CN117551660A - Rice blast resistance related gene OsMAPKKK19 and application thereof - Google Patents
Rice blast resistance related gene OsMAPKKK19 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering. In particular to cloning, functional verification and application of a related gene OsMAPKKK19 for mediating rice blast resistance, which discovers and proves the positive regulation and control effect of the OsMAPKKK19 gene in rice blast resistance reaction for the first time, the resistance of rice to rice blast can be obviously enhanced by improving the expression level of the OsMAPKKK19 through a genetic engineering method, and the field agronomic character statistics discovers that over-expression of the OsMAPKKK19 does not influence the normal growth and development of rice.
Description
Technical Field
The invention relates to the field of rice disease resistance breeding, in particular to a rice blast resistance related gene OsMAPKKK19 and application thereof.
Background
Rice (Oryza sativa) is one of the most prominent food crops in the world. However, stable production and yield increase of rice are always threatened by various diseases, wherein rice blast caused by rice blast germ (Magnaporthe oryzae) is one of three diseases of rice, and rice areas all over the world occur, which not only affects the yield of rice, but also affects the quality of rice. Practice proves that breeding and popularizing new rice varieties with durable and broad-spectrum resistance is one of the most effective methods for preventing and controlling rice blast.
Long-term threat from pathogenic bacteria plants evolved a complex and elaborate set of immune systems that recognize and defend against pathogenic bacteria, which can be divided into two layers. The immune response elicited by PAMPs (pathogen-associated molecular patterns), the conserved component of pathogenic bacteria, is called PTI (PAMP-triggered immunity), which is the first layer of innate immunity in plants, with broad spectrum and persistence; however, some pathogens successfully infect hosts and release effector (effector) into the host cell to inhibit PTI. In the face of a new round of challenges, plants evolve a second layer of defense system, and through the intracellular disease-resistant proteins, effector factors are specifically identified, downstream disease-resistant related reactions are activated, blocking of PTI by the effector factors is relieved, so that the capability of resisting pathogen infection is regained, and the second layer of immune reactions, also called ETI (effector triggered immunity), are very specific.
More and more rice blast resistance genes have been cloned, such as NLR proteins Pigm, pi9, pik, pi-zt, pib, etc., and non-NLR proteins such as Bsr-d1, pid2, ptr, etc., some of which have been widely used in disease-resistant breeding. However, the mutation rate of Pyricularia oryzae is high, and some resistance genes are rapidly disabled, so that cloning of a novel broad-spectrum resistance gene is required to satisfy the emergence of novel Pyricularia oryzae strains.
In the early experiments of the inventor, the gene OsMAPKKK19 is up-regulated after being induced by rice blast bacteria, and the excessive expression in tobacco can induce the tobacco leaves to generate cell death, and the rice blast resistance of rice can be obviously enhanced by improving the expression level of the gene OsMAPKKK19 in the rice through genetic engineering, so that the gene OsMAPKKK19 has the foundation for improving the rice blast resistance of the rice.
Disclosure of Invention
In order to clone a novel broad-spectrum resistance gene to meet the appearance of a novel rice blast bacterial strain, the invention provides a rice blast related gene OsMAPKKK19 and performs functional identification on the gene.
The technical scheme of the invention is as follows:
the invention provides a rice blast resistance related gene OsMAPKKK19, wherein the CDS sequence of the OsMAPKKK19 gene is shown as SEQ ID NO.1, and the total length of the CDS sequence is 2073bp.
The invention provides a protein coded by a rice blast resistance related gene OsMAPKKK19, the amino acid sequence of which is shown as SEQ ID NO.2, and 690 amino acids are coded.
The invention also provides application of the rice blast resistance related gene OsMAPKKK19 or the protein encoded by the rice blast resistance related gene OsMAPKKK19 in improving plant disease resistance, and overexpression of the related gene OsMAPKKK19 or the protein encoded by the related gene OsMAPKKK19 in plants is performed to activate plant immune response.
The invention also provides application of the rice blast resistance related gene OsMAPKKK19 or a protein encoded by the rice blast resistance related gene OsMAPKKK19 in improving rice blast resistance, and after being infected by rice blast bacteria, the expression of the protein encoded by the related gene OsMAPKKK19 or the related gene OsMAPKKK19 is obviously increased.
