CN117230087A - Rice blast resistance related gene OsCAMTAPL and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular breeding, and in particular relates to a rice blast resistance related geneOsCAMTAPLAnd applications thereof. Rice plantOsCAMTAPLThe CDS sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The research of the invention finds that whether knocking out or over-expressingOsCAMTAPLThe genes all lead to remarkable improvement of the disease resistance of the rice, which shows that the genes play an important role in the rice blast resistant path. The invention proves for the first time thatOsCAMTAPLGene involved in rice blast resistance can be obtained byOsCAMTAPLGeneThe method is applied to genetic engineering genetic breeding of crops, cultivates rice blast resistant crop varieties, reduces pesticide spraying and grain loss, reduces environmental pollution, ensures grain safety, has reference significance for disease resistance related researches of other crops, and can be applied to genetic improvement for improving the resistance of related agricultural crops such as rice and the like.

Description

A rice blast resistance-related gene OsCAMTAPL and its application

Technical field

The invention relates to the field of agricultural biotechnology. Specifically, it relates to the application of OsCAMTAPL, a gene related to rice blast resistance, or its encoded protein, methods for improving rice blast resistance, and gene knockout and overexpression materials. Methods for constructing and improving rice varieties resistant to Magnaporthe oryzae.

Background technique

Rice is one of the important food crops in the world. Rice blast is a rice disease caused by the semi-biotrophic fungus Magnaporthe oryzae of the phylum Ascomycota and the class Faecalimycetes, which seriously affects the yield and quality of rice. Magnaporthe oryzae harms the entire growth period of rice and can cause about 10-30% reduction in rice production around the world every year. It is commonly known as rice cancer. In severe cases, it can lead to rice harvest failure, and the pathogen can also harm other crops, such as wheat, barley, etc. It is an important harmful pathogen in the world.

Due to changes in farming systems, monoculture planting of crops, iteration of rice varieties, and application of fertilizers and drugs in recent years, the damage caused by rice blast has shown an increasing trend. Moreover, due to the short generation of blast fungus, most disease-resistant rice varieties lose resistance within 2-3 years of promotion, seriously endangering rice grain production. The short promotion period of rice disease-resistant varieties is a common problem in the rice production process. A serious problem, enhancing the long-lasting resistance of rice is an urgent problem in rice improvement.

Ca ²⁺ (Calciumions) ions, as intracellular second messengers, play an important role in the disease resistance response of rice. The influx of Ca ²⁺ through the plasma membrane is the key to activating the natural immune signal of rice and is the key to the early infection of rice by Magnaporthe oryzae. Key factors in signaling. Early immune signals can trigger changes in intracellular free Ca ²⁺ concentration and spatiotemporal patterns. Changes in free Ca ²⁺ concentration will be recognized by many Ca ²⁺ -binding proteins, which then transmit immune signals downstream, among which there is an important class of transcription factors. The family plays an important role in the downstream of immunity, called CAMTAs (Calmodulin-bindingtranscription activators). Arabidopsis AtCAMTA1, AtCAMTA2 and AtCAMTA3 have overlapping functions in plant immunity. AtCAMTA3 function is the most important, and its loss of function leads to Constitutive activation in immune responses and dwarf plant growth. The atcamta1/2/3 triple mutant exhibits extreme autoimmunity and severe growth arrest, indicating that these three CAMTAs are redundant negative regulators of Arabidopsis immunity. Through homologous comparison, a gene OsCAMTAPL with a similar structure to the CAMTAs family was discovered in rice. In the early stage of the experiment, through homologous gene cloning, it was found that both gene knockout and overexpression of OsCAMTAPL in rice can significantly improve rice blast resistance. Therefore, the OsCAMTAPL gene has potential application value in breeding disease-resistant rice materials. After searching, there is no report on the role of OsCAMTAPL gene in rice blast resistance in the existing reports. In view of this, the present invention is proposed.

Contents of the invention

In view of the problems encountered in rice production, the present invention provides the application of the OsCAMTAPL gene or the protein encoded by it in improving rice resistance to rice blast. Gene knockout technology is used to search for resistance genes that play a role in rice. Based on this, the present invention provides the rice blast resistance gene OsCAMTAPL and performs functional identification of the gene, which aims to screen, cultivate and identify rice resistance with the help of the OsCAMTAPL gene. Plasmodium species.

