weakened the broad-spectrum resistance to rice bacterial blight, and plants that overexpressed the OsCBM1 gene enhanced the broad-spectrum resistance to rice bacterial blight. The present invention has important significance for the improvement of rice, an important food crop, in terms of resistance to bacterial blight. Summary of the invention

[0007] In view of this, the present invention provides the use of OsCBM1 gene in improving the disease resistance of rice.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] Application of OsCBM1 gene in improving rice resistance to bacterial blight, the nucleotide sequence of the OsCBM1 gene is shown in SEQ ID NO: 1.

[0010] Furthermore, the protein encoded by the OsCBM1 gene is shown in SEQ ID NO: 2;

[0011] Furthermore, the CDS sequence corresponding to the protein encoded by the OsCBM1 gene is shown in SEQ ID NO: 3;

[0012] Furthermore, the method includes constructing the OsCBM1 gene on an overexpression vector and transforming rice to obtain a transgenic strain with enhanced resistance to bacterial blight of rice.

[0013] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0014] The invention proves that bacterial blight infection can induce the expression of OsCBM1 gene.

[0015] The present invention confirms that inhibiting or overexpressing the OsCBM1 gene in rice enables rice to show resistance changes to different bacterial blight pathogens. Through systematic research, the present invention finds that the OsCBM1 gene has an important regulatory function in rice resistance to bacterial blight.

[0016] The present invention confirms that OsCBM1 affects the resistance of rice to bacterial blight pathogen by indirectly affecting the content of SA.

[0017] The invention confirms that OsCBM1 affects the activities of SOD, POD and CAT enzymes to regulate the resistance of rice to bacterial blight pathogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The accompanying drawings show the expression levels of the OsCBM1 gene at different time points after the rice variety Zhonghua 11 of the present invention was inoculated with the rice bacterial blight pathogen PXO99;

[0020] Figure 2 The accompanying drawings show the identification of the expression level of OsCBM1 gene in the rice strains with suppressed expression and overexpression of OsCBM1 gene according to the present invention;

[0021] Figure 3 The attached figure shows the diseased length of a single plant after inoculation of bacterial blight pathogen in rice strains overexpressing OsCBM1 gene, rice strains inhibiting expression of OsCBM1 gene and wild type according to the present invention; Figure 3 A is a real picture of the diseased strain after inoculation with PXO99; Figure 3 B is the summary of the disease length of individual plants inoculated with different bacterial blight pathogens (PXO99, PXO341, ZHE173, and FuJ23);

[0022] Figure 4The accompanying drawings show the growth of bacterial leaf blight fungi in leaves of rice strains overexpressing OsCBM1 gene, rice strains inhibiting expression of OsCBM1 gene and wild type after inoculation with bacterial leaf blight fungi PXO99;

[0023] Figure 5 The accompanying drawings show the changes in SA content in leaves of the rice strains overexpressing the OsCBM1 gene of the present invention, the rice strains inhibiting the expression of the OsCBM1 gene, and the wild type after inoculation with the bacterial blight pathogen PXO99;

[0024] Figure 6 The accompanying drawings show the changes in SOD enzyme activity in leaves of the OsCBM1 gene overexpression rice strain of the present invention, the OsCBM1 gene inhibition expression rice strain, and the wild type before and after inoculation with bacterial blight pathogen PXO99;

[0025] Figure 7 The accompanying drawings show the changes in POD enzyme activity in leaves of the OsCBM1 gene overexpression rice strain, the OsCBM1 gene suppression expression rice strain, and the wild type before and after inoculation with bacterial blight pathogen PXO99;

[0026] Figure 8 The accompanying drawings show the changes in CAT enzyme activity in leaves of the OsCBM1 gene overexpressing rice strains, the OsCBM1 gene inhibiting expression rice strains and the wild type before and after inoculation with bacterial blight pathogen PXO99. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] Analysis of the expression pattern of OsCBM1 gene in rice treated with Xanthomonas solani

