CN116041460A - Rice Xa48 (t) protein and application of coding gene thereof - Google Patents
Rice Xa48 (t) protein and application of coding gene thereof Download PDFInfo
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- CN116041460A CN116041460A CN202211116593.9A CN202211116593A CN116041460A CN 116041460 A CN116041460 A CN 116041460A CN 202211116593 A CN202211116593 A CN 202211116593A CN 116041460 A CN116041460 A CN 116041460A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention belongs to the field of molecular biology technologyThe application includes enhancing resistance of rice to bacterial leaf blight, enhancing resistance of rice to rice blast or breeding rice disease resistance, treating target plants by RNAi interference technology, gene knockout technology and gene overexpression technology, selecting Xa48 (T) gene with HNY as background, knocking out plant T 3 Generation, RNAi interference plant T 3 Generation and Japanese-fine-based Xa48 (T) gene over-expression plant T 3 The rice bacterial leaf blight inoculation investigation is carried out on the plants, the results show that the resistance of the rice plants to the bacterial leaf blight is obviously reduced after knocking out and interfering the gene Xa48 (t), and the resistance of the rice plants to the bacterial leaf blight is obviously enhanced after over-expressing the candidate gene Xa48 (t).
Description
Technical Field
The invention belongs to the technical field of molecular biology, and in particular relates to riceXa48(t) use of the protein and the coding gene thereof.
Background
Gram-negative bacterium Monilinia flavescens rice varietyXanthomonas oryzae pv. Oryzae，Xoo) The length is about 0.7 to 2.0 [ mu ] m, the width is 0.4 to 0.7 [ mu ] m, and the optimal growth temperature is 25 to 30 ℃. Infection withXooThe disease type of the rice after the process is mainly divided into 3 types of leaf withering type, wilting type and wilting type, and the yield reduction of the rice at different degrees can be caused. In recent years, bacterial leaf blight of rice is highly developed in regions in the south of the Yangtze river, and also occurs in other provinces, and is considered to be one of the main agricultural diseases in China. Along withXooSmall continuous evolution variation, multiple disastrous weather caused by climate, global warming and the like, and continuous change of domestic rice varietiesMore etc., the disease presents a trend toward reepitfall.
In agricultural production, chemical agent control, biological control, improved cultivation technology, cultivation of disease-resistant varieties and other methods are generally adopted for controlling bacterial leaf blight.
The chemical agent has the characteristics of quick response and wide control range for preventing and treating bacterial leaf blight, and rice bacterial leaf blight has the characteristics of popularity and multiple occurrence, and the long-term use of the chemical agent reduces the bearing capacity of soil environment and causes irreversible influence on the environment. The method for preventing and treating bacterial leaf blight by using the improved cultivation technology has higher requirement and longer time labor investment, and the labor cost for preventing and treating bacterial leaf blight by using the method is too high because the number of workers engaged in agricultural production in China is smaller.
Years of agricultural production practices show that the discovery of new genes for resisting bacterial leaf blight and the cultivation of disease-resistant germplasm with broad-spectrum resistance are the most reliable methods for preventing bacterial leaf blight, can be fundamentally achieved, and are characterized in thatXooAfter invasion, plants carrying disease-resistant genes can still grow normally, so that the yield of the rice is maintained at the original level basically. Up to now, the objective genes are polymerized and utilized mainly by conventional crossbreeding in agricultural production, and along with the updating of transgenic technology means, the gene editing breeding and transgenic breeding modes gradually provide new views for breeding disease-resistant varieties.
Yunnan rice seeds are rich in resources, and one of the Oryza Glutinosa (HNY) of the local indica rice seeds from Geng Ma county of Yunnan province is used as Yunnan plateau due to the characteristic of high bacterial leaf blight resistanceXooIs characterized by specific variety identification. Current research Yu Tengqiong et al uses HNY and other multiple identified varieties to collect 180 or more from regions in Yunnan, acquired in 2003, 2005, 2007, 2009XooThe strain pathotype identification, HNY shows medium to high resistance to all the reference strains, the local rice HNY can still keep the stability of the resistance of the strain in the near 10 years when facing to high-speed differentiation and Yunnan highland bacterial blight bacteria with rich pathogenic types and population types, which indicates that the strain has durable resistance to the carried resistance gene in the long-term growth and evolution process, has very high research value, and is especially aimed atThe isolated resistance genes of the inventionXa48(t) there have been no reports on the correlation.
Disclosure of Invention
The main purpose of the invention is to provide a riceXa48(t) protein and its coding gene, pairXa48(t) fine positioning and separation cloning of the gene, analyzing the related functions of the gene, and creating disease-resistant germplasm carrying the gene, enriching the rice disease-resistant gene resource library.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a rice Xa48 (t) protein, wherein the amino acid sequence of the rice Xa48 (t) protein is shown as SEQ ID NO.1, and the application comprises the steps of enhancing the resistance of rice to bacterial leaf blight, enhancing the resistance of rice to rice blast or breeding rice disease resistance.
The invention further provides a gene for encoding the Xa48 (t) protein of rice, and the nucleotide sequence is shown as SEQ ID NO. 2.
Further, a primer pair for amplifying the Xa48 (t) gene is provided, and the primer pair can be a primer pair shown as Xa48 (+) and Xa48 (-), or can be other primer pairs which can amplify the gene and are designed by the existing primer design software.