The technical solutions adopted by the present invention are as follows:

A rice blast resistance-related gene OsCAMTAPL, the CDS sequence of the resistance-related gene OsCAMTAPL is shown in SEQ ID NO.1.

Further, the amino acid sequence of the protein encoded by the above-mentioned resistance-related gene OsCAMTAPL is shown in SEQ ID NO. 2.

Application of the above-mentioned resistance-related gene OsCAMTAPL in regulating rice resistance to rice blast.

A method of regulating rice's resistance to rice blast: when it is necessary to improve rice's resistance to rice blast, the OsCAMTAPL gene in rice is knocked out or overexpressed; the CDS sequence of the OsCAMTAPL gene is shown in SEQ ID NO.1 .

Application of the above resistance-related gene OsCAMTAPL in rice breeding: rice blast-resistant rice lines are obtained by screening rice plants with high or low expression of the rice OsCAMTAPL gene; the CDS sequence of the OsCAMTAPL gene is as SEQ ID NO.1 shown.

A method for constructing transgenic rice resistant to rice blast: transforming the rice OsCAMTAPL gene into a rice plant to obtain transgenic rice with high expression of the rice OsCAMTAPL gene, or knocking out the OsCAMTAPL gene in rice to obtain transgenic rice with low expression of the rice OsCAMTAPL gene; The CDS sequence of the OsCAMTAPL gene is shown in SEQ ID NO.1.

The present invention discovered and proved for the first time the key role of the OsCAMTAPL gene in rice blast resistance; knocking out the OsCAMTAPL gene through genetic engineering methods in the rice variety Zhonghua 11 (ZH11) improved the rice's resistance to rice blast, and compared with the wild type In comparison, the number and area of lesions and the biomass of M. oryzae in the OsCAMTAPL mutant were significantly reduced, and the expression levels of rice disease resistance-related genes increased before and after inoculation; increasing the expression level of OsCAMTAPL genes will also significantly increase the resistance of rice to M. oryzae. Resistance. The rice blast resistance gene OsCAMTAPL provided by the invention can be used to cultivate high-resistant rice varieties.

The beneficial effects of the present invention are:

(1) In the present invention, various identification methods such as spray inoculation method, punch inoculation method, and blast fungus growth detection were used to prove that the deletion of OsCAMTAPL gene significantly increases the disease resistance of rice to rice blast; after inoculation The detection of disease course-related genes proved that the OsCAMTAPL gene deletion led to the upregulation of plant disease resistance-related gene expression, indicating that the OsCAMTAPL gene is an excellent candidate gene for crop molecular design and a potential resistance gene for creating rice resistant to rice blast.

(2) By constructing OsCAMTAPL gene overexpression rice material, the present invention found that the OsCAMTAPL overexpression rice material also showed disease resistance phenotype after inoculation, and the biomass statistics of Magnaporthe oryzae were consistent with the phenotype. The above results all indicate that the OsCAMTAPL gene is an excellent candidate gene for creating transgenic rice and crop molecular design with enhanced resistance to rice blast. It has important theoretical value and broad application prospects, and can be used for genetic improvement of resistance to rice and other related agricultural crops. .

Description of drawings

Figure 1 shows the knockout of OsCAMTAPL gene through genetic engineering to design target sites and identification of mutants in Example 1 of the present invention.

Figure 2 is a diagram showing the disease resistance phenotype identification of plants after OsCAMTAPL gene knockout in Example 2 of the present invention.

Figure 3 is a graph showing the detection of disease resistance-related disease course genes in OsCAMTAPL and ZH11 in Example 3 of the present invention.

Figure 4 is a diagram showing the identification and disease resistance phenotype identification of rice plants inoculated with OsCAMTAPL overexpression material in Example 4 of the present invention.

Figure 5 shows the subcellular localization of OsCAMTAPL protein in Example 5 of the present invention.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

Example 1: Knockout of OsCAMTAPL in rice and acquisition of mutants

(1)Materials and methods

The target site for knockout was selected based on the rice OsCAMTAPL gene sequence (the CDS sequence of the rice OsCAMTAPL gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2). 11 (ZH11) background, the present invention selects two target sites to be knocked out twice respectively through CRISPR/Cas9 technology, and obtains T ₀ generation rice through Agrobacterium-mediated genetic transformation of rice callus, which is obtained through genotype analysis. Independent genetic strains.

