TW200826836A - Method of using plant transgenics realized through tomato spotted wilt virus (TSWV) (Tospovirus genus) RNA replicase high retention region to provide anti-TSWV effect and application thereof - Google Patents

Abstract

This invention relates to a method of using plant transgenics realized through tomato spotted wilt virus (TSWV) ( tospovirus genus) RNA duplication high retention region to provide anti-TSWV effect and an application thereof, wherein replicase high retention region of watermelon silver spot virus is used for breeding and selecting transgenic plant with extensive resistance, whereby the transgenic plant so provided can simultaneously resist at least five kinds of different and genetically distant TSWV effectively.

Description

200826836 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method and application for a tomato spotted wilt virus, in particular to a high-retention region of an RNA replicase using a tomato spotted virus It has a wide range of methods against the genus Fusarium oxysporum virus and its application in plants. [Prior Art] The virus of the genus Tomato spotted genus (Tbs/JOF/rz/s genus) is spread by the thrips in a resilient manner and distributed throughout the world, and can infect more than 900 families (more than 900 species). Species) Monocotyledonous and dicotyledonous plants, which endanger many important economic crops and are a globally important plant pathogenic virus, and thus have received the attention of international scholars. Tomato spotted wilt virus belongs to the genus of the only infected plant in the family of viruses. The other genus viruses of this family are the pathogens of humans, animals, poultry and other animals. They are spherical virus particles with lipid mantle and contain three negative electrodes. Sexual and bipolar genomic RNA, because of its extremely unstable viral particles and large genetic variability, is difficult to carry out research and is difficult to control, and is a plant pathogenic virus of global concern. Moreover, the virus has large variability, so it is difficult to prevent and control traditional disease-resistant breeding methods. Plant natural disease resistance genes can only resist a small number of closely related viruses, and breeding is not effective. As mentioned above, the tomato spotted wilt virus belongs to many important economic crops in the world, and it is not geographically distributed. The host range of the tomato spotted wilt virus, the species of the thrips, and the characteristics of the virus are also different. 5 200826836 The Asian region mainly uses the watermelon spot virus found in Taiwan, iffatenneIon silver, to help tt!e virus. It is mainly caused by the virus in Hongqing Group, which harms Cucurbitaceae and Solanaceae crops. Taiwan is located in the tropics, (4) belts, and the climate is suitable for the breeding of the virus of the genus Verticillium virus (ie 15 horses) and becomes a hotbed of the genus Phytophthora virus. The damage caused by the above-mentioned watermelon silver spot virus is particularly serious. It is an important cause of harm to Taiwan melon crops, which seriously affects the quality and output value of crops. The neighboring countries in the Tsuen Wan, such as the Japanese, Indian, and Thai, have different reports of the virus damage of the genus Panicillium virus, which mainly harms the melons and (4) crops. It is known that the tomato spotted wilt virus is harmful to melons, solanae, etc. Cash crops cause serious damage to melons and tomato cultivation. Due to different climatic patterns, the temperate regions in Europe and the United States have different crop types and are suitable for different species of thrips. However, there are different distributions of the tomato spotted wilt virus, mainly due to tomato spotted worms. s, TSWV) serogroup virus-based, with TSWV as its typical representative, caused serious damage to the cultivation of Solanaceae and flower crops. The concept of pathogen-derived resistance (PDR) was proposed in 1985 and was confirmed by the coat protein of the tobacco mosaic virus (卯TMV) in transgenic tobacco plants. Later, people used this gene transfer strategy to protect many kinds of plants to prevent plant viruses. In the past, using plant genetic engineering technology to grow transgenic plants was the most useful strategy for controlling viral diseases in the tomato spotted wilt virus. Prevention Party 6 200826836 Faces Many researchers have attempted to transfer the nucleocapsid gene (N gene) of TSWV into tobacco cells to form N-gene transgenic tobacco, and to obtain different degrees of resistance after inoculation of TSWV. Sexual performance. When nucle〇capsid pr〇tein (Np) accumulates in plant cells, transgenic tobacco is resistant to GRSV and INSV other than TSWV, indicating its resistance to protein. In addition, low transgenic performance Highly specific RNA vector resistance can also be obtained. However, these resistances are limited to viruses that provide genes. A related virus with a similar nucleic acid sequence or nucleic acid sequence, therefore, its applicability is greatly limited. Therefore, the existing method against the tomato spotted wilt virus belongs to its highly specific resistance in a variety of harmful tomato spotted wilt virus. Those who can play a preventive role are limited to viruses with similar kinship. If they are effective against a variety of tomato spotted wilt viruses, they still need to find a solution. [Disclosed from the above] The method for resisting the tomato spotted wilt virus belongs to its highly specific resistance, which is limited to viruses with similar kinship, and is limited to a variety of harmful Fanzhuang Verticillium virus. The main purpose of the present invention is The present invention provides a method for using a highly reproducible region of an RNA replicase of the genus Pombidium virus (Aspw/rw) to transfect a plant into a plant body, and has a method against the tomato spotted wilt virus, and the transgenic plant obtained by the method has broad resistance. It can effectively prevent and control a variety of viruses: the spotted wilt virus. 