[0030] The nucleotide sequence of the OsCBM1 gene (5485 bp) is shown in SEQ ID NO: 1;

[0031] The protein encoded by the OsCBM1 gene (366 residues) is shown in SEQ ID NO: 2;

[0032] The CDS sequence (1101 bp) corresponding to the protein encoded by the OsCBM1 gene is shown in SEQ ID NO: 3;

[0033] In order to verify whether the OsCBM1 gene is involved in regulating the disease resistance of rice. The present invention inoculated the rice variety Zhonghua 11 with bacterial blight pathogen at the booting stage, and used quantitative reverse transcription-PCR (qRT-PCR) technology to analyze the expression pattern of the OsCBM1 gene. The results showed that the OsCBM1 gene could be induced to express by the bacterial blight pathogen. This result suggests that the OsCBM1 gene may be involved in regulating the resistance of rice to the bacterial blight pathogen, and the excessive expression of the OsCBM1 gene may improve the disease resistance of rice. Figure 1 .

[0034] Example 2

[0035] Acquisition and identification of materials with suppressed expression and overexpression of OsCBM1 gene

[0036] Using the cDNA of Nipponbare as a template, the partial fragment of OsCBM1 gene was transferred into the suppression vector, and the constructed vector was: pDS1301-p35S-OsCBM1. Using the cDNA of Zhonghua 11 as a template, the full-length fragment of OsCBM1 gene was transferred into the overexpression vector, and the constructed vector was: PU1301-OsCBM1.

[0037] The present invention introduces a pDS1301-p35S-OsCBM1 vector containing a strong promoter 35S (AJ007626) driving a partial sequence of the OsCBM1 gene and a PC1301-35S-OsCBM1 vector containing a strong promoter 35S (AJ007626) driving a full-length fragment of the OsCBM1 gene into a rice variety Nipponbare through an Agrobacterium-mediated genetic transformation method, and obtains multiple independent transgenic families. Among them, the suppression material selects family 54 and family 55, and the overexpression material selects family 38 and 41, and the expression level of the OsCBM1 gene is detected by qRT-PCR technology.

[0038] Flag leaves at the booting stage were collected from the fields in Changsha, and RNA was extracted according to the instructions of TransZol (Quanshijin). 1-5 μg of total RNA was treated with DNaseI (Invitrogen, USA) for 15 minutes to remove genomic DNA contamination, and then reverse transcribed using oligo(dT)15 oligo primer and M-MLV reverse transcriptase (Promega, USA). Real-time quantitative PCR analysis kit was used. Green PCR MasterMix (Dalian Tokara Company) was used, and real-time quantitative PCR reaction was performed on ABI 7500 Real-Time PCR system (Applied Biosystems, USA) according to the instruction manual of the kit. The expression level of rice endogenous actin gene was used to measure and normalize the RNA content of the samples.

[0039] OsCBM1 gene specific PCR primers for qRT-PCR analysis:

[0040] CBM1-qRT-F: 5′-CATTGGGACGAACTACTGGG-3′, as shown in SEQ ID NO: 2;

[0041] CBM1-qRT-R: 5′-GTTCACAAGACACACGGGTG-3′, as shown in SEQ ID NO: 2;

[0042] Actin gene PCR primers:

[0043] ACTIN1-qRT-F: 5′-TGCTATGTACGTCGCCATCCAG-3′, as shown in SEQ ID NO: 2;

[0044] ACTIN1-qRT-R: 5′-AATGAGTAACCACGCTCCGTCA-3′, as shown in SEQ ID NO:2.

[0045] The results of qRT-PCR showed that the expression level of OsCBM1 gene in positive plants of the transgenic families with suppressed expression was significantly lower than that in the wild-type control, and the expression level of OsCBM1 gene in positive plants of the transgenic families with overexpression was significantly higher than that in the wild-type control. Figure 2 .