(2)Results and analysis

Referring to Figure 1, the two target sites selected for knockout based on the rice OsCAMTAP gene sequence are 5’-GGATTCTGATGCTCAGTACA-3’ and 5’-GTGAGAAGCTACTCACTTTG-3’. Through genotype analysis, two mutant lines, oscamtapl-1 and oscamtapl-2, were found. Among them, oscamtapl-1 has a base deletion at CDS166bp of the OsCAMTAP gene, and oscamtapl-2 has a base insertion at CDS1064bp of the OsCAMTAP gene. Both deletions cause frameshift mutations, leading to the premature appearance of the terminator, and are mutants with loss of function of the OsCAMTAPL protein.

Example 2: Identification of disease resistance phenotype of OsCAMTAPL gene rice mutants

(1)Materials and methods

Rice cultivation method: hull the rice, soak in 75% ethanol for 5 minutes, discard the ethanol, and rinse with sterile water three times; continue soaking with 50% pasteurization solution for 30 minutes; in ultra-clean Taichung, discard the disinfectant and rinse with sterile water Seed 3 times, discard the sterile water; spread the seeds evenly in the MS culture medium, usually 15-20 seeds/dish, cultivate in the greenhouse at 28°C for one week before transplanting, place in a long-day incubator, and transplant again after one week Continue culturing outdoors while ensuring adequate light and moisture.

Preparation of M. oryzae spore liquid: Place the preserved M. oryzae filter paper pieces in CM solid medium and incubate at a constant temperature of 28°C to grow M. oryzae. After ten days of growth, the M. oryzae is removed with a sterile scalpel in a clean Taichung Cut the culture medium with mycelium into small pieces and transfer it to the rice bran culture medium to continue culturing. After Magnaporthe oryzae mycelium covers more than 3/4 of the area of the rice bran culture medium, use a sterile scraper to gently scrape off the bacteria on the surface of the culture medium in a clean Taichung. Silk, continue culturing in a light incubator for 3-5 days to promote sporulation; prepare 0.2% Tween-20 solution, pour into the rice bran culture medium, gently scrape the rice bran culture medium with a scraper to elute the spores, and filter the spores , Haemocytometer counting.

Spray inoculation method: Spray the prepared Magnaporthe oryzae spore liquid with a concentration of 1×10 ⁵ /ml evenly on the 3-week-old rice to ensure that all rice leaves are covered with the bacterial liquid. In a high-humidity environment, incubate in the dark After 24 hours of treatment, continue culturing under 12 hours of light and 12 hours of darkness. During this period, the environment is maintained at high humidity, and the disease phenotype is observed after 3 days;

Punch inoculation method: Take a rice seedling that has grown for about one month and has a leaf width greater than 1cm. Use a punch to gently press the rice leaves to form a wound, but cannot penetrate it; take 10 μl with a concentration of 2×10 ⁵ / ml of Magnaporthe oryzae spore liquid was added dropwise to the surface of the perforated leaves, and the leaves at the perforated area were sealed with tape, making sure that the spore liquid did not flow out; the inoculated rice was darkened in a greenhouse at 26°C for 24 hours, and then left for 12 hours. Continue culturing for 7 days under light and 12 hours of darkness and high humidity conditions, and count the size of leaf lesions; detection of blast fungus biomass: select equal amounts of leaves of the mutant and wild type with the same disease trend, extract rice genomic DNA, and determine the concentration , using Ubiquitin of rice and MoPot2 of Magnaporthe oryzae as internal reference genes, the growth of Magnaporthe oryzae in rice leaves was detected through qPCR at the DNA level.