7 200826836 In order to achieve the above object, the anti-Panza variabilis virus of the present invention The technical means adopted by the method of the virus is that the method comprises the following steps: (a) Cloning against a tomato spotted wilt virus RNA according to a high-retention region of the RNA replicase by a selection method At least one gene fragment; 1 (b) re-sequencing the at least one gene fragment into at least one suitable vector to form at least one transgene; and translating the at least one transgene into a single transgene Planting into a plant host of the tomato spotted wilt virus to form a transgenic plant; (d) inoculating the tomato spotted wilt virus for resistance screening; (e) obtaining the transgenic plant. Preferably, the tomato spotted wilt virus is a WSMoV virus. More preferably, the RNA is dependent on nucleotides 3975 to 4928 of the viral complementary strand of the RNA replicase south retaining region wsm〇V L RNA. More preferably, the selection method is by RT-PCR, and seqid Ν〇·:1 and SEQ ID NO.:2 are used as primers for selection to obtain a gene fragment. More preferably, the method of colonization is carried out by RT-PCR using seqid Ν〇··3 and SEQ Π) NO· :2 as primers to obtain a gene fragment. More preferably, the selection method adopts RT-? (^ method to select seqid • and SEQ ID NO. 2· as a primer to obtain a gene fragment 0 200826836 More preferably, the selection is The method adopts RT-? (^ method to select seqid NO.:5 and SEQ ID NO.:6 as primers to obtain a gene segment. Preferably, the method of selection includes RT- In the manner of pcR, SEQ ID Ν0·:7 and SEq ID Ν0·:8 are used as primers to select a gene fragment having an inker sequence. Further preferably, the re-colonization method is The method comprises the following steps: (a) re-sorting the gene fragment into a pGEM-7zf(+) vector having a glucoamylase (Glucuronidase, GUS) gene; (b) using the gene fragment together with the Gus as a leader sequence The gene is re-selected into a PBI121 binary vector to form a transgene; / (c) the transgene is obtained. Further preferably, the re-cultivation means comprises: (a) the linked sequence The gene fragment is re-selected into a pGEM-7zf(+) vector with the GUS gene; (b) The transgene is formed by the fragment and the gene fragment having the ligation sequence together with the GUS gene as a leader sequence and a pBI12l binary vector, and then directly subjected to 1 igation; (c) obtaining the transgene. The planting method is to transfer the transgene into the plant host of the tomato spotted virus by the Agrobacterium transformation method. More preferably, the plant host of the tomato spotted virus belongs to the grass (Nicotiana benthanu.ana). The transgenic genus 仪 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 The host is melon, and the transgenic plant is melon. More preferably, the plant host of the tomato spotted virus belongs to watermelon, and the transgenic plant is watermelon. Preferably, the transgenic plant further utilizes at least one plaque. A genus of genus genus (4) is vaccinated with sputum and added with u to obtain a transgenic plant that has been screened. More preferably, at least one tomato spotted genus virus WSM〇v. More preferably, the at least one tomato spotted wilt virus line TSWV, GRSV, INSV or PCFV. More preferably, the at least one tomato spotted wilt virus line TSWV, GRSV, INSV and PCFV. Preferably, the at least one tomato spotted wilt virus further comprises WSMoV. Another main object of the present invention is to provide a high-retention region of RNA replicase using the tomato spotted wilt virus for transgenic into a plant body. The use of a method against a genus Panicillium virus includes providing a transgenic plant obtained by the method as a means of antiviral. Preferably, the transgenic plant is tobacco. Preferably, the transgenic plant is a tomato. Preferably, the transgenic plant is melon. Preferably, the transgenic plant is a watermelon. 200826836 It can be seen from the above that the steps of the present invention and the application thereof can provide a highly reservable region of the RNA replicase of the genus Tomato spotted genus (TbspoF/rys), which is transgenic into plants and has resistance to tomato spotted wilt virus. The method of virus makes the transgenic plants obtained by the method have broad resistance, and can effectively control a plurality of tomato spotted wilt viruses, and by using the method, the transgenic plants obtained by the method are provided as Anti-virus means. [Embodiment] In order to understand the technical features and practical functions of the present invention in detail, and in accordance with the contents of the specification, the following further describes the following preferred embodiments: [abbreviation and noun] GRSV : Groundnut ringspot virus, INVV INSV: Impatiens necrotic spot virus, Papaver plaque virus PCFV: Peanut chlorotic fan-spot virus, Peanut yellow leaf spot virus PBNV: r/rws, Peanut bud gangrene