Xa48(+):acaacatggccaacccagaggattt Xa48(-):atacacagccgcagcccactctccc
Further, the above genes or primer pairs can be applied to: (1) identifying or assisting in identifying bacterial leaf blight resistance of rice; (2) Preparing a product for identifying or assisting in identifying rice bacterial leaf blight resistance; (3) Screening or breeding single plants or lines or varieties of rice with bacterial leaf blight resistance; (4) Screening or breeding single plants or strains or varieties of rice with bacterial leaf blight; (5) Preparing a product for screening or breeding single plants or lines or varieties of rice with bacterial leaf blight resistance; (6) And (3) preparing a product for screening or breeding single plants or lines or varieties of rice with bacterial leaf blight. It will be appreciated that bacterial blight resistance genes that have been isolated and cloned in the prior art are:Xa1、Xa2、Xa3 /Xa26、Xa4、xa5、Xa7、Xa10、xa13、Xa14、Xa21、Xa23、xa24(t)、xa25、Xa27、Xa31、xa41、Xa45、Xa47(t) etc., and the novel disease resistance genes of the present inventionXa48(t) can be used alone or in combination with these genes for identification, assisted identification, screening or breeding of rice with good traits, and specific methods of application can be referred to the methods of the prior art by those skilled in the art, in particular the applications related to bacterial leaf blight genes which have been found.
Further, the application of the rice disease-resistant breeding comprises any one of the following applications: (1) MAS breeding of bacterial leaf blight resistance genes, detecting genetic conditions of target genes in any offspring group through closely linked molecular markers, selecting offspring excellent germplasm with target genetic information, and improving breeding efficiency; single gene MAS breeding and multiple gene MAS breeding are common in agricultural production. Single gene MAS breeding is to introduce a single target gene into an excellent strain, such as Wu Hao, introduce Xa7 into variety "meib", identify offspring population by Xa7 specific markers, create new maintainer line material carrying bacterial blight resistance Xa7, and multi-gene MAS polymerization breeding is to polymerize 2 or more than 2 excellent genes according to the needs of agricultural production, thereby creating germplasm material with a plurality of excellent traits. For example: liu Wuge and the like are polymerized to form a plurality of rice blast resistance genes and bacterial leaf blight resistance genes, so that a plurality of sterile line excellent xenogenic substances with broad-spectrum resistance are cultivated, and the Xa48 (t) protein coding genes discovered by the invention can be used for MAS breeding by adopting a method in the prior art;
(2) Transgenic breeding of bacterial leaf blight resistance genes; it will be appreciated that in a plant breeding history, researchers have found that homologous genes of a single gene in other varieties may play a unique role, thereby breaking cross-species and distant hybridization incompatibility problems using transgenic breeding techniques. For example Gao Lifen, an Xa21 gene system CX8621 is constructed by using agrobacterium-mediated transgenic technology, and the strain has high resistance to bacterial leaf blight of rice; zhang Xiaogong and the like, broad-spectrum disease-resistant genes Xa23 are introduced into target materials by using a transgenic technology, and a transgenic line carrying Xa23 and inheriting stably is finally obtained through multi-generation inoculation identification.
(3) The gene editing technology can change the original genome sequence by precisely modifying the target genome, and the technical means has great prospect in plant breeding at present. The TALENs, ZFN and CRISPR are the main technical means of the gene editing system and are relatively mature, and the invention can also be used on the basis of the prior artXa48And (t) carrying out gene editing breeding on the gene.
Furthermore, the invention also provides a method for regulating and controlling the bacterial leaf blight resistance of rice, which specifically comprises the step of increasing the expression level of a Xa48 (t) protein coding gene of the rice, wherein the nucleotide sequence of the Xa48 (t) protein coding gene is shown as SEQ ID NO. 2.
Further, the invention also provides a method for cultivating bacterial leaf blight resistant rice strains, which comprises the following steps:
s1: taking rice HNY expressing Xa48 (t) protein coding gene as a male parent; hybridization is carried out by taking Minghui 63, IRBB1 and yellow jade as female parent respectively;
s2: f1 generation seeds are harvested by hybridization, sowing and growing are continued until booting stage inoculation investigation and screening are carried out; culturing plants with the resistance phenotype of the medium resistance to seed collection to obtain F2 generation seeds, inoculating rice white leaf blight again, and screening the phenotype;
s3: and (3) continuously sowing rice with good resistance phenotype, growing to the booting stage, inoculating bacterial leaf blight bacteria, continuously selecting hybrid offspring with genetic stability, and harvesting F3 generation seeds.
RNAi interference technology, gene knockout technology and gene over-expression technology implemented by the invention treat target plants, and HNY is selected as backgroundXa48(T) Gene, knockout plant T 3 Generation, RNAi interference plant T 3 Substituted and against Japanese sunXa48(T) Gene-overexpressing plant T 3 The generation is a research object, and rice bacterial leaf blight inoculation investigation is carried out on the plants, and the results show that the knockout and the interference are realizedGeneXa48After (t), the resistance of the rice plant to bacterial leaf blight is obviously reduced, and the candidate gene is overexpressedXa48After (t), the resistance of rice plants to bacterial leaf blight is obviously enhanced.
According to the invention, through analysis of salt tolerance, drought tolerance and rice blast resistance of rice, the gene Xa48 (t) has no obvious effect on the aspect of salt tolerance and drought tolerance of rice, but has a certain effect in the process of rice blast resistance.
By analysis ofXa48(t) the action relationship with other disease-resistant related genes and hormone genes, and the gene is primarily defined to be controlled by JA pathway, so that PR gene expression is caused, and a plurality of types of disease-resistant reactions are mediated.
By hybridization of HNY with high-quality materials, 3 excellent bacterial blight-resistant strains with good resistance and multiple bacterial blight-resistant gene polymerization are created, and more than 1000 high-quality offspring rice seeds are harvested, so that the strain capable of being used for offspring population detection is developedXa48(t) a related molecular marker.
In conclusion, through excavation, separation cloning, functional analysis and germplasm creation research of bacterial leaf blight resistance genes, the research field of disease resistance genes is widened, disease resistance gene resource libraries are enriched, and a rich theory and material foundation is provided for rice disease resistance breeding.