(2)Results and analysis

See Figure 2. Figure 2a shows the inoculation of Magnaporthe oryzae strain Zhong1 using the spray inoculation method. Observation and photos were taken 3 days after inoculation. ZH11 is the wild type, and oscamtapl-1 and oscamtapl-2 are two alleles of OsCAMTAPL created through CRISPR/Cas9 technology. Protein function loss mutant, Bar=1cm; Figure 2c shows the inoculation of Magnaporthe oryzae strain Zhong1 using the punch inoculation method. Observation and photos were taken 7 days after the inoculation, Bar=1cm; Figures 2b and 2d show the spray and punch inoculation methods respectively. For the detection of M. oryzae biomass on diseased leaves, the relative gene expression levels were calculated using the 2- ^△△CT method;

Judging from the phenotypes of spray inoculation and punch inoculation, after the OsCAMTAPL protein function was deleted, the number and size of mutant lesions were significantly reduced compared with the wild type (Figure 2a, 2c), and the biomass of M. oryzae decreased ( As shown in Figures 2b and 2d), it shows that the functional loss of OsCAMTAPL protein significantly improves the disease resistance level of rice; Figure 2a shows that compared with ZH11, both oscamtapl-1 and oscamtapl-2 show increased resistance to blast fungus and reduced lesions. ; Figure 2c shows that compared to ZH11, the plaque area of oscamtapl-1 and oscamtapl-2 is reduced, indicating that oscamtapl-1 and oscamtapl-2 are more disease resistant; Figures 2b and 2d show that the biomass of M. oryzae in the mutant is significantly lower than that in the wild type.

Example 3: Detection of disease resistance-related gene expression levels in mutant oscamtapl and wild-type ZH11

(1)Materials and methods

Take rice seedlings that have grown for 2-3 weeks. Three seedlings form a group. The mutant oscamtapl-1 and wild-type ZH11 were inoculated with rice blast fungus for 24 hours. The sample RNA was extracted, reverse transcribed into cDNA, and real-time fluorescence quantitative PCR was used. Detect the expression of disease resistance-related genes in ZH11 and oscamtapl-1, and use Ubiquitin as the internal reference gene to detect the expression of OsOPR1, OsPR5, OsPR10 and OsWRKY45 transcripts;

(2)Results and analysis

Referring to Figure 3, real-time quantitative PCR was used to detect the difference in the transcript levels of OsOPR1, OsPR5, OsPR10 and OsWRKY45 in the wild type and oscamtapl-1 mutant after inoculation with Zhong124h.

As can be seen from Figure 3, compared with the wild type, the expression of two disease course-related genes, OsOPR1 and OsOPR10, is up-regulated to a certain extent in oscamtapl-1, indicating that the resistance of rice to rice blast increases after the OsCAMTAPL gene mutation. The OsCAMTAPL gene regulates rice resistance to rice blast in a negative regulatory manner.

Example 4: Overexpression of OsCAMTAPL improves rice resistance to blast disease

Using the pCXUN vector as the backbone, construct the UBi:CAMTAPL-HA overexpression vector of the full-length OsCAMTAPL CDS sequence (the vector construction primers are UBCAMTAPL-F:5'-GATTACGCTCCAATACTTATGTTTGGGGATGTT-3' and 5'-UBCAMTAPL-R:ATTCGGATCCCCAATACTCACCAAATGGCAGA-3') , on the background of japonica rice variety Zhonghua 11 (ZH11), OsCAMTAPL overexpression rice materials OE-OsCAMTAPL-1 and OE-OsCAMTAPL-2 were obtained through rice-mediated callus genetic transformation, and were multiplied to the T2 generation for subsequent experiments.

(1)Materials and methods

Detection of the expression level of OsCAMTAPL overexpression plants: Extract RNA from three plants each of ZH11, OE-OsCAMTAPL-1, and OE-OsCAMTAPL-2, reverse transcribe into cDNA, and use real-time fluorescence quantitative PCR to measure the expression level of the OsCAMTAPL gene in the overexpression plants. Detection: The overexpression level of OsCAMTAPL gene is detected in rice materials with a normal growth cycle of about three weeks; at the same time, the total rice protein is immunoblotted to detect the protein expression of OsCAMTAPL-HA;

Identification of rice blast resistance of OsCAMTAPL gene overexpression plants: rice wild-type material ZH11, overexpression materials OE-OsCAMTAPL-1, OE-OsCAMTAPL-2, rice culture and inoculation methods are the same as in Example 1, and the growth cycle of the inoculated materials is one In March, Punch inoculation was carried out, and the biomass of M. oryzae was quantitatively analyzed on the diseased materials.

