A method for creating a rice material capable of blocking the transmission of rice stripe virus by small brown planthoppers

Abstract

This invention provides a method for creating rice materials that can block the transmission of rice stripe virus by planthoppers, belonging to the field of agricultural science and technology. Based on the characteristic that the rice stripe virus (RSV) glycoprotein NSvc2-N mediates viral infection of the planthopper midgut, NSvc2-N is selected from this glycoprotein. S (720 bp, encoding the soluble region of NSvc2-N, i.e., amino acids 30-268), was integrated into the rice genome using transgenic technology. The resulting transgenic rice plants significantly reduced the virus acquisition rate, virus content in the insects, and virus transmission rate of planthoppers, demonstrating that the NSvc2-N expressed in the transgenic plants... S The protein can competitively bind to the viral infection site located in the midgut of the planthopper, thereby blocking the planthopper from transmitting the virus. This method fills the gap in technology that uses the plant itself to block the planthopper from transmitting the virus. Compared with traditional "insect control and disease prevention" methods, this method is simple, economical, green, and efficient, and can be applied to antiviral breeding, disease control research, and research on the mechanism of insect virus transmission.

Description

Technical Field

[0001] This invention relates to a method for inserting rice stripe virus glycoprotein into the rice genome using transgenic technology. NSvc2-NS The method of using genes to block the transmission of viruses by planthoppers can be applied to agricultural production and belongs to the field of agricultural science and technology. Background Technology

[0002] Rice stripe virus ( Rice stripe virus RSV belongs to the genus Cervaviridae (RSV). Tenuivirus The main insect vector for its propagation is the gray planthopper ( ). Laodelphax striatellus Once infected, the rice planthopper can carry the virus for life and can transmit it through eggs. The RSV genome consists of four RNAs: RNA1, RNA2, RNA3, and RNA4, encoding a total of seven proteins. Among them, the viral replicase is RdRp, NS2 and NS3 are gene silencing repressors, NSvc2 is a glycoprotein, NCP is a nucleocapsid protein, SP is a disease-specific protein, and NSvc4 is a motility protein. The main symptoms of RSV infection in rice can be broadly classified into two types: leaf curling and leaf expansion. Leaf curling is characterized by typical "false heart decay" symptoms, mainly manifested as chlorosis at the base of the central leaf, twisting, and drooping in an arc shape; in severe cases, the central leaf will die. Leaf expansion symptoms are mainly manifested as yellow mottled chlorotic stripes on the leaves; the diseased leaves do not twist or curl, eventually drooping and withering. This disease can cause reduced rice yield or even total crop failure. At the beginning of this century, RSV (Rice Stripe Virus) broke out on a large scale in the rice-growing areas of East my country, with an epidemic period lasting for more than ten years, causing severe yield losses and posing a huge threat to food security. As rice is an important food crop in my country, its safe production is related to national food security. Therefore, the prevention and control of rice stripe virus is a long-term and indispensable task.

[0003] Currently, the most common control measures for rice stripe virus disease are the use of resistant varieties and pest management. Resistant varieties have played a crucial role in controlling the disease, but they cannot completely block virus infection of plants or vector-borne transmission; the disease cycle can still be maintained in the field. Pest management can control vector numbers and reduce virus transmission, but the use of chemical pesticides can cause environmental pollution and lead to pesticide resistance in insects. Therefore, developing green and efficient disease control technologies remains key to disease control. Plant genetic transformation utilizes molecular biology and genetic engineering techniques to insert and integrate exogenous gene fragments into the plant genome, ensuring their stable inheritance in offspring. In recent years, extensive research on transgenic rice has been conducted both domestically and internationally, resulting in the conversion of several superior agronomical traits into rice materials and the acquisition of numerous transgenic plants.

[0004] NSvc2 is a glycoprotein encoded by the RSV RNA2 gene fragment. Previous studies have shown that the NSvc2 protein is cleaved in rice, breaking down into two proteins: the NSvc2-N-terminal and the NSvc2-C-terminal, both involved in RSV acquisition by the planthopper. During RSV acquisition, the NSvc2-N-terminal protein binds to both viral particles and midgut receptors in the planthopper, initiating endocytosis to allow RSV to enter the midgut cells of the vector. Therefore, the NSvc2-N-terminus is crucial for the transformation of the planthopper from a non-virulent insect to a virus-carrying insect (PLoSPathogens, 2019, 15(3): e1007655). Utilizing this characteristic of the NSvc2-N-terminal protein in RSV transmission, a targeted method can be designed to introduce the NSvc2-N gene (or a portion thereof) into the rice genome. This will cause the rice plant to produce the glycoprotein, which competitively binds to the midgut virus infection site of the vector, thereby blocking the transmission of the virus by the planthopper. This strategy can be widely applied and referenced in antiviral breeding. Summary of the Invention

