Application of rice RNA helicase 52B gene in regulating plant antiviral activity

Abstract

The application belongs to the field of plant biological control, and specifically discloses application of a rice RNA helicase RH52B gene in regulating plant resistance to viruses. The nucleotide sequence of the RH52B gene is shown as SEQ ID NO. 1. Through CRISPR-Cas9-mediated genome editing technology, rh52b knockout mutants are generated in the background of wild-type rice Nipponbare, and compared with Nipponbare plants, it is found that the down-regulation of rh52b expression in rice due to knockout can significantly increase the plant resistance to RSV virus. The application provides a new method for cultivating crops with enhanced resistance to viruses by using genetic engineering means, and has important significance for regulating plant resistance and breeding, and has a broader application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological control. Specifically, this invention relates to a plant RNA helicase and its application in antiviral properties. Background Technology

[0002] my country is a world-renowned grain producer, and rice is its largest grain crop, accounting for over 40% of the country's total planting area and yield, and is one of the staple crops in southern China. The yield and quality of rice directly affect national food security and economic development. Plants face numerous challenges during their growth, such as unfavorable climate conditions, poor soil, attacks from various viruses, bacteria, fungi, and pests, nutrient imbalances and excessive or insufficient watering due to improper field management, and competition from weeds. These factors seriously threaten plant growth, especially pests and diseases, which have a significant impact on crop yields. Statistics show that pests and diseases seriously threaten global agricultural production and food security, causing up to 30% of food losses annually. Plant virus infections have become the second leading cause of disease in agricultural production, causing enormous economic losses worldwide each year. Of the more than 200 plant viruses transmitted by insects, accounting for over 80% of all plant viruses, they cause global economic losses of US$20 billion annually. In my country, major insect-borne plant viruses include rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), and wheat yellow dwarf virus (BYDV). These viruses are difficult to detect, trace, and control. Once they occur, they can become epidemic diseases. Furthermore, the lack of effective control measures for plant viruses, the over-reliance on and misuse of chemical pesticides, leading to pesticide residues, severe soil damage, increasing ecological pollution, the emergence of pesticide-resistant pests, and making it difficult to effectively control viral diseases with chemical pesticides.

[0003] Rice stripe virus is widespread in China, posing a serious threat to the country's food security. It is a viral disease transmitted by the rice planthopper, commonly known as "cancer of rice." Infected plants often suffer from withered panicles or small, deformed panicles that fail to produce grains. After the jointing stage, the disease manifests as yellow-green stripes on the lower part of the flag leaf. While all rice varieties do not suffer from heart rot, the panicles are deformed and produce very few grains. The rice stripe virus is transmitted solely through insect vectors; other routes of transmission are not possible. The primary vector is the rice planthopper, which, once infected, can transmit the virus for life and via its eggs. The virus multiplies within the insect and can also be transmitted through eggs. The virus infects more than 50 species of grasses, including rice, wheat, barley, oats, corn, millet, foxtail millet, and others. However, apart from rice, other hosts play a relatively minor role in the infection cycle.

[0004] Different rice varieties exhibit significant differences in resistance to various viruses and their vectors, but most currently cultivated varieties are generally not resistant. Rice seedlings are relatively susceptible to disease from the tillering stage, with resistance increasing daily after jointing. Planting susceptible host crops in the preceding crop, excessive weed growth in the field, mixed planting of single and double seasons, transplanting of early, mid, and late-maturing varieties, early sowing of early rice, and sparse planting of single plants all create favorable ecological conditions for the accumulation of virus-transmitting insects and toxins, thus promoting the occurrence and spread of diseases. Warm winters and springs, with high temperatures and dryness in the early stages of rice growth, are conducive to the survival and activity of overwintering generations and virus-transmitting vectors during seedling growth, promoting the spread and transmission of viruses. RNA silencing, plant hormones, autophagy, PTI and ETI innate immune pathways are the main pathways for plant antiviral activity. RNA, as a crucial component of the central dogma of genetics, participates in various life processes, playing a vital role in the complex and multifaceted interactions between plant viruses and hosts, between plant viruses and virus-transmitting vectors, and between plant viruses, vector insects, and host plants. RNA helicase is an essential molecule in RNA metabolism, often acting as a molecular chaperone in protein translation and affecting the normal functioning of the body.

[0005] However, the interaction between plant viruses and their host plants is complex. Especially over the long course of this interaction, viruses have evolved diverse infection mechanisms. The limited number of existing disease-resistance genes means that viral infection cannot be effectively controlled, seriously threatening crop yield and resistance. Therefore, the discovery of new disease-resistance genes has become a crucial antiviral strategy. Screening for new resistance genes and cultivating new disease-resistant transgenic crops is of great significance for improving crop yield and resistance. Summary of the Invention

[0006] To fill the gap in the existing technology, this invention provides an application of the rice RNA helicase gene RH52B in plant antiviral activity, and specifically provides the following technical solution:

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

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

[0009] A third aspect of the invention provides the application of the gene or protein described above in regulating plant antiviral activity, 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 in the target plant as described above, thereby downregulating the expression of the rice RNA helicase RH52B gene in the target plant and obtaining plants that can significantly reduce RSV virus infection.

[0011] A fourth aspect of the present invention provides a method for cultivating transgenic plants resistant to RSV virus. The method employs gene editing technology to knock out the rice RNA helicase RH52B gene in the target plant, thereby downregulating the expression of the rice RNA helicase RH52B gene in the target plant, and thus obtaining transgenic plants resistant to RSV virus.