[0046] Example 3

[0047] Correlation analysis and functional verification of OsCBM1 gene suppression and overexpression rice lines

[0048] (1) Analysis of broad-spectrum resistance of OsCBM1 gene-suppressed and OsCBM1 gene-overexpressed rice lines to different bacterial blight pathogens

[0049] In the summer field in Changsha, Hunan, China, rice lines with suppressed expression of OsCBM1 gene, rice lines with overexpression of OsCBM1 gene and wild-type control were inoculated with bacterial blight pathogens. The results showed that when rice lines with overexpression of OsCBM1 gene were inoculated with different pathogenic races of bacterial blight pathogens (PXO99, PXO341, ZHE173, FuJ23, all bacterial blight pathogens were donated by the International Rice Research Institute) at the booting stage, the length of diseased individual plants in OsCBM1 gene-overexpressing rice lines 38 and 41 was significantly shorter than that in wild-type (p<0.01). However, the length of diseased individual plants in OsCBM1 gene-suppressed rice lines 54 and 55 was significantly longer (p<0.01). Figure 3 .

[0050] The above results showed that the rice lines overexpressing the OsCBM1 gene had a broad-spectrum disease resistance to different bacterial blight pathogens (PXO99, PXO341, ZHE173, and FuJ23) compared with the wild type, while the rice lines with suppressed expression of the OsCBM1 gene were more susceptible to different bacterial blight pathogens (PXO99, PXO341, ZHE173, and FuJ23) compared with the wild type.

[0051] (2) Analysis of the number of bacterial blight pathogens in rice lines with OsCBM1 gene suppression and overexpression

[0052] The growth of Xanthomonas spp. in leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 were analyzed in the field in summer in Changsha, Hunan, China, at the booting stage. The strains with suppressed expression of OsCBM1, rice lines with overexpression of OsCBM1, and wild-type control were inoculated with the strain PXO99 (gift from the International Rice Institute) at the booting stage in the field. Leaf materials (leafs 6 cm below the inoculation cut) were collected at different times after inoculation. Three leaves (representing three experimental replicates) were collected from the same material. The leaf materials were processed according to the reported method and the growth of bacteria was analyzed. The main analysis steps are as follows: disinfect the leaf surface with 75% alcohol for 1 minute, dry it, place it in a mortar and add 1ml of sterile distilled water to grind it into a homogenate, then dilute it with double sterile water into different concentration gradients, and repeat three PSA culture dishes (200g potato, 20g agar, 20g sucrose, and deionized water to 1000ml) for each concentration gradient. Count the number of bacterial colonies after growing in the dark at 22-25℃ for 2-3 days. The bacterial growth curve was drawn based on the LOG value of the number of bacterial leaf blight colonies on each leaf. The growth analysis of bacterial leaf blight bacteria showed that after inoculation with the pathogenic race PXO99 of bacterial leaf blight bacteria, the number of bacterial leaf blight bacteria in the leaves of the OsCBM1 gene overexpressing rice strain was significantly lower than that of the wild-type control. The results are shown in Figure 2. Figure 4 .

[0053] (3) Analysis of SA content in rice lines with OsCBM1 gene suppression and overexpression

[0054] The content of SA in leaves of rice strains with suppressed expression of OsCBM1 and rice strains with overexpression of OsCBM1 were inoculated with bacterial blight pathogenic race PXO99 at the booting stage in Changsha, Hunan, China in summer. The rice strains with suppressed expression of OsCBM1, rice strains with overexpression of OsCBM1 and wild-type control were inoculated with bacterial blight pathogenic race PXO99 (donated by International Rice) at the booting stage in the field, and leaf materials were collected 12 hours after inoculation. The content of SA was detected by liquid chromatography. The main analysis steps are as follows: take 0.1g (accurate to 0.001g) of the sample, grind it with liquid nitrogen, add 1mL of pre-cooled methanol water (20mL water + 80mL methanol), place it in a 4℃ refrigerator for extraction for 16h, shake it 2-3 times during the period, wash it twice with methanol water, take 4mL of the above liquid through a mixed anion exchange solid phase extraction column, elute it twice with 2mL1% formic acid methanol solution, blow it to near dryness with 35℃ nitrogen, dilute it to 0.5mL with water, vortex mix it, filter it with a filter membrane, and measure it on a machine (LCMS-8060). According to the mass spectrometry peak intensity data corresponding to the standard solution of concentration (100ng / mL, 50ng / mL, 25ng / mL, 10ng / mL, 5ng / mL, 1ng / mL, 0.5ng / mL, 0.1ng / mL, 0.05ng / mL, 0.01ng / mL), the concentration of the sample is converted. The analysis of SA content showed that after inoculation with bacterial blight pathogenic race PXO99, the SA content in the leaves of OsCBM1 gene overexpressing rice lines was significantly higher than that of wild type control. Figure 5 .

