Application of rice RNA helicase RH52B gene in regulation and control of plant virus resistance

Abstract

The invention belongs to the field of plant biocontrol, and particularly discloses application of a rice RNA helicase RH52B gene in regulation and control of plant virus resistance. The nucleotide sequence of the RH52B gene is as shown in SEQ ID NO. 1. Through a CRISPR-Cas9 mediated genome editing technology, an rh52b knockout mutant generated under the background of wild rice Nipponbare is compared with a Nipponbare plant for analysis, and it is found that expression down-regulation caused by knockout of the rh52b in rice can significantly increase the anti-RSV virus of the plant. The invention provides a new method for cultivating and enhancing the virus resistance of crops by using a genetic engineering means, and has important significance and wider application prospect in regulating the plant resistance and breeding for disease resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biological control, and specifically relates to a plant RNA helicase and its application in antiviral performance. Background Art

[0002] my country is a world-renowned grain producer, and rice is the largest grain crop in my country. Its planting area and output account for more than 40% of the country's grain, and it is one of the staple crops in the southern region. The yield and quality of rice are directly related to the country's food security and economic development. Plants face many challenges in the process of growth, such as unfavorable climatic conditions, poor soil, invasion by different types of viruses, bacteria, fungi or pests, nutrient imbalance and excessive or insufficient water caused by improper field management, weed competition and other problems. The above factors seriously threaten plant growth, especially pests and diseases, which have an impact on crop yields that cannot be underestimated. According to statistics, pests and diseases seriously threaten global agricultural production and food security, and up to 30% of grain losses are caused by pests and diseases each year. Plant virus infection has become the second largest disease in agricultural production, causing huge economic losses worldwide every year. Among them, there are more than 200 insect-borne plant viruses, accounting for more than 80% of plant viruses, causing economic losses of 20 billion US dollars per year worldwide. In my country, the main insect-borne plant viruses include rice stripe leaf blight (RSV), southern rice black-streaked dwarf disease (SRBSDV), wheat yellow dwarf disease (BYDV), etc. These viruses are difficult to detect, trace, and prevent. Once they occur, they may become epidemic diseases. In addition, there is a lack of effective control measures for plant viruses, and excessive reliance on and abuse of chemical pesticides have caused pesticide residues, serious soil damage, increasingly serious ecological and environmental pollution, the emergence of drug-resistant pests, and it is difficult to effectively control viruses and other diseases through chemical pesticides.

[0003] Rice stripe leaf blight is widespread in China, posing a serious threat to my country's food security. Rice stripe leaf blight is a viral disease transmitted by the gray planthopper, commonly known as cancer on rice. Diseased plants often wither and produce panicles or panicles that are small, deformed and unfruitful. After jointing, the disease occurs and yellow-green stripes appear on the lower part of the sword leaf. All types of rice do not wither, but the heading is deformed and there are few fruits. Rice stripe leaf blight virus is only transmitted by vector insects and not by other means. The vector insect is mainly the gray planthopper. Once infected, it can transmit the virus throughout life and through eggs. The virus proliferates in the insect body and can also be transmitted through eggs. The virus infects more than 50 species of plants in the Gramineae family, including rice, wheat, barley, oats, corn, millet, sorghum, foxtail grass, etc. However, except for rice, other hosts play a small role in the infection cycle.

[0004] Different rice varieties have obvious differences in resistance to various viruses and their vectors, but most of the existing major cultivated varieties are generally not resistant to diseases. Rice is more susceptible to diseases from the seedling stage to the tillering stage, and its resistance increases after jointing. Planting susceptible host crops before rice fields, weeds in the fields, mixed planting of single and double seasons, transplanting of early, medium and late maturing varieties, early sowing of early rice, and sparse planting of single plants will create favorable rice field ecological conditions for the accumulation of virus-transmitting insects and virus sources, thus facilitating the occurrence and spread of diseases. Warm winter and spring, high temperature and dryness in the early stage of rice growth are conducive to the survival and activity of virus-transmitting vectors during the wintering generation and rice seedling growth, and promote the spread and spread of viruses. RNA silencing, plant hormones, cell autophagy, PTI and ETI natural immune pathways are the main ways for plants to resist viruses. As an important part of the central dogma of genetics, RNA participates in a variety of life processes and plays an important role in the interaction between plant viruses and hosts, the interaction between plant viruses and virus-transmitting vectors, and the complex and multi-faceted interactions between plant viruses, vector insects and host plants. RNA helicase is an important molecule that is indispensable in the RNA metabolism process. It often participates in the protein translation process as a molecular chaperone, affecting the normal execution of body functions.