RdRp: RNA-dependent RNA polymerase, RNA-dependent RNA replicase 1SW· Tomato spotted wi 11 virus, sold spotted, horse π 200826836 virus WSUoV: Fatennelon si Iver mottle virus, watermelon silver spot virus due to isolation of tomato spotted virus The strains are numerous and the host range is wide, and it is difficult to distinguish between the symptoms and the host range. Therefore, the use of nucleocapsid protein (NP) serum phylogenetic relationship and nucleotide sequence analysis has become an important classification basis. Currently known 16 species of tomato spotted wilt virus can be divided into three main serogroups, namely TSWV serogroup, including five viruses such as TSWV, GRSV, TCSV, CSNV and ZLCV; WSMoV serogroup, including WSMoV, PBNV Five viruses, WBNV, CCSV and CaCV; IYSV serogroup, including IYSV and TYRV. And four other serotypes of INSV, MYSV, PYSV and PCFV. The tomato spotted wilt virus is a globular virus with a lipid protein envelope and is about 80-110 nm in diameter. There are three linear single-stranded RNAs (ssRNAs) in the virus granules, which are named L, Μ and S RNA according to their molecular weights. The two sequences of the three genomic RNAs are complementary and paired with each other. They are tightly wrapped with NP to form a pseudo-circular structure. Three protein RNAs within the viral particle can be translated into six protein products. The L RNA is negatively polarized and has an open reading frame (ORF), which corresponds to the production of a large protein, which is considered to be an RNA replicase. Comparing the L protein sequences of four different viruses TSWV, INSV, PBNV, WSMoV, and other infected animal viruses of Fangke, it was found that this protein has a high degree of similarity, and it contains a high 12 repertoire of 200826836 degree retention of RNA replicase ( Chu ei a:, 2001· Phytopathology 91:36 368·). In addition, according to the degenerate primers designed by the nucleotide sequence of L RNA, RT-PCR can be successfully applied to greenhouse and field materials including at least TSWV, GRSV, INSV, WSMoV and PCFV. Detection of Verticillium virus (Chu ei aA, 2001.