(2)Results and analysis

See Figure 4. Figure 4a shows the identification of the OsCAMTAPL expression level in the OsCAMTAPL gene overexpression plant OE-OsCAMTAPL. Rice seedlings with a growth cycle of one month were used as materials, and ZH11 was used as the negative control. RNA samples were extracted and reverse transcribed into cDNA for real-time Fluorescence quantitative PCR detection and analysis; Figure 4b shows the detection of OsCAMTAPL-HA protein in randomly selected plants from the two lines OE-OsCAMTAPL-1 and OE-OsCAMTAPL-2; Figure 4c shows the inoculation of Magnaporthe oryzae using the punch inoculation method. Strain Zhong1, observed and photographed 7 days after inoculation, Bar=1cm; Figure 4d shows the statistics of the biomass of Magnaporthe oryzae in the diseased materials;

As can be seen from Figure 4, compared with the wild type, the expression level of the OsCAMTAPL gene in the over-expression plants was significantly increased, with an average of more than a hundred times (Figure 4a). The over-expression material met the experimental requirements; Western blotting detected the expression of OsCAMTAPL-HA The protein expression was normal, and no bands were detected in the wild type (Figure 4b); the results of the Punch inoculation experiment showed that compared with the wild type, the area of lesions in OsCAMTAPL overexpressing plants was reduced after inoculation, and the bacterial biomass detection on the diseased leaves was also It shows that the growth of Magnaporthe oryzae in over-expressed plants is less than that of wild type (Figure 4c, 4d), indicating that over-expression of OsCAMTAPL can improve the resistance of rice to rice blast.

Example 5: Analysis of subcellular localization and transcriptional activity of OsCAMTAPL protein

(1)Materials and methods

Subcellular localization: clone the CDS sequence of the rice OsCAMTAPL gene and connect it to the pCAMBIA2300-35S-GFP vector through the infusion method to obtain the pCAMBIA2300-35S-OsCAMTAPL-GFP plasmid. The obtained plasmid is combined with the pCAMBIA2300-35S-GFP empty vector plasmid. Transform Agrobacterium and transiently infect Nicotiana benthamiana, and observe the subcellular localization of cells under a confocal laser microscope.

(2)Results and analysis

Referring to Figure 5, it shows that the GFP protein is widely distributed in the cytoplasm, cell membrane and nucleus. The OsCAMTAPL-GFP fusion protein is widely distributed in the nucleus, and only a few are on the cell membrane.

The above are only examples of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the content of the description of the present invention, or directly or indirectly applied in other related technical fields, shall be regarded as Likewise, it is included in the patent protection scope of the present invention.

Claims (6)

1. A rice blast resistance-related gene OsCAMTAPL , characterized in that: the CDS sequence of the resistance-related gene OsCAMTAPL is shown in SEQ ID NO. 1. 2. The rice blast resistance-related gene OsCAMTAPL according to claim 1, characterized in that: the amino acid sequence of the protein encoded by the resistance-related gene OsCAMTAPL is shown in SEQ ID NO. 2. 3. Application of the resistance-related gene OsCAMTAPL according to claim 1 in regulating rice resistance to rice blast. 4. A method for regulating rice's resistance to rice blast, characterized in that: when it is necessary to improve rice's resistance to rice blast, the OsCAMTAPL gene in rice is knocked out or overexpressed; the CDS sequence of the OsCAMTAPL gene is such as SEQ Shown as ID NO.1. 5. Application of the resistance-related gene OsCAMTAPL in rice breeding as claimed in claim 1, characterized in that: rice strains resistant to rice blast are obtained by screening rice plants with high or low expression of the rice OsCAMTAPL gene; the OsCAMTAPL The CDS sequence of the gene is shown in SEQ ID NO.1. 6. A method for constructing transgenic rice resistant to rice blast, characterized by: transferring the rice OsCAMTAPL gene into the rice plant to obtain transgenic rice with high expression of the rice OsCAMTAPL gene, or knocking out the OsCAMTAPL gene in the rice to obtain low-expression rice OsCAMTAPL Gene transgenic rice; the CDS sequence of the OsCAMTAPL gene is shown in SEQ ID NO.1.