Drawings
FIG. 1 is a graph showing the phenotypic identification effect of transgenic plants; (note: A: HNY andXa48(t) knock-out plant (900-9) lesion length comparison; b: HNY is compared with the length of the plant disease spots of JG 30; c: HNY compared to RNAi interference plant (G8-6) lesion length; d: nippon sunny and JapaneseXa48(t) overexpressing plants (H9-7) lesion length contrast);
FIG. 2 shows the effect of the relative expression amount of Xa48 (t) after spraying the exogenous hormone (note: A: after spraying GAXa48(t) relative expression in HNY; b: after JA sprayingXa48(t) relative expression in HNY; c: after spraying SAXa48(t) relative expression in HNY);
FIG. 3 is a graph showing the relative expression level of AOS1 gene under Xoo stress;
FIG. 4 is a graph showing the relative expression level of LOX gene under Xoo stress;
FIG. 5 shows analysis of relative expression levels of PAL genes under Xoo stress;
FIG. 6 is a graph showing the relative expression level of PR1A gene under Xoo stress;
FIG. 7 shows analysis of PR10 gene relative expression under Xoo stress.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The invention identifies a local rice seed from Yunnan Geng Ma county, oryza sativa (Haoneoyang, HNY) shows the resistance level above to bacterial leaf blight, uses HNY and a local variety of Jing 30 (Jing ang 30, JG 30) to hybridize and selfe to find that HNY is a local variety carrying a pair of dominant disease-resistant genes, uses BSA method to position the disease-resistant gene carried by HNY in the 0.12 Mb section between 22887903-23002998bp on the long arm end of chromosome 11 of rice, separates Xa48 (t) carried by clone HNY, uses HNY near isogenic line H197 (HNY near isogenic line H197 is the early stage of hybridization back to BC with Jing 30 (JG 30) as female parent and recurrent parent, HNY as male parent) 4 F 1 Instead, the BC is obtained by selfing 2 times 4 F 3 The generation stabilizing material, namely the near isogenic line with the number of H197) and the JG30 filial generation segregation population are used for fine positioning of Xa48 (t) and screening candidate genes. The resistance gene segment Xa48 (t) is further screened and obtained by the method of ORFs prediction, qRT-PCR expression level detection and transgenic plant functional complementation verification, the amino acid sequence of the resistance gene segment Xa48 (t) is shown as SEQ ID NO.1, and the nucleotide sequence of the gene encoding the resistance gene segment Xa48 is shown as SEQ ID NO. 2.
Example 1
Construction of rice RNAi plants
The construction reference of rice RNAi interference plants is set as two steps of target gene vector construction and genetic transformation (Xiaoping, wang Nan, kuang Xiaoming, etc.), and RNA interference is utilized to research the function of rice OsROSES1 gene [ J ]. University of West south university (Nature science edition), 2020, 42 (06): 1-10). Designing a specific primer by taking a sequence corresponding to Xa48 (t) as a template, performing PCR amplification by taking cDNA of HNY as a template (table 1), constructing a pBWA (V) HS: xa48 RNA interference vector, performing BsaI digestion on an amplified product (table 2), converting the digested ligation product into competent DH5 alpha (incubating competent cells pre-stored at-80 ℃ C. On ice to be nearly liquid, freezing by using liquid nitrogen for 1 minute, bathing at 37 ℃ C. And repeating the steps); a single colony with better growth condition is selected for 10 forward sequencing, and positive monoclonal conforming to the target is used for subsequent genetic transformation.
Selecting full and normal HNY seeds, sterilizing with 75% alcohol, transporting the plasmids and HNY seeds which are correctly identified in the early stage to a primary biotechnology company, entrusting the company to cultivate and screen HNY callus, infecting the callus by using a correctly identified competent cell suspension, transferring the callus with infection resistance to a newly configured culture medium (the culture medium and the culture process are completed by the primary biotechnology company) under a sterile environment, carrying out secondary screening until rooting, packaging and sealing, and culturing and growing in a greenhouse environment. DNA extracted from rice leaves growing to seedling stage is taken to detect Xa48 (T) gene, and RNAi plant T is detected correctly 0 Standing at room temperature for hardening seedlings for 2-3 days after the substitution and return, and transplanting into a greenhouse. RNAi T 0 Culturing the strain of the generation until the booting stage is inoculated with Xoo, then investigating the length of the lesion, taking tender leaves of the strain conforming to the target property, extracting DNA, and detecting the target gene Xa48 (t) by using a specific primer. RNAi plants are screened and marked, and seeds are harvested until the next year after the rice ears are mature, and the plants are sown again. The process is repeated for 2 times, and RNAi strain T is finally harvested 3 The seeds were used for this study after sowing and growth.
Note that: the enzyme digestion connection reaction system is that enzyme digestion connection is carried out for 2 hours at 37 DEG C
Example 2
Construction of rice over-expression plants
The construction of the rice over-expression plant comprises two steps of target gene vector construction and genetic transformation. Vector construction methods reference (Chen S, songakumarn P, liu J, et al A versatile zero background T-vector system for gene cloning and functional genomics [ J ]. Plant Physiol, 2009, 150 (3): 1111-1121): specific primers H1 (+) and H1 (-) are designed by taking a Japanese sunny sequence corresponding to a target gene Xa48 (t) on NCBI as a template, PCR amplification is carried out (table 3), an amplified product is named pBWA (V) HS-H1, double enzyme digestion treatment is carried out (restriction enzyme Eco31I is used for carrying out enzyme digestion on a vector, rDNAH1 is used for carrying out enzyme digestion on the vector for the second time, a reaction system is shown in tables 4 and 5), and the enzyme digestion product is recovered and purified, TA is connected to construct the vector, and the connection method is the construction of rice RNAi plants. Single colonies with better growth conditions are selected and sent to the primary and distant biotechnology limited company for sequencing, and positive monoclonal meeting the target is used for subsequent genetic transformation.