[0005] This invention addresses the aforementioned research background by using rice as the research subject and employing transgenic technology to insert a foreign gene—a glycoprotein from the RSV virus—into rice. NSvc2-NS The gene is 720 bp in length, with 1–717 bp originating from RSV. NSvc2 Nucleotides 88-804 of the gene have a 3-base stop codon added at the 3' end (see the nucleotide sequence listing for details). This gene encodes the soluble region at the N-terminus of NSvc2 (corresponding to amino acid sequences 30-268 of the NSvc2 protein). After obtaining transgenic rice materials, a feeding and transmission experiment showed that the virus acquisition rate, virus content in insects, and virus transmission rate of planthoppers were significantly lower than those of the control group (wild-type rice) after feeding on the transgenic rice. This indicates that the rice materials prepared using this method have the function of blocking RSV transmission by planthoppers. This invention provides a method for preparing transgenic rice using viral glycoproteins that can bind to the vector infection site to block virus transmission by planthoppers, filling the gap in technology for blocking virus transmission by planthoppers using the plant itself. The rice materials obtained using this method can reduce the virus-carrying rate of planthoppers, thereby blocking virus transmission. This method is simple, economical, green, and efficient, and can be applied to antiviral breeding, disease control research, and research on the mechanism of insect virus transmission. Attached Figure Description

[0006] Figure 1: Schematic diagram of the structure of the foreign gene inserted into transgenic rice.

[0007] Figure 2: Exogenous genes in transgenic rice plants NSvc2-NSThe RT-PCR detection results (M: standard molecular weight of DNA; 1: wild-type Nipponbare rice sample; 2-13: transgenic rice plant sample).

[0008] Figure 3: Western blot results of glycoprotein NSvc2-NS in transgenic rice plants (1: wild-type Nipponbare rice sample; 2-5: transgenic rice plant samples; rice Rubisco as internal control protein).

[0009] Figure 4: Results of Dot-ELISA detection of RSV-carrying rate of planthoppers after feeding on transgenic and wild-type rice (control group) leaves (data are shown as mean ± SD, ** indicates in the mean ± SD). P (There was a significant difference between groups at the level <0.01).

[0010] Figure 5: After feeding on transgenic rice and wild-type rice (control group), planthoppers were fed RSV-infected leaves. Quantitative real-time RT-PCR was used to detect the RSV virus in the planthoppers after the cycle period. NCP Gene expression levels (data are shown as mean ± SD, ** indicates at) P (There was a significant difference between groups at the level <0.01).

[0011] Figure 6: Results of RSV-transmission success rate of planthoppers after feeding on transgenic and wild-type rice (control group) leaves, followed by a cycle of single-plant virus transmission experiments. (Data are shown as mean ± SD, ** indicates within...) P (There was a significant difference between groups at the level <0.01). Detailed Implementation

[0012] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0013] Example 1: Using NSvc2-NS transgenic rice to block RSV transmission by planthoppers

[0014] 1. NSvc2-NS Cloning of genes

[0015] Primers were designed based on the reported nucleotide sequence of rice stripe virus RNA2 (GenBank accession number: AY186789): NSvc2-NS-F: 5'-GGATCCATGAGGAACAGGGAGAAGGGAAC-3' and NSvc2-NS-R: 5'-GGTACCTCAAGAGTACAATGAATATTT-3'. Total RNA was extracted from infected rice leaves and amplified by RT-PCR. NSvc2-NSThe gene was cloned into the rice expression vector pBWA(V)HS to construct the transgenic expression vector pBWA(V)HS-NSvc2-NS (Figure 1), and sequenced to verify it.

[0016] 2. Preparation of transgenic rice materials

[0017] The constructed pBWA(V)HS-NSvc2-NS vector was transformed into Agrobacterium, and Agrobacterium was then mixed with rice (variety: Nipponbare). Oryza sativa L. cv. Rice transformation was performed by co-culturing mature embryo callus tissue from Nipponbare embryos. After screening with hygromycin, resistant callus was selected and transferred to differentiation medium. The callus differentiated into seedlings in a constant-temperature incubator. Once the seedlings reached approximately 1 cm in height, they were transferred to rooting medium to strengthen them, thus obtaining T0 generation transgenic seedlings. DNA was extracted from the seedlings, and positive plants were screened by PCR. These positive seedlings were then transplanted to experimental fields for further growth. No significant abnormalities were observed in the plant growth, ultimately yielding T1 generation transgenic plant seeds.