Phytopathology 91: 361-368.). Each of the embodiments of the present invention is capable of being translated by a region having high retention on the L RNA of WSMoV (L RNA complementary strand sequence nt 3975-4928, containing five copies of the RNA replicase) Sexual and non-translatable, and the construction of double-stranded RNA are constructed in Ti vector, and the constructed transgenic fragments are transferred to tobacco cells to produce transgenic tobacco by Agrobacterium infection, and inoculated with WSM〇 under greenhouse conditions. And other genus Fusarium oxysporum viruses, such as TSWV, GRSV, INSV, PCFV, etc., to screen for transgenic plants that have extensive resistance to different species of tomato spotted wilt virus. [Virus Source] WSMoV and PCFV are collected from watermelons and peanuts in Taiwan. The tswv New York strain (tSWV-NY) was isolated from tomato and was provided by R. Provvidenti of New Y〇rk State Experifflent, located in Geneva. GRSV was collected from Brazil in Brazil, from New York. Presented by New York State Experiment Agricultural Station, 13 200826836 by D. Gonsalves. INSV is an isolate of Impatiens from the United States (INSV-M), located at North Carolina State University in Raleigh ( Provided by J. Moyer of North Carolina State University. Example 1 Example 1 of the present invention is an RNA-replicating enzyme (RdRp) of WSMoV. High-retention region gene fragment selection: (1) First extraction with ULTRASPECTM RNA The group (Biotex laboratories, Houston, TX) extracted and isolated the total RNA of the WSMoV-infected terminal grass. (2) Based on the nucleotide sequence of WSMoV L RNA, the specific primer pair of the high-retention region gene fragment was designed: WL3975 (Ncol): (5'-GCCATGGAGCACACATACAAGCATATCGCC-3') [SEQ ID NO.: 1] and WL4928c (5acI): (5,-GAGCTCGAGTCGTTCTCTTCTCCTGGCAGC-3') [SEQ ID NO.:2] WL3975UcoI) primer and WL4928c (5^cI) primer correspond to the 3975-3998 nucleotide and 4907-4928 nucleoside of the viral complementary strand (viral complementary strand, 200826836 vc strand) of WSMoV L RNA, respectively. The acid is accompanied by a c〇I cleavage position (CCATGG, bottom line) and a &d cleavage position (GAGCTC, bottom line). (3) Then, using rt-PCR, the above-mentioned wL3975(tVcoI) primer and WL4928c(&cI) primer were used to synthesize the first strand cDNA by reverse transcriptase, and then PCR was performed with Taq polymerase. A high-retention region gene fragment of the replicase on the L RNA (expected size is 954 bp), and the PCR product was analyzed by 1.2% colloidal electrophoresis, and a fragment was obtained, which corresponds to the high retention region of the aforementioned WSM〇v This region includes a portion of five RNA-dependent RNA replicase enzymes. (4) The gene fragment obtained after the above amplification was incubated with the τορο TA Cloning Kit (Invitrogen, Carlsbad, CA) and transferred to the competent cell. TOP l〇F, (Invitrogen, Carlsbad, CA) . After screening by kanamycin and X-Gal/IPTG blue and white, the selected transgenic cells are propagated and their plastid DNA is purified by alkali treatment. The DNA sequence is ABI377-1 9 automatic DMA sequencing system (ABI 377 -1 9 automatic DMA sequencing system) (Perkin-Elmer

Applied Biosystems, Foster City, CA) After sequencing, via GCG BESTFIT program (Wisconsin Package version 9. 0, Genetics