Selecting full and normal-color Japanese sunny seeds, sterilizing with 75% alcohol, transporting plasmids and Japanese sunny seeds which are correctly identified in the early stage to a primary biotechnology company, entrusting the company to cultivate and screen Japanese sunny callus, infecting the Japanese sunny callus by using a correctly identified competent cell suspension, transferring the infected callus to a newly configured rooting culture medium (the culture medium and the culture process are completed by the primary biotechnology company) under a sterile environment, carrying out secondary screening until rooting, packaging and sealing, and culturing and growing in a greenhouse environment. Extracting DNA from the over-expression plant growing to seedling stage and the leaf of contrast HNY, designing Xa48 (T) gene specific primer for amplification, and detecting the true or false of the over-expression plant to obtain the over-expression plant T 0 Instead, standing at room temperature for hardening seedlings for 2-3 days after returning, and transplanting into a greenhouse. Overexpression of plant T 0 And culturing the strain of the generation, inoculating Xoo during the booting stage, investigating the length of the lesion, taking and screening the over-expression strain which accords with experimental expectation, and harvesting seeds after the rice is mature until the next year for re-sowing. The process is cycled for 2 times, and the Xa48 (T) gene over-expression strain T is finally harvested 3 The seeds were used for this study after sowing and growth.
Note that: PCR reaction system in the construction of the over-expression plant vector: pre-denaturation for 5min (94 ℃); denaturation for 30 sec (94 ℃), annealing for 45sec (50 ℃), extension for 44sec (72 ℃), 30 cycles of this process; the final extension is carried out for 10min (72 ℃), and the amplified product is stored at 4 DEG C
Note that: the enzyme digestion connection reaction system is that enzyme digestion connection is carried out for 2 hours at 37 DEG C
Note that: the enzyme digestion connection reaction system is that enzyme digestion connection is carried out for 2 hours at 37 DEG C
Example 3
Construction of Rice Gene knockout Strain
Construction of the CRISPR/Cas9 vector of Xa48 (t) xylcrisprcas 9Pubi-H-osu3 (references Ma X, zhang Q, zhu Q, et al, a robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants [ J ]. Mol Plant, 2015, 8 (8): 1274-1284): specific primers G5-F and G5-R were designed using the corresponding Japanese sunny sequence of the target gene Xa48 (t) on NCBI as templates and PCR amplification was performed (Table 6), and the amplification system is shown in Table 4. The targets obtained by successful amplification were constructed as final vectors Hu-cas9pl-G5 (PT 2), respectively. A digestion reaction system (table 7) is prepared by taking T4 Buffer, a target, eco31I and T4 ligase synthesized by Bofar biological company as raw materials, carrying out digestion linking for 2h at 37 ℃, converting into escherichia coli competent cells through electric shock (incubating competent cells stored at-80 ℃ in advance on ice to be nearly liquid, freezing by using liquid nitrogen for 1 min in a water bath at 37 ℃ and repeating the steps), selecting correctly grown monoclonal thalli to coat a monoclonal antibody LB medium (kanamycin) after successful conversion, selecting and growing single bacterial plaques after a logarithmic growth period, carrying out sequencing identification by Bofar biological company, and storing the correctly identified competent cells in an environment at-80 ℃.
The genetic transformation of the transgenic plants is as follows: selecting full and normal HNY seeds, sterilizing with 75% alcohol, transporting the plasmids and HNY seeds which are correctly identified in the early stage to a primary biotechnology company, entrusting the company to cultivate and screen HNY callus, infecting the callus by using a correctly identified competent cell suspension, transferring the callus with resistance after infection to a newly configured rooting culture medium (the culture medium and the culture process are completed by the primary biotechnology company) under a sterile environment, carrying out secondary screening until rooting, packaging and sealing, and culturing and growing in a greenhouse environment. Taking rice leaves growing to a seedling stage to extract DNA (deoxyribonucleic acid) for knockout gene detection, and correctly detecting a gene knockout plant T 0 Placing the seedlings at room temperature for hardening for 2-3 days after the seedlings are sent back, and transplanting the seedlings into a greenhouse for use. Knockout of Gene T 0 And culturing the generation strain, inoculating Xoo during the booting stage, investigating the length of the lesion, taking tender leaves of the strain meeting the target property, extracting DNA, and detecting the existence of the target gene Xa48 (t) in the gene knockout plant by using a specific primer. Screening and marking gene knockout plants which meet the experiment expectations, and harvesting seeds until the second year after the rice ears are mature for re-sowing. The process is cycled for 2 times, and finally the Xa48 (T) gene knockout strain T is harvested 3 The seeds were used for this study after sowing and growth.
Note that: the amplification procedure was 94℃for 5min pre-denaturation, 94℃for 30 sec,50℃45sec,72℃for 16 sec (30 cycles), 72℃for 10min
Note that: enzyme digestion connection reaction system: 37 ℃ for 20 min 1 time, 37 ℃ for 10min for 5 times, 20 ℃ for 10min for 5 times, 37 ℃ for 20 min for 1 time, and 80 ℃ for 5min for 1 time.