[0055] (4) Analysis of superoxide dismutase (SOD) content in rice lines with OsCBM1 gene suppression and overexpression

[0056] In the summer field in Changsha, Hunan, China, the content of superoxide dismutase (SOD) in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 were inoculated with the pathogenic race PXO99 of bacterial leaf blight at the booting stage. The rice lines with suppressed expression of OsCBM1, rice lines with overexpression of OsCBM1 and wild-type control were inoculated with the pathogenic race PXO99 of bacterial leaf blight (donated by International Rice) at the booting stage in the field, and the leaf materials were collected 12 hours after inoculation. The content of SOD was detected by SOD detection kit (BC5165, SOD EC 1.15.1.1, Beijing Solebow Company) combined with microplate reader method. The main analysis steps are as follows: 0.1g of plant tissue was taken in a centrifuge tube, 500-1000mL of enzyme extract was added, and the mixture was homogenized in an ice bath. Centrifuge at 8000g for 10 min at 4℃; take the supernatant into a new centrifuge tube and detect it with a microplate reader (spark 10M). Compared with the wild-type control, the SOD enzyme activity of the OsCBM1 gene overexpressing rice strain was enhanced before and after inoculation with the bacterial blight pathogen PXO99. The results are shown in Figure 6 .

[0057] (5) Analysis of peroxidase (POD) content in rice lines with OsCBM1 gene suppression and overexpression

[0058] The POD content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in Changsha, Hunan, China, at the booting stage. The POD content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in the summer. The POD content in rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in the booting stage. The POD content in rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in the summer ... Centrifuge at 8000g for 10 min at 4°C; take the supernatant into a new centrifuge tube and test it with a microplate reader (spark 10M). Compared with the wild-type control, the POD enzyme activity of the OsCBM1 gene overexpressing rice strains was enhanced before and after inoculation with the bacterial blight pathogen PXO99. The results are shown in Figure 7 .

[0059] (6) Analysis of catalase (CAT) content in rice lines with OsCBM1 gene suppression and overexpression

[0060] The CAT content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in Changsha, Hunan, China, at the booting stage. The CAT content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in the summer. The CAT content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 was analyzed in the field in the booting stage. The CAT content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 were analyzed in the field in the summer. The CAT content in the leaves of rice lines with suppressed expression of OsCBM1 and rice lines with overexpression of OsCBM1 were ... Centrifuge at 8000g for 10 min at 4°C; take the supernatant into a new centrifuge tube and detect it with a microplate reader (spark10M). Compared with the wild-type control, the CAT enzyme activity of the OsCBM1 gene overexpressing rice strain was enhanced before and after inoculation with the bacterial blight pathogen PXO99. The results are shown in Figure 8 .

[0061] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of OsCBM1 gene in improving rice resistance to bacterial blight, characterized in that: The nucleotide sequence of the OsCBM1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The protein encoded by the OsCBM1 gene is shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that: The CDS sequence corresponding to the protein encoded by the OsCBM1 gene is shown in SEQ ID NO:

3.

4. The use according to claim 1, characterized in that: The method comprises constructing the OsCBM1 gene on an overexpression vector and transforming rice to obtain a transgenic strain with enhanced resistance to rice bacterial blight.

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