The invention has the beneficial effects that:
(1) According to the invention, through analysis and screening of rice and rice blast fungus interaction expression patterns, MAPKKK gene OsMAPKKK19 which is induced to be expressed by rice blast fungus infection is found, the expression level of the MAPKKK gene OsMAPKKK19 is up-regulated by approximately 5 times compared with that of water treatment after being induced by rice blast fungus infection for 48 hours, the function of the rice blast resistance gene OsMAPKKK19 is identified, and through Punch inoculation and rice blast fungus growth detection, the over-expression of the OsMAPKKK19 can enhance the rice blast resistance, the positive regulation effect of the OsMAPKKK19 gene in rice blast resistance reaction is discovered and proved for the first time, and the OsMAPKKK19 gene is an excellent candidate gene for creating transgenic rice and crop molecular design with enhanced rice blast resistance, so that the method has important theoretical value and wide application prospect;
(2) In production practice, the expression level of the cloned OsMAPKKK19 gene in the flower 11 (ZH 11) in the rice variety can be improved by a genetic engineering method, the resistance of the rice to rice blast can be obviously enhanced, and the rice blast resistance gene OsMAPKKK19 provided by the invention can be applied to the genetic improvement of related agricultural crops such as rice and the like by researching main agronomic characters such as plant height, effective tillering number, grain length, grain width and the like of an OsMAPKKK19 gene over-expression plant through field agronomic character statistics, and the like, and the functional deletion of the OsMAPKKK19 is found not to influence the growth and development of the rice.
Drawings
The sequence table SEQ ID NO.1 is the CDS sequence of the cloned OsMAPKKK19 gene;
the sequence table SEQ ID NO.2 is the amino acid sequence of the protein encoded by the cloned OsMAPKKK19 gene of the invention;
FIG. 1 is a graph showing the expression analysis of OsMAPKKK19 gene in example 2 of the present invention after infection with Pyricularia oryzae;
FIG. 2 is a graph showing the detection of cell death caused by overexpression of the OsMAPKKK19 gene in tobacco leaves in example 3 of the present invention;
FIG. 3 is a graph showing the identification of disease resistance phenotype of rice cultivar of example 4 over-expressing OsMAPKKK19 in flower 11 (ZH 11);
FIG. 4 is a diagram showing the main agronomic trait analysis of the overexpression of OsMAPKKK19 in rice cultivar ZH11 in example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples, but is not limited thereto, wherein rice cultivar ZH11 and Pyricularia oryzae strain Guy11 are the subjects of the present invention.
Example 1
In the embodiment, a rice blast resistance related gene OsMAPKKK19 is provided, the CDS sequence of the OsMAPKKK19 gene is shown as SEQ ID NO.1, and the total length of the CDS sequence is 2073bp; the protein coded by the rice blast resistance related gene OsMAPKKK19 has an amino acid sequence shown in SEQ ID NO.2, and codes 690 amino acids.
Example 2
Expression of OsMAPKKK19 Gene induced by Pyricularia oryzae infection
(1) Materials and methods
The rice cultivation method comprises the following steps: after the wild rice ZH11 is soaked for germination acceleration, the wild rice ZH11 is directly planted in a flowerpot containing nutrient soil, and is cultivated in a greenhouse with illumination at 28 ℃ for 14 hours and darkness for 10 hours, and proper moisture is ensured during cultivation, so that diseases and insects are avoided;
preparing a rice blast fungus spore liquid: placing the preserved Pyricularia oryzae filter paper sheet in CM solid culture medium, culturing at 28deg.C in dark at constant temperature, cutting the culture medium with Pyricularia oryzae into small pieces with sterile scalpel at ultra clean bench when Pyricularia oryzae growth area reaches about half of culture dish area, and transferring into rice bran culture medium for continuous culture. After rice blast fungus hypha covers more than 3/4 area of rice bran culture medium, the mycelium on the surface of the culture medium is gently scraped off by a sterile glass sheet on an ultra clean bench, and the culture is continued for 3-5 days by illuminating an incubator at 25 ℃ to promote spore production. Preparing 0.2% Tween-20 solution, pouring into rice bran culture medium, slightly scraping with glass sheet, eluting spores, filtering spores, and counting with blood cell counting plate until spore concentration reaches 1×10 5 Individual/ml;
spraying inoculation and sampling: the prepared concentration is 1×10 5 The bacterial liquid of each/ml is evenly sprayed on the rice leaves with the seedling age of 20 days, so that the rice leaves are ensured to be fully hung with spore liquid, and the rice leaves are continuously cultivated under the conditions of 26 ℃ and 12h illumination and 12h darkness after being subjected to dark treatment at 26 ℃ for 24 hours in a high humidity environment, and the environment is kept high humidity during the period. Samples were taken before and 24h and 48h after spraying, respectively, while samples treated with water served as controls. And extracting total RNA of the sample by adopting a Trizol method, reversely transcribing the total RNA into cDNA, and carrying out quantitative PCR analysis by utilizing a specific primer of the OsMAPKKK19 gene.