In the present invention, examples 1, 2 and 3 were carried outFurther defineXa48(t) correlation with bacterial leaf blight resistance, RNAi intervention technique, gene knockout technique and Gene overexpression technique of the present invention treat target plants, selecting HNY as backgroundXa48(T) Gene, knockout plant T 3 Generation, RNAi interference plant T 3 Substituted and against Japanese sunXa48(T) Gene-overexpressing plant T 3 Instead, these plants were investigated for rice bacterial leaf blight inoculation (FIG. 1). The results show that: the disease-spot ratio of 146 rice bacterial leaf blight inoculated on the control material HNY is 7.146%, the disease-spot ratio of 123 rice bacterial leaf blight inoculated on the JG30 (adamantine 30) is 30.32%, the disease-spot ratio of 18.76% after rice bacterial leaf blight inoculated on the Japanese 87 strain, and the disease-spot ratio of 16.77% for 21 rice bacterial leaf blight inoculated on the Xa48 (t) gene knockout plants 900-9; the RNAi interference plant G8-6 of Xa48 (t) gene has a total of 96 plants inoculated with rice bacterial leaf blight and the disease-spot ratio of 14.11%, and the overexpression plant H9-7 has a total of 71 plants inoculated with rice bacterial leaf blight and the disease-spot ratio of 6.45%. The rice plant resistance to bacterial leaf blight is obviously reduced after knocking out and interfering the gene Xa48 (t), and the rice plant resistance to bacterial leaf blight is obviously enhanced after over-expressing the candidate gene Xa48 (t).
Example 4
Salt stress, drought stress and rice blast inoculation experiment
Salt tolerance determination: 100 rice seeds were cultivated in water with or without 150 mmol NaCl. Germination was carried out at 28 ℃/25 ℃ (day/night) for 6 days with a photoperiod of 12 hours, and the seedling height and fresh weight of each line were determined in triplicate and averaged. Analysis of experimental data refers to an analytical method of first-friend formation and the like (first-friend formation, yan Youwei, feng Mengshi, and the like, salt resistance identification of the rice bud stage and the seedling stage of the transgenic OsEBP-89 gene [ J ]. Tianjin agriculture science, 2014, 20 (10): 1-4), and salt tolerance of plants is judged according to relative salt damage rate.
Drought tolerance assay: 10 seedlings with the growth condition similar to that of 4 weeks are taken and placed in the same growth environment, drought stress is carried out for 12 days, and water is poured again to restore normal water supply for 7 days. And (3) after 12 days of rehydration growth, taking the plants with green and healthy young leaves as survival plants, and calculating the survival rate of the plants, namely the survival plants account for the total number of the treated plants.
The rice blast inoculation identification method comprises the steps of preparing quantitative oat agar culture medium, sterilizing for 15 minutes, placing rice blast bacteria on the oat culture medium under a sterile environment, culturing until mycelia are fully distributed on a culture dish (usually for 5-7 days), carefully wiping aerial mycelia, performing moisturizing culture, and waiting for spore production in a dark environment. Preparing a rice blast fungus spore suspension, diluting and subpackaging until spraying is carried out to inoculate rice blast fungus on rice plants in a 3.5-4.0 leaf period. And (3) placing the material in a plant artificial climate box at 24-26 ℃ for dark culture for 18-20 hours, transferring to a greenhouse for routine management, investigating the disease condition according to disease index standards after 8 days, investigating and judging the anti-infection condition, and repeating the test for 2 times.
The invention example 4 further investigated the effect of Xa48 (t) on salt stress, drought stress and rice blast resistance and, based on the results of the fine localization and isolation cloning studies, the effect of Xa48 (t) on other resistance functions.
In the salt stress test, the strength of salt tolerance of plants was determined based on the relative salt damage rate by using plants overexpressing Xa48 (t) in the presence of Nippon and Nippon as the background (Table 8). The results show that the over-expression plant with 150 mmol NaCl stress has no obvious difference in root length, seedling height and comprehensive relative salt damage rate compared with the control Japanese sunny.
Note that: h9-3 is strain 3 of Xa48 (t) overexpressing plants against the background of Nipponbare; h9-5 is strain 5 of Xa48 (t) overexpressing plants against the background of Nipponbare; h9-11 is line 11 of Xa48 (t) overexpressing plants against Japanese sunny background; h9-16 is line 16 of Xa48 (t) overexpressing plants against Japanese sunny background
In drought stress experiments, plant survival rates were calculated from plants grown in water for 12 days with green healthy young leaves as surviving plants using Nippon and Xa48 (t) overexpressing plants in the background of Nippon as subjects, and the results are shown in the following Table (Table 9): the survival rate of the over-expressed plants H9-7 and H9-16 is lower than 80% of that of a control, the survival rates of H9-3 and H9-9 are 100% higher than that of the control Japanese sunny, and the average survival rate of the over-expressed strains is not obviously different from that of the control.
Note that: h9-3 is strain 3 of Xa48 (t) overexpressing plants against the background of Nipponbare; h9-7 is line 5 of the Xa48 (t) expressing plant against the sun; h9-9 is line 11 of Xa48 (t) overexpressing plants against Japanese sunny background; h9-16 is line 16 of Xa48 (t) overexpressing plants against the Japanese sunny background.
In rice blast inoculation experiments, the experimental results of the RNAi interference plants of Xa48 (t) on the background of Nippon and on the background of HNY, HNY and Xa48 (t) on the background of Nippon are shown in Table 10: the incidence rate of rice blast of Xa48 (t) gene over-expressed plants is generally lower than that of control Japanese sunny, xa48 (t) gene knockout plants and Xa48 (t) gene RNAi interference plants are higher than that of control HNY, and the Xa48 (t) gene enhances the resistance of rice plants to rice blast.
Note that: h9-3 is strain 3 of Xa48 (t) overexpressing plants against the background of Nipponbare; h9-5 is strain 5 of Xa48 (t) overexpressing plants against the background of Nipponbare; h9-11 is line 11 of Xa48 (t) overexpressing plants against Japanese sunny background; h9-16 is line 16 of Xa48 (t) overexpressing plants against the sun; g9-1, G5-4, G4-1, G8-6 are transgenic plants numbered 1 of strain 9, transgenic plants numbered 4 of strain 5, transgenic plants numbered 1 of strain 4, transgenic plants numbered 6 of strain 8 of Xa48 (t) after RNAi in the background of milli-glutinous.