[0012] The experiments of this invention demonstrate that the rice RNA helicase RH52B gene plays a crucial regulatory role in rice resistance to RSV, and knocking out this gene to reduce its expression enhances rice resistance. Therefore, this gene can be used to regulate rice virus resistance, providing important insights for rice resistance breeding. This invention provides important gene resources for cultivating new disease-resistant crop varieties using genetic engineering techniques, and has significant application prospects for improving crop disease resistance. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0014] Figure 1A The structure of the RH52B gene is shown; the Nipponbare gDNA sequence below it is the original sequence of the knockout site, and the red-marked base rh52b#67 is the inserted base that caused the frameshift mutation in the RH52B gene. Figure 1B Agarose gel image for genotyping of rh52b#67 after PCR amplification; Figure 1C The sequencing alignment diagram of rh52b#67 after the PCR stock solution was sent to BGI for sequencing.

[0015] Figure 2 Phenotypic images of 4-week-old plants of rh52b#67 mutant and wild-type rice Nipponbare NPB are shown, with wild-type rice Nipponbare on the left and rh52b#67 mutant on the right.

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

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

[0018] Figure 5 The amount of RSV-CP mRNA accumulated in plants after inoculation with RSV for rh52b mutants. Detailed Implementation

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

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

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

[0022] The wild-type rice variety used in the following examples is Nipponbare. The rh52b mutant and the wild-type rice genomes show only a frameshift mutation in the RH52B gene; the remaining genes are identical.

[0023] Example 1: Application of the rice RNA helicase OsRH52B gene in regulating plant antiviral resistance

[0024] I. Identification of rh52b mutant

[0025] Genomic DNA was extracted from rh52b mutant positive seedlings and amplified by PCR using CRISPR-RH52B(500)-F and CRISPR-RH52B(500)-R primers.

[0026] The main reagents used were: KOD One™ PCR Master Mix purchased from Toyobo, DNA marker and gelred nucleic acid dye purchased from Polymer Biotech, DNA gel extraction kit purchased from TransGen Biotech, and primers synthesized by BGI Genomics 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 as follows:

[0031]

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

[0033]

[0034] After PCR amplification, 5 μL of amplification product was added to 1 μL of loading buffer and electrophoresed on a 1.0% agarose gel containing gelred nucleic acid dye. 1×TAE was used as the electrophoresis buffer, and electrophoresis was performed at 150V for 20 minutes. The gel was then photographed with a gel imaging system, and positive samples were sent to BGI Genomics Co., Ltd. for sequencing.

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

[0036] II. Phenotype of 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 using a special experimental camera.

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

[0039] III. Phenotypic characteristics of rh52b mutant after inoculation with rice stripe virus (RSV)

[0040] The RH52B gene mutant rh52b#67 and wild-type rice seeds of Nipponbare were sown. After the plants grew to 10 days, they were inoculated with RSV virus. The RSV phenotype in the mutant and wild-type plants was observed after two weeks of culture. Wild-type rice NPB inoculated with RSV virus and the mutant rh52b were placed on the same horizontal line, and the entire plant was photographed using a specialized laboratory camera.

[0041] The results of photographing the entire plant after RSV infection are as follows: Figure 3 As shown in the image, the results of photographing plant leaves after infection with plant virus RSV are as follows. Figure 4As shown, the overall growth of the RH52B gene mutant rh52b#67 after inoculation with RSV virus was more vigorous than that of wild-type rice NPB plants, with milder disease symptoms and smaller-scale striped leaf blight symptoms on the leaves, exhibiting a clear disease resistance phenotype.

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

[0043] IV. qPCR detection of RSV-CP mRNA accumulation in rh52b mutant after RSV inoculation

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

[0045] Total RNA was extracted from the plant, ensuring that all reagents and consumables were RNase-free during the experiment. The extracted RNA was then subjected to mRNA reverse transcription. mRNA reverse transcription involves two steps: removal of genomic DNA and mRNA reverse transcription.

[0046] Step 1: Removal of genomic DNA

[0047]

[0048] After reacting at 42°C for 2 minutes, place on ice.

[0049] Step 2: Reverse transcription reaction.

[0050]

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

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

[0053] The cDNA obtained from reverse transcription was diluted 10-fold and then subjected to real-time quantitative PCR using a 2×ChamQ SYBR qPCRMaster Mix from Vazyme. The reaction system is as follows:

[0054]

[0055] Real-time PCR experiments were performed using an ABI Stepone Plus instrument.

[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 an internal control for mRNA qPCR. After qPCR detection, the relative expression level of RSV-CP mRNA was calculated using a formula, and the results are as follows. Figure 5 As shown, after RSV infection, the accumulation of RSV-CP mRNA in the RH52B gene mutant rh52b#67 was significantly downregulated compared to that in wild-type rice NPB.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a rice RNA helicase RH52B gene in regulating plant antiviral resistance, characterized in that, The gene nucleotide sequence is shown in SEQ ID NO.

1. The virus is rice stripe virus (RSV), and the plant is rice. Gene editing technology is used to knock out the rice RNA helicase RH52B gene in the target plant, thereby downregulating the expression of the rice RNA helicase RH52B gene in the target plant, thus obtaining plants that can reduce RSV virus infection.

2. A method for cultivating transgenic plants resistant to RSV virus, characterized in that, Gene editing technology is used to knock out the rice RNA helicase RH52B gene as described in claim 1 in the target plant, thereby downregulating the expression of the rice RNA helicase RH52B gene in the target plant, and thus obtaining a transgenic plant resistant to RSV virus, wherein the plant is rice.

Images