[0005] However, the interaction between plant viruses and host plants is complex. Especially in the long process of plant virus-host interaction, viruses have evolved a variety of infection methods, and the number of existing disease resistance genes is limited, so virus infection cannot be effectively prevented and controlled, which seriously threatens crop yield and resistance. Therefore, the discovery of new disease resistance genes has become a very important antiviral method. Screening new resistance genes and cultivating new disease-resistant transgenic crops are of great significance to improving crop yield and resistance. Summary of the invention

[0006] In order to fill the gap in the prior art, the present invention provides an application of a rice RNA helicase gene RH52B in plant antiviral activities, and specifically provides the following technical solutions:

[0007] In a first aspect of the present invention, a rice RNA helicase gene RH52B is provided, the gDNA sequence length of which is 6296 bp, the CDS sequence length of which is 1917 bp, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] In a second aspect of the present invention, a protein encoded by the rice RNA helicase gene RH52B is provided, the amino acid sequence of which is 638 aa in length, and the specific amino acid sequence is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention provides a use of the above-mentioned gene or protein in regulating plant resistance to viruses, wherein the virus is rice resistance to rice stripe virus (RSV). Preferably, the plant is rice.

[0010] In one embodiment, the above application uses gene editing technology to knock out the rice RNA helicase RH52B gene as described above in the target plant, so that the expression of the rice RNA helicase RH52B gene in the target plant is downregulated, thereby obtaining a plant that can significantly reduce the infection of RSV virus.

[0011] The fourth aspect of the present invention provides a method for cultivating transgenic plants resistant to RSV virus, wherein the method uses gene editing technology to knock out the rice RNA helicase RH52B gene in the target plant, so that the expression of the rice RNA helicase RH52B gene in the target plant is downregulated, thereby obtaining transgenic plants resistant to RSV virus.

[0012] The experiment of the present invention proves that the rice RNA helicase RH52B gene has an important regulatory effect on rice resistance to RSV, and knocking out the gene to reduce its expression can enhance rice resistance. Therefore, the gene can be used to regulate rice resistance to viruses, which has important implications for rice resistance breeding. The present invention provides important gene resources for cultivating new disease-resistant crop varieties using genetic engineering methods, which is used to improve the disease resistance of crops and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0014] Figure 1A The structure of RH52B gene is shown; the Nipponbare gDNA sequence below is the original sequence of the knockout site, and the red bases in rh52b#67 are inserted bases and cause the frameshift mutation of RH52B gene; Figure 1B This is the agarose gel image of the genotype identification of rh52b#67 after PCR amplification; Figure 1C This is the sequencing comparison diagram of rh52b#67 after the PCR stock solution was sent to BGI for sequencing;

[0015] Figure 2 The phenotype of the rh52b#67 mutant plant and the wild-type rice Nipponbare NPB at 4 weeks old, with the wild-type rice Nipponbare on the left and the rh52b#67 mutant on the right;

[0016] Figure 3 The overall growth and disease status of the rh52b mutant and wild-type Nipponbare NPB rice plants 14 days after RSV infection;

[0017] Figure 4 The damage to the leaves of the rh52b mutant and wild-type Nipponbare NPB rice plants 28 days after RSV infection;

[0018] Figure 5 This is the amount of RSV-CP mRNA accumulated in the plants after the rh52b mutant was inoculated with RSV. DETAILED DESCRIPTION

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] In the following examples, m / v refers to g:ml unless otherwise specified.

[0022] The wild-type rice variety used in the following examples is Nipponbare. In the genomes of the rh52b mutant and the wild-type rice, only the RH52B gene has a base insertion frameshift mutation, and the other genes are the same.

[0023] Example 1 Application of Rice RNA Helicase OsRH52B Gene in Regulating Plant Antiviral Resistance

[0024] I. Identification of rh52b mutants

[0025] The genomic DNA of rh52b mutant positive seedlings was extracted and PCR amplified using Crispr-RH52B(500)-F and Crispr-RH52B(500)-R primers.

[0026] The main reagents used were: KOD OneTM PCR Master Mix purchased from Toyobo, DNA marker and gelred nucleic acid dye purchased from Polymer Corporation, DNA gel recovery kit purchased from Quanshijin Company, and primers synthesized by BGI Biotechnology Co., Ltd.

[0027] Primer sequences used:

[0028] Crispr-RH52B(440)-F:5'-TGTGGTTTGTTTCAGTGATCGAG-3'

[0029] Crispr-RH52B(978)-R:5'-CTTGTCTCAACAGGGATGTCTTC-3'

[0030] The PCR system used was:

[0031]

[0032] The reaction conditions for the first round of PCR amplification are:

[0033]

[0034] After PCR amplification, take 5 μL of amplified product and add 1 μL of loading buffer for electrophoresis on a 1.0% agarose gel with Gelred nucleic acid dye, use 1×TAE as the electrophoresis buffer, electrophoresis at 150V for 20 minutes, and use a gel imager to image the gel, record the positive samples and send them to BGI Biotechnology Co., Ltd. for sequencing.

[0035] The PCR products were sequenced and analyzed, and some of the sequencing results are shown in Figure 1. Figure 1A The structure of RH52B gene is shown; the Nipponbare gDNA sequence below is the original sequence of the knockout site, and the rh52b#67 marker base (base A at the 7th position of the 3' end) is the inserted base and causes the frameshift mutation of RH52B gene; Figure 1B This is the agarose gel image of the genotype identification of rh52b#67 after PCR amplification; Figure 1C This is the sequencing comparison diagram of rh52b#67 after the PCR stock solution was sent to BGI for sequencing.