(2) Results and analysis
Referring to FIG. 1, the OsMAPKKK19 gene in rice was significantly higher than the expression level in the water-treated sample at 48h after Pyricularia oryzae treatment, indicating that the expression of OsMAPKKK19 gene was induced by Pyricularia oryzae infection.
Example 3
Overexpression of the OsMAPKKK19 gene in tobacco leaves causes cell death
(1) Materials and methods
The OsMAPKKK19 gene and the positive control genes 1 and 2 were ligated to pCAMBIA1300-GFP vector, and tobacco was injected after transformation of Agrobacterium. After two days, taking the leaf after the cell death of the tobacco leaf, and photographing. The photographed leaves are placed at the bottom of a 50mL centrifuge tube, a proper amount of trypan blue dye solution is added, the leaves are immersed, and the leaves are taken out and kept stand for 1h after being boiled in boiling water for 5-10 min. Pouring trypan blue dye liquor, adding clear water for rinsing, adding chloral hydrate decolorization liquor after rinsing, decolorizing for 6 hours, replacing the decolorization liquor every two hours, and photographing and observing after the leaves are transparent;
(2) Results and analysis
Referring to FIG. 2, the areas injected with OsMAPKKK19-GFP in Nicotiana benthamiana leaves showed gray dead spots as the areas injected with positive control 1 and positive control 2, while the areas injected with GFP protein showed normal green (FIG. 2 a); after trypan blue staining, the grey areas on the leaves were stained blue (fig. 2 b), indicating that the grey areas were indeed cell death;
in addition, GFP, positive control 1, positive control 2 and OsMAPKKK19-GFP proteins were all normally expressed in the tobacco (FIG. 2 c), which indicates that overexpression of the OsMAPKKK19 gene in tobacco induces cell death of tobacco leaves and activates the immune response of tobacco.
Example 4
Overexpression of OsMAPKKK19 increases rice resistance to rice blast
(1) Materials and methods
Detection of expression level of OsMAPKKK19 in OsMAPKKK19 overexpressing plant: respectively taking 5-strain blades of ZH11, OE-OsMAPKKK19-1 and OE-OsMAPKKK19-2 with seedling age of 3 weeks, extracting total RNA, performing reverse transcription to cDNA, and detecting the expression level of OsMAPKKK19 in the over-expressed plant by using real-time fluorescence quantitative PCR;
punching and inoculating: taking ZH11, OE-OsMAPKKK19-1, OE-OsMAPKKK19-2 grown for about 1 month, lightly pressing with a puncher at 1/3 position of blade from blade tip to form wound, and collecting 10 μl with concentration of 1×10 5 Per ml of Pyricularia oryzae spore liquid, drop-wise onto the surface of leaf at the perforation site, and seal with tape. Will be inoculated with bacteriaThe rice is placed in a greenhouse at 26 ℃ for dark treatment for 24 hours, then is placed in 12 hours of illumination and 12 hours of darkness and is continuously cultivated for 6 to 8 days under the high humidity condition, and then the leaf spot size is counted;
determination of Magnaporthe grisea biomass: the method comprises the steps of selecting equivalent leaves containing complete lesions, extracting genome DNA of rice, measuring concentration, taking the Ubiquitin of the rice and the post 2 of the rice blast fungus as internal reference genes, and detecting the growth amount of the rice blast fungus in the rice diseased leaves through qPCR of DNA level.