Example 5
Disease-resistant germplasm creation
HNY is used as male parent, and Minghui 63, IRBB1 and yellow jade which are used as female parent are used for hybridization. Hybrid harvesting F 1 And continuing sowing and growing the generation seeds until the booting stage is inoculated for investigation and screening. Culturing the plants with the resistance phenotype of more than medium resistance to seed collection to obtain F 2 Seed generation, re-inoculating rice white leaf cake and screening phenotype. Sowing rice with good resistance, growing to gestation period, inoculating bacterial leaf blight bacteria, researching genetic stability of filial generation and harvesting F 3 Seed generation.
Evaluation of bacterial leaf blight resistance of Rice in booting stage
Population of offspring to be crossed F 3 Instead of growing to the booting stage, bacterial leaf blight is inoculated, the length of the rice lesion (the length from the inoculation position to the middle stem margin of the wilt leaves) is investigated after 21 days, and the average lesion length or average lesion area of each plant is calculated. Inoculating and identifying the filial generation by using bacterial strain of rice PXO99, wherein the disease spot length and the disease spot ratio are shown in Table 11:
note that: minghui 63 is a indica type conventional rice, bred by Sanming city agricultural science institute, and carries a bacterial leaf blight resistance geneXa26(t); IRBB1 is a bacterial leaf blight resisting gene bred by International institute of riceXa1Is a near isogenic line of (2); yellow jade, indica type conventional rice carrying bacterial leaf blight resistance geneXa1，Xa12。
The minimum disease spot ratio of HNY XMinghui 63 after inoculation of rice bacterial leaf blight is 11.3%, the next is HNY Xyellow jade is 12.3%, and the highest disease spot ratio in the offspring group is HNY XIRBB 1 is 13.6%. According to the classification standards described above, the disease spot ratio of the three materials inoculated with PXO99 is obviously lower than that of a disease-sensitive variety JG30, and the three materials all belong to the resistance to bacterial leaf blight, which shows that the filial generation carrying Xa48 (t) has the resistance to the bacterial leaf blight, and the filial generation population materials can be directly used as disease-resistant strain materials for bacterial leaf blight in rice production.
Agricultural character investigation of rice hybrid offspring material
The results of investigation and statistics of the average values (plant height, rice spike length, spike neck length, total grain number per spike, solid grain number per spike, and seed setting rate) of 5 agronomic traits of 28 plants in total of 3 hybrid offspring against bacterial leaf blight are shown in Table 12:
final statistical co-harvest of hybrid offspring F 3 Seed 893 grains, wherein HNY ×Minghui 63 total 414 grains, HNY ×topaz total 230 grains, HNY ×irbb1 total 249 grains; hybrid offspring F 3 Seed 641, wherein HNY ×Minghui 63 total 314, HNY ×topaz total 130 and HNY ×irbb1 total 197. According to the table, the agronomic traits of the hybrid offspring were shown to be in the normal range, and it was seen that Xa48 (t) did not alter other agronomic traits between varieties.
Example 6
Xa48(t) analysis of relationship between the gene and the disease resistance-related Gene
In order to investigate whether Xa48 (t) mediates plant endogenous hormone genes and other disease-resistant related genes to participate in disease-resistant reaction, the invention designs specific primers for PR1A genes and PR10 genes in key genes AOS1, LOX genes, PAL genes and PR genes of rice endogenous hormone signal paths according to literature data as shown in table 13, and performs qRT-PCR analysis on Xa48 (t) knocked-out plants with HNY, JG30, nippon and HNY as background and Xa48 (t) overexpressed plants with Nippon as background, and the relation between Xa48 (t) and the genes is investigated, and the result is as follows:
after bacterial leaf blight bacteria are inoculated, the AOS1 gene and the PR10 gene can be positively regulated by Xa48 (t), compared with a control, the relative expression quantity of the AOS1 gene and the PR10 gene is down-regulated after Xa48 (t) is knocked out, and the relative expression quantity of the AOS1 gene and the PR10 gene is up-regulated after Xa48 (t) is over-expressed (figures 3 and 7), which shows that Xa48 (t) can positively regulate and trigger the AOS1 gene and the PR10 gene to participate in plant disease resistance reaction together.
After bacterial leaf blight bacteria are inoculated, the correlation between the LOX gene, the PAL gene and PR1A and Xa48 (t) is not large, and compared with a control, the correlation between the relative expression quantity of the LOX gene and the PAL gene and Xa48 (t) is not obvious after the gene Xa48 (t) is knocked out and overexpressed (figures 4, 5 and 6), so that the correlation between the Xa48 (t) mediated disease resistance reaction and the LOX gene and the PAL gene is not obvious.
Example 7
Analysis of relationship between Xa48 (t) and exogenous hormone
In order to explore whether Xa48 (t) is regulated and controlled by plant exogenous hormone genes JA (jasmonic acid), SA (salicylic acid) and GA (gibberellin), the invention utilizes the mixed dilution of response exogenous hormones SA, JA and GA to dilute the mixture to the final concentration of: JA 100 umol/L, SA 100 umol/L, GA umol/L, the diluted liquid was dispensed into a spray can and evenly sprayed on rice plants, and then qRT-PCR analysis was performed, the primer sequences are shown in Table 14, and the relationship between Xa48 (t) and rice response exogenous hormone was investigated (FIG. 2), and the results were as follows: xa48 (t) is positively regulated by GA, JA and SA, and the relative expression quantity of the gene is obviously increased after 2-8 h are treated.
The partial operations in the above embodiments are specifically:
1. cultivation and field management of rice materials
Putting the rice seeds to be tested into a culture dish subjected to pre-sterilization treatment, soaking and accelerating germination by using distilled water, standing in a dark room temperature environment until the seeds are exposed to white, selecting germinated seeds, putting the germinated seeds into a seedling water culture box for root accelerating, regulating the temperature of an illumination incubator to 28 ℃, standing and culturing, and adding water to the top of the seedling water culture box during 2-3 days. And (3) when the rice is cultured to a three-to-four-leaf stage, the rice is moved to a room temperature environment and placed for 3 days for hardening seedlings, then the rice is transplanted to a field for transplanting seedlings, and the rice is cultured to a booting stage for inoculation Xoo identification investigation.