Computer Group, Madison, WI) aligns to confirm the correctness of the clone as a template for subsequent construction. 15 200826836 Example 2 The second embodiment of the present invention is the construction of translatable and non-translatable transgenes. (1) Principle: Since the L RNA of WSMoV is a negative RNA molecule, the complementary strand RNA corresponding to the viral strand is used as a protein. The expression template is such that the RNA transcribed from the DNA fragment obtained by using the complementary strand RNA as a template is in a translatable form; otherwise, the transcript of the DNA using the viral strand RNA itself as a template is a non-translated RNA fragment. Alternatively, a stop codon sequence (TAA) may be inserted at the 5' end of the DNA fragment of the translatable protein or a single nucleotide acid may be removed to cause a frame shift to be constructed as a non-translated body. (2) Translatable gene fragment: As shown in the first figure, the WL3975 (iVcoI) primer and the WL4928c (5^cI) primer are amplified with the nucleotide sequence 3975-4928 of the WSMoV L RNA virus complementary strand. According to the above principle, the gene fragment can be translated into a region containing 5 RNA-dependent RNA polymerase portions, and is translatable. (3) Design WLt3975UcoI) primer and WLst3975UcoI) primer: According to the WSMoV high-retention region fragment as template, the following primer was designed: WLt3975(yVcoI): (5, -GCCATGGAAT^T^GAGCACACATACAAGCATATCGCC-3,) [SEQ ID NO· :3] 200826836 WLst3975(^coI): (5, -GCCATGGA7^rKGAGCACACATACAAGCATATCGCC-3,) [SEQ ID NO.: 4] WLt3975UcoI) The primer and the WLst3975 (#coI) primer are added to the reading at the 5' end. The stop codon in the in-frame and the stop codon of the -1 frame-shift (TAATAG) are indicated in italics. (4) Non-translatable gene fragment: WLt3975UcoI) primer and WLst3975UcoI) primers were combined with the aforementioned WL4928c (5\3cI), and the gene fragment containing the high-retention region was selected by RT-PCR and TA cloning as described above. Since the two fragments are designed with two sets of stop codons at the 5' end, and the fragment amplified by the WLst3975d〇I) primer and the 叽4928〇(5^1) primer has a 5' stop codon, In addition, its reading frame is displaced, so it is non-translatable. (5) Design WL4928cUcoI) primer and ^3975 (5^(1) primer: WL4928c(^coI): (5, -CCATGGGTCGTTCTCTTCTCCTGGCAGC-3, ) [SEQ ID NO. 17 200826836 yiL3QT5<iSacl): (5,-GAGCTCGAGCACACATACAAGCATATCGCC -3, ) [SEQ ID NO.: 6] WL4928cd〇I) The primer and the ^3975 (5^6:1) primer have a restriction enzyme cleavage position opposite to the WL3975C σΐ) primer and the WL4928c (&cI) primer. . (6) Inverse non-translatable gene fragment: relative to the aforementioned WL3975UcoI) primer and ^49280 (5^(1) primer-increasing fragment, WL4928cd〇I) primer and Zha 3975 (&匚1) primer Increase the same area and set the opposite cloning site to create an antisense construct. (7) Confirm the sequence and add the GUS leader sequence: About the above (a) WL3975UcoI) primer and Zha 4928〇 (&^:1) primer fragment amplification (b) WLt3975UcoI) primer and WL4928c (&cI) primer increase Fragment (c) WLst3975UcoI) primer and WL4928c (&cI) primer-amplified fragment (d) WL4928c (#coI) primer and WL3975C heart cl) primer fragment amplification fragment is sequenced with TOPO TA selection kit To confirm that the sequence is correct. 18 200826836 Subsequently, the restriction enzyme enzymes #col and SacI were cut, and then cloned into the pGEM-7zf(+) vector (Promega, Madison, USA) having the GUS gene to obtain a GUS leader sequence. (8) Preparation of WLm, WLmt, WLmts and WLmAs: Finally, individual fragments carrying the GUS leader sequence are then spliced to / with sunflower mosaic virus / ffosa / c κ / ri / s, with /, such as 1 and SacI. CaMV) 35S promoter and a NOS terminator pBI121 binary vector (Clontech, Mountain View, CA), CaMV35S as a promoter, NOS as a terminator, corresponding to the aforementioned Each fragment was constructed as (a) WLm (b) WLmt (c) WLrats (d) WLraAs, which is a transgene with high transcriptional efficiency in plants. Example 3 Construction of the three-line double-stranded RNA of the present invention: (1) Preparation of WLm-linker fragment: Design WL4928c primer and WL3975WCO-linker primer: WL4928c (J^aI):