[0018] 3. Detection of NSvc2-NS glycoprotein expression in transgenic rice plants

[0019] Transgenic rice seeds of different lines (T1 generation) were rinsed with deionized water, soaked at 25℃ (with 20µg / mL hygromycin added) for 2 days, germinated for 1 day, and then sown in 1 L beakers (15 seeds per beaker) and grown in a 28℃ light incubator. When the seedlings reached approximately 10 cm in height, RNA was extracted, and the expression of the NSvc2-NS gene was detected by RT-PCR. Figure 2 Simultaneously, leaf proteins were extracted, and the expression of NSvc2-NS protein in rice plants was detected by Western blot. Ultimately, four transgenic lines with high NSvc2-NS protein expression levels were obtained. Figure 3 ), for use in subsequent experiments.

[0020] 4. Effects of transgenic rice plants on the virus acquisition rate of planthoppers

[0021] The vector population of planthoppers was screened and preserved by the Plant Virus Laboratory of the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. Seedlings of the NSvc2-NS high-expression transgenic line identified in step 3 were transplanted to the field and grown to a height of 30 cm for virus transmission experiments. Third-instar nymphs of non-virulent planthoppers fed on transgenic rice plants, with wild-type Nipponbare rice serving as a control. After 3 days, the planthoppers were transferred to RSV-infected leaves and fed with the virus for 2 days. After feeding, they were transferred to healthy rice plants (variety: Wuyujing 3) for a 12-day recovery period. The virus-carrying rate of the planthoppers was determined by dot-ELISA after the recovery period, with four replicates. The results showed that the virus acquisition rate of planthoppers after feeding on transgenic rice plants was 19.7%, significantly lower than the virus acquisition rate of the control group (50.7%). P <0.01)( Figure 4 This indicates that feeding on NSvc2-NS transgenic rice plants can significantly inhibit the acquisition of RSV virus from diseased rice leaves by planthoppers.

[0022] 5. Effects of transgenic rice plants on the virus content in planthoppers

[0023] RNA was extracted from the planthoppers that had completed the cycle in step 4, and quantitative real-time RT-PCR was performed according to the method described by Li et al. (Acta Virologica, 2012, 56(1):67-71) to analyze the viral load in the planthoppers of the treatment and control groups. The target gene was RSV nucleocapsid protein (NCP), and the primers used were qNCP-F: 5'-TGCAGAAGGCAATCAATGACAT-3' and qNCP-R: 5'-TGTCACCACCTTTGTCCTTCAA-3', housekeeping gene. β- Actin As internal controls, the primers were qActin-F: 5'-TCTTGAGATTGGACTTGGC-3' and qActin-R: 5'-GTAGCACAGTTTCACCTTG-3'. After obtaining the CT values, 2 -ΔCt Method for calculating viruses NCP The relative expression levels of genes. Results showed that after feeding on transgenic rice plants, the planthoppers contained [a certain amount of] gene expression. NCP Gene expression was significantly reduced, decreasing by 87.9% compared to the control group. P <0.01)( Figure 5 This indicates that consuming diseased leaves after feeding on NSvc2-NS transgenic rice plants can significantly reduce the RSV virus content in planthoppers.

[0024] 6. Effects of transgenic plants on the virus transmission rate of planthoppers

[0025] After the planthoppers in step 4 had completed the cycle period, a single-plant, single-sprout virus transmission experiment was conducted. One 8 cm tall rice seedling (variety: Wuyujing 3) was transferred to a glass test tube, one seedling per tube, and one planthopper was simultaneously introduced into each tube for virus inoculation. One day later, the seedlings were transplanted into the field and covered with insect-proof netting. The disease incidence rate in the rice was recorded after 25 days. The experiment was conducted in quadruplicate. The results showed that after feeding on transgenic rice plants, the virus transmission rate of the planthoppers was significantly reduced compared to the control group, with a reduction of approximately 59.4%. P <0.01)( Figure 6 This indicates that consuming NSvc2-NS transgenic rice plants can significantly reduce the virus-transmitting ability of planthoppers.

[0026] Based on the above results, it can be concluded that transgenic rice plants that produce RSV glycoprotein NSvc2-NS can largely block the transmission of rice stripe virus by planthoppers.

Claims

1. A method for creating rice materials that block the transmission of rice stripe virus by planthoppers, characterized in that: The soluble region at the N-terminus of the rice stripe virus glycoprotein NSvc2— NSvc2-NS Gene fragments were integrated into the rice genome through transgenic technology. The resulting transgenic rice lines expressing NSvc2-NS protein significantly reduced the ability of planthoppers to acquire and spread RSV, thereby achieving the goal of blocking the transmission of the virus by planthoppers. The NSvc2-NS The transgenic recipient rice material for the gene fragment is Nipponbare. The NSvc2-NS The nucleotide sequence of the gene fragment is shown in SEQ ID No. 1.

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