2. Culture medium and other reagents
Nutrient agar medium (NA medium) formulation: yeast extract 1 g, sucrose 12 g, agar powder 18 g, peptone 5 g, beef extract 3 g; distilled water is added to 1L, the pH is regulated to 6.8-7.0, and the mixture is stirred uniformly, and then the mixture is injected: the optimal growth pH value of bacterial leaf blight bacteria is 6.8-7.0, and the bacterial leaf blight bacteria are autoclaved for 15 min at 121 ℃.
The formula of the oat culture medium is as follows: 3g of oat, 10g of sucrose and 16-20 g of agar powder g, adding distilled water to about 1L, adjusting the pH of the mixed solution to 6.8-7.0, subpackaging, and sterilizing at 121 ℃ for 15 min.
LB medium: active yeast extract 3 g; tryptone 12 g; solid sodium chloride 8 g; distilled water 1L, pH value is regulated to 6.9-7.0, and single or compound antibiotics can be added according to experimental requirements.
In the embodiment, mix, plant RNA extraction kit and real-time fluorescent dye reagent used for PCR amplification are provided by Nanjinouzan biological company; the plasmid extracting set and the RNA reverse transcription kit are provided by Tiangen scientific biology Co., ltd; TAE, TBE, CTAB the screening process from RNAi plant, over-expression plant and callus of gene knockout plant to seedling is completed by Boyu biotechnology company, which adopts conventional rice culture technique.
3. Cultivation, inoculation and investigation of pathogenic bacteria
The Xoo strain is usually stored in an environment of-80 ℃, 20-ul dispersed and dripped on NA culture medium for rejuvenation at 28 ℃ by a pre-sterilized pipetting gun, bright yellow colonies are formed on the surface of the culture medium after 3 days of culture, bacterial leaf blight bacteria are suspended by sterile water, and diluted to OD 600 The value is 0.7-1.0.
The rice material in booting stage is inoculated with the bacterial wilt, the sterilized clean scissors are dipped with the bacterial liquid, the position with the length of 2 cm to 3cm of the tip end of the rice sword blade is used as the optimal inoculation port, and 7 sword blades are inoculated by a single plant for inoculation. And re-dipping the bacterial liquid when the bacterial liquid is inoculated for 3 times or the bacterial liquid of the scissor edge is slightly dry. After inoculation, the rice plants were placed in an environment of 28-30 ℃ and grown for 21 days. After determining The disease condition, the length of The lesion (length from The inoculation site to The middle stalk margin of The wilt leaves) after 21 days of inoculation of The rice was investigated, and The average lesion length or average lesion area per plant was calculated (Hong Y, liu Q, cao Y, et al, the OsMPK15 negatively regulates magnaporthe oryza and Xoo disease resistance via SA and JA signaling pathway in rice [ J ]. Frontiers in plant science, 2019, 10:752).
4. Extraction of genomic DNA from rice
Using the CTAB method: cutting young rice leaves as much as possible, placing the young rice leaves into a centrifuge tube, adding 2 pre-sterilized steel balls, symmetrically placing the centrifuge tube into a freezing crusher for freezing and vibrating treatment to powder, adding 800 mu L of 1.5 XCTAB buffer solution preheated at 65 ℃ and slightly reversing the solution for 6-8 times repeatedly, placing the solution into a water bath kettle for mild soaking for 30 min, taking out the centrifuge tube at intervals of 5min, and slightly reversing the top and bottom of the centrifuge tube for several times; adding the following steps of: 1, about 600-800 mu L of the prepared chloroform/isoamyl alcohol mixed solution is slowly shaken until milky floccule is suspended in the mixed solution; centrifuging at-4deg.C for 10min (8000-10000 r), and slightly sucking supernatant 600 ul by pre-sterilized pipette, wherein plant tissue is not easy to adsorb; adding equal volume of isopropanol into the supernatant by a pipetting gun, slightly blowing and shaking, standing and refrigerating the mixed solution in an environment of-20 ℃ for preferably 30-90 min; 10000-12600 r, wherein white solid DNA is attached to the bottom of the centrifuge tube, and the supernatant is discarded; repeatedly adding 400-600 mu L of 75% ethanol twice to rinse the solution to be transparent for the last time, and sucking out waste liquid at the bottom of the centrifuge tube; standing the centrifuge tube in an air circulation environment overnight; finally, adding proper sterile ddH according to the precipitation amount 2 O-dissolution (typically 30-50 ul).
5. PCR reaction system and amplification program
Other PCR reactions not specifically described in the present invention, the reaction systems are shown in the following table, and the amplification procedure is: pre-denaturation for 2 min (98 ℃); denaturation for 10 sec (98 ℃), annealing for 15sec (Tm ℃) and extension for 15sec (72 ℃), 33 cycles of this process; the final extension was carried out for 5min (72 ℃) and the amplified product was stored at 4 ℃.
PCR reaction system: the amount (50. Mu.L system), 2X Flash Hot Start MasterMix (Dye) 25. Mu.L; forward primer (10. Mu.M) 2. Mu.L; reverse primer (10. Mu.M) 2. Mu.L; 1. Mu.L of Template DNA; ddH 2 O20. Mu.L, note: when the target fragment cannot be amplified and obtained, the gradient method can be used for changing the annealing temperature to explore the optimal annealing temperature of the target fragment。
6. Agarose gel electrophoresis experiments
120 g ul distilled water is measured and 1.2g agarose is added, and the mixture is heated until the solid particles are dissolved; cooling to 50deg.C, adding 1ul nucleic acid dye, mixing, and pouring into gel tank (preventing bubbles from floating on the surface of colloid during pouring); sucking the PCR product of 3-5 ul with a pipette, and performing 180V electrophoresis for about 30 min until the strips are uniformly dispersed; the gel was slightly moved in an ultraviolet projection imager and a suitable aperture focus scan was selected for taking a picture.