(2) Results and analysis
Referring to FIG. 3, FIG. 3a shows the detection of OsMAPKKK19 expression levels in OsMAPKKK19 overexpressing plants, using 3 week old seedlings as material, extracting total RNA, reverse transcribing to cDNA, and qRT-PCR analysis using primers specific for OsMAPKKK19, where OE-OsMAPKKK19-1 and OE-OsMAPKKK19-2 are two different strains, and wild-type ZH11 is a control; FIG. 3b shows that ZH11, OE-OsMAPKKK19-1 and OE-OsMAPKKK19-2 were inoculated with Pyricularia oryzae strain Guy11 by a well-perforated inoculation method, and after inoculation for 7 days, observed and photographed, bar=1 cm; FIG. 3c is a quantitative analysis of the amount of Pyricularia oryzae growth in the disease leaves by the punch inoculation method;
as can be seen from FIG. 3, the expression level of the OsMAPKKK19 gene in the over-expressed plants is up-regulated by 2-3 times (FIG. 3 a) relative to the wild-type ZH11, and the over-expressed materials meet the experimental requirements; the perforation inoculation experiment result shows that compared with a wild type control, the area of the lesion is smaller after the plant with the over-expressed OsMAPKKK19 gene is inoculated, and the amount of the rice blast in the diseased leaves is smaller (as shown in figures 3b and 3 c), which proves that the over-expressed OsMAPKKK19 gene can improve the resistance of rice to rice blast, and proves that the OsMAPKKK19 gene positively regulates and controls the resistance of rice to rice blast.
Example 5
Agronomic trait statistical analysis of OsMAPKKK19 gene overexpression plants
(1) Materials and methods
The wild ZH11 and two OsMAPKKK19 gene over-expression lines are respectively sown with 100 seeds, after the seeds germinate, seedlings are raised in paddy fields, after the seedlings grow for 25 to 30 days, the seedlings are transplanted in the paddy fields, and the row spacing and the plant spacing are kept basically consistent. Photographing plants in a grouting period, and selecting 15 plants of ZH11 and OsMAPKKK19 gene over-expression plants with consistent growth vigor in a mature period to carry out agronomic character statistics, wherein agronomic character indexes comprise plant height, effective tiller number, grain length, grain width and the like of rice;
(2) Results and analysis
As shown in FIG. 4, the statistical result shows that the plant with the over-expressed OsMAPKKK19 gene has no difference from the wild type in the aspects of main agronomic characters including plant height, effective tiller number, grain length, grain width and the like, which indicates that the over-expressed OsMAPKKK19 gene does not influence the growth and development of rice.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (4)
1. A rice blast resistance related gene OsMAPKKK19, characterized in that: the CDS sequence of the OsMAPKKK19 gene is shown as SEQ ID NO.1, and the total length of the CDS sequence is 2073bp.
2. The protein encoded by the rice blast resistance-related gene OsMAPKKK19 according to claim 1, wherein the protein is characterized in that: the amino acid sequence of the protein coded by the OsMAPKKK19 gene is shown as SEQ ID NO.2, and 690 amino acids are coded.
3. Use of a rice blast resistance related gene OsMAPKKK19 according to claim 1 or a protein encoded by a rice blast resistance related gene OsMAPKKK19 according to claim 2 for improving plant disease resistance, wherein the related gene OsMAPKKK19 or the protein encoded by the related gene OsMAPKKK19 is overexpressed in plants to activate plant immune response.
4. The use of a rice blast resistance-related gene OsMAPKKK19 according to claim 1 or a protein encoded by a rice blast resistance-related gene OsMAPKKK19 according to claim 2 for increasing rice blast resistance, characterized in that the expression of the protein encoded by the related gene OsMAPKKK19 or the related gene OsMAPKKK19 is significantly increased after infection by Pyricularia oryzae.
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| CN117947094A (en) * | 2024-03-26 | 2024-04-30 | 云南农业大学 | Method for improving rice blast resistance by Pi-Pprs42 gene and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117947094A (en) * | 2024-03-26 | 2024-04-30 | 云南农业大学 | Method for improving rice blast resistance by Pi-Pprs42 gene and application |
| CN117947094B (en) * | 2024-03-26 | 2024-06-04 | 云南农业大学 | Method for improving rice blast resistance by Pi-Pprs42 gene and application |
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