[0036] 2. Phenotype of the rh52b mutant

[0037] The RH52B gene mutant rh52b#67 and wild-type rice Nipponbare seeds were sown. When the plants were 4 weeks old, the wild-type rice NPB and the mutant rh52b were placed on the same horizontal line and the whole plant was photographed with a special camera for the experiment.

[0038] The overall photo of the plant is as follows Figure 2 As shown, the overall growth of the RH52B gene mutant rh52b#67 plants was almost the same as that of the wild-type plants.

[0039] 3. Phenotype of the rh52b mutant after inoculation with Rice stripe virus (RSV)

[0040] The RH52B gene mutant rh52b#67 and wild-type rice Nipponbare seeds were sown. After the plants grew to 10 days old, RSV virus was inoculated. After two weeks of cultivation, the RSV virus phenotypes on the mutant and wild-type plants were observed. Wild-type rice NPB and mutant rh52b infected with RSV virus were placed on the same horizontal line, and the whole plant was photographed with a special camera for the experiment.

[0041] The results of the overall photo of the plant after infection with the plant virus RSV are as follows Figure 3 As shown, the results of photographing plant leaves after infection with plant virus RSV are as follows Figure 4After inoculation with RSV virus, the overall growth of the RH52B gene mutant rh52b#67 was more vigorous than that of the wild-type rice NPB plants, with milder disease, and a smaller range of stripe leaf blight symptoms on the leaves, showing an obvious disease-resistant phenotype.

[0042] The above results indicate that knocking out the RH52B gene can enhance plant resistance to viruses.

[0043] 4. QPCR detection of RSV-CP mRNA accumulation in rh52b mutant after RSV virus inoculation

[0044] The RH52B gene mutant rh52b#67 and wild-type rice Nipponbare seeds were sown, and RSV virus was inoculated after the plants grew to 10 days. After the infected plants were cultured for two weeks, the total RNA of the mutant and wild-type plants was extracted.

[0045] Extract total plant RNA and ensure that all reagents and consumables are RNase-free during the experiment. The extracted RNA is reversed to mRNA. mRNA reverse transcription includes two steps: removing genomic DNA and mRNA reverse transcription.

[0046] Step 1: Removal of genomic DNA

[0047]

[0048] After reacting at 42°C for 2 min, the mixture was placed on ice.

[0049] Step 2: Reverse transcription reaction.

[0050]

[0051] The reverse transcription reaction was carried out at 37°C, 15 min; 85°C, 5 s; 4°C, ∞.

[0052] After mRNA was converted into cDNA, the expression level of RSV-CP was detected by QPCR.

[0053] After the cDNA obtained by reverse transcription was diluted 10 times, real-time fluorescence quantitative PCR reaction was performed using 2×ChamQ SYBR qPCR Master Mix from Vazyme. The reaction system is as follows:

[0054]

[0055] ABI stepone plus instrument was used for quantitative PCR experiments.

[0056] Primer sequences used:

[0057] OsEF1a-qF:5'-ACATTGCCGTCAAGTTTGCTG-3'

[0058] OsEF1a-qR: 5'-AACAGCCACCGTTTGCCTC-3'

[0059] RSV-CP-qF: 5'-AGCCAGTGCCTCTCACATATC-3'

[0060] RSV-CP-qR: 5'-TCAATGACATCTCCAAAGATGCG-3'

[0061] The reaction procedure is as follows:

[0062]

[0063] EF1a was used as the internal reference for mRNA QPCR. After QPCR detection, the relative expression of RSV-CP mRNA was calculated using the formula. The results are shown in Figure 5 After RSV infection, the mRNA accumulation of RSV-CP in the RH52B gene mutant rh52b#67 was significantly downregulated compared with that in the wild-type rice NPB.

[0064] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A rice RNA helicase RH52B gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A protein encoded by a rice RNA helicase RH52B gene, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Use of the gene according to claim 1 or the protein according to claim 2 in regulating plant antiviral properties, characterized in that: The virus is rice resistant to rice stripe virus (RSV).

4. The use according to claim 3, characterized in that: The plant is rice.

5. The use according to claim 3 or 4, characterized in that: The rice RNA helicase RH52B gene as claimed in claim 1 is knocked out in the target plant by gene editing technology, so that the expression of the rice RNA helicase RH52B gene in the target plant is downregulated, thereby obtaining a plant that can significantly reduce the infection of RSV virus.

6. A method for cultivating transgenic plants resistant to RSV virus, characterized in that: The rice RNA helicase RH52B gene as claimed in claim 1 is knocked out in the target plant by gene editing technology, so that the expression of the rice RNA helicase RH52B gene in the target plant is downregulated, thereby obtaining a transgenic plant resistant to RSV virus.

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