7. Extraction and reverse transcription of RNA
The extraction of RNA in the study is carried out according to the specification steps of the kit R701, so that the condition that the used articles are not polluted by RNase in the experimental process is ensured, and the extraction method refers to the accompanying specification of the product: cutting and split charging rice leaves into a pre-sterilized mortar, rapidly grinding into powder by using liquid nitrogen or a freezing crusher, adding 500 mu LRNA-easy lysate, and carrying out severe shaking to enable a sample to be fully cracked; 200. Mu.L ddH was added to the lysate 2 Mixing and standing O (RNase-free); 15 Centrifuging at high speed for min to enable the powdery leaves to sink to the bottom; taking 650 mu L of isopropanol, slightly blowing and mixing; centrifuging at high speed for 5min after the milk yellow floccule is suspended, and discarding the supernatant; rinsing twice with 75% ethanol of 300-600 ul until the rinsing liquid is transparent; after 9000r centrifugation for 3 min, white sediment is attached to the bottom of the centrifugal tube; pouring the waste liquid carefully, placing the mouth of the centrifuge tube at an open bevel for 5min at room temperature, and adding appropriate ddH according to the precipitation amount 2 O (RNA-Free) was blown to pellet dissolution (low temperature or ice handling) using a pipette and the resulting RNA solution was stored at-80 ℃.
The cDNA required in this study was obtained by RNA reverse transcription, which was performed using the procedure described in kit KR116, and the cDNA solution obtained from the reaction was stored at-20℃until use. RNA reverse transcription procedure: 42. incubating for 18min at the temperature; 95. the cDNA solution obtained by reverse transcription is preserved at-20 ℃ for standby at-3 min. RNA reverse transcription reaction system: the amount (20. Mu.L system) of R Buffer 2. Mu.L; total RNA 1. Mu.L; ddH 2 O (RNA-Free) 12 μL；KRT Buffer2 μL；FRTE Mix1 μL；FQP Mix1 μL；FQP Mix2 μL。
Claims (10)
1. The application of the rice Xa48 (t) protein is characterized in that the amino acid sequence of the rice Xa48 (t) protein is shown as SEQ ID NO.1, and the application is any one of the following:
(1) Enhancing the resistance of rice to bacterial leaf blight;
(2) Enhancing the resistance of rice to rice blast;
(3) And (6) breeding rice disease resistance.
2. A gene encoding the Xa48 (t) protein of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 2.
3. A primer set for amplifying a gene of the rice Xa48 (t) protein of claim 2.
4. Use of a gene according to claim 2 or a primer pair according to claim 3 in any of the following:
(1) Identifying or assisting in identifying bacterial leaf blight resistance of rice;
(2) Preparing a product for identifying or assisting in identifying rice bacterial leaf blight resistance;
(3) Screening or breeding single plants or lines or varieties of rice with bacterial leaf blight resistance;
(4) Screening or breeding single plants or strains or varieties of rice with bacterial leaf blight;
(5) Preparing a product for screening or breeding single plants or lines or varieties of rice with bacterial leaf blight resistance;
(6) And (3) preparing a product for screening or breeding single plants or lines or varieties of rice with bacterial leaf blight.
5. The use of the rice Xa48 (t) protein according to claim 1, wherein the use of rice disease-resistant breeding comprises any of the following:
(1) MAS breeding of bacterial leaf blight resistance genes;
(2) Transgenic breeding of bacterial leaf blight resistance genes;
(3) And (3) carrying out gene editing breeding on the bacterial leaf blight resistance genes.
6. A method for regulating resistance of rice to bacterial leaf blight is characterized by improving expression level of Xa48 (t) protein coding genes of rice, wherein the nucleotide sequence of the Xa48 (t) protein coding genes is shown as SEQ ID NO. 2.
7. The method for regulating resistance to bacterial leaf blight of rice as defined in claim 6, wherein the method for increasing expression level of Xa48 (t) protein-encoding gene of rice comprises: constructing Xa48 (t) protein coding genes on a transgenic vector, and transferring the transgenic vector into other rice by a genetic transformation method to obtain an over-expressed bacterial leaf blight-resistant rice strain.
8. The method for regulating resistance to bacterial leaf blight of rice as defined in claim 6, wherein the method for increasing expression level of Xa48 (t) protein-encoding gene of rice comprises: spraying exogenous hormones JA, SA and GA for regulating and controlling.
9. The method for controlling bacterial leaf blight resistance of rice according to claim 6, wherein rice overexpressing Xa48 (t) protein encoding gene is crossed with other rice lines to cultivate bacterial leaf blight-resistant rice lines.
10. The method according to claim 9, wherein the method for cultivating the bacterial leaf blight resistant rice strain comprises the following steps:
s1: taking rice HNY expressing Xa48 (t) protein coding gene as a male parent; hybridization is carried out by taking Minghui 63, IRBB1 and yellow jade as female parent respectively;
s2: f1 generation seeds are harvested by hybridization, sowing and growing are continued until booting stage inoculation investigation and screening are carried out; culturing plants with the resistance phenotype of the medium resistance to seed collection to obtain F2 generation seeds, inoculating rice white leaf blight again, and screening the phenotype;
s3: and (3) continuously sowing rice with good resistance phenotype, growing to the booting stage, inoculating bacterial leaf blight bacteria, continuously selecting hybrid offspring with genetic stability, and harvesting F3 generation seeds.
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