(5, -ATGCTCTAGAGTCGTTCTCTTCTCCTGGCAGC-3, ) [SEQ 200826836 ID NO. :7] WL3975#co-linker: (5, -CCGGCCATGG, 6Y^7GAGCACACATACAAGCA-3,) [SEQ ID NO.: 8] On the other hand, borrow The WL4928c(i7?aI) primer and the WL3975YCO-1 i nker increase the amplitude to obtain a WLm-linker fragment, which includes all bits and a Ycol cleavage bit and a join sequence (1 inker sequence, also known as spacer, the above indication Is the sequence of italics). (2) Preparation of WLmds: The WLm-linker fragment was then cloned and sequenced in a Τ0Ρ0 ΤΑ selection set to confirm its sequence. As shown in the first figure, the WLm-linker fragment cut, the WLm fragment cut with the version "ol/SacI, and the pB 11 21 cut with /Aal/SacI were mixed and directly bonded. Production of inverted WLmds structures. As described above, WLmds will correspond to the WSMoV viral vector (ie, the WLm-linker fragment cut using Bal/iVcoI) and its complementary strand (ie, the WLm fragment cut with VcoI/AcI). The DNA sequences which are described above as a ligation sequence are ligated to allow their transgenic transcripts to self-pair to form a double-stranded RNA (dsRNA). 20 200826836 Example 4 Breeding of four-line transgenic plants in the present invention: The transgenes formed by the above-mentioned WLm, WLmt, WLmts, WLmAs and WLmds are all transferred to Agrobacterium by direct transformation of liquid nitrogen (transf〇rm). (dfrMacier/(10) Disarmed strain LBA 4404 cells, and plant gene transfer. The above-mentioned transgenic construction system uses the leaf-disk transformation method, with Horsh et al. The Agrobacterium transformation method proposed in 985 was sent to tobacco cells separately; the Agrobacterium transformed cell with the transgenic vector was LB medium containing kanamycin and streptomycin at 28 °C overnight. Culture; take health from the leaves of the grass after sterilizing with water and water and rinsing with sterile water, cut into 〇·5 cm squares and place them in the Agrobacterium liquid that has been cultured overnight, and then transfer the transformed cells to MS104 medium. Cultured for two days; the msi〇4 medium contains MS salts (Gibco BRL, Gaithersburg, MD), 〇·1% vitamin B5, 3% sucrose, 1 " g/mi 6-aniline oxime (6-benzylamino purine, BA) ^0.1^ g/ml naphthaleneacetic acid (NAA), 〇·8% lye. Then move to 200/zg/ml kanamycin and 5〇〇# g /ffll Carbencillin in the screening of MS104 medium until the shoot grows. The shoot is cut and transferred to the hormone-free medium (ie 21 200826836 contains 200 // g In the MS medium of /ml kanamycin, the rooted plant is planted in a cultivation medium containing vermiculite, and planted under isolated greenhouse conditions for subsequent analysis. Example 5 Confirmation of the five-line transgenic lines of the present invention:

(1) Genomic DNA extraction kit (Genomic DNA)

Purification Kit) (GeneMark, Tainan, Taiwan) extracts the total DNA of non-GMO and transgenic tobacco according to the instructions of the manufacturer of the kit. (2) PCR was performed on WL3975Uc〇I) and WL4928c (5^cI) with specific primers to detect the presence of the transgene. In addition, PCR was performed using PNPTII: (5 -ATGATTGAACAAGATGGATTGCAC-3') [SEQ ID Ν0::9] and ΜΝΡΤΙΙ: (5 GAAGAACTCGTCAAGAAGGCGATA-3, ) [SEQ ID NO.: 10] as primer pairs to detect whether Mark the gene neomycin ph〇sph〇transferase (NPTII) to confirm whether it is a transgenic plant. Take 50 ng of extracted DNA as a template and perform ρπ with the above primer pair. The steps of the PCR temperature cycle program are as follows: 22 200826836 Temperature time effect 1.94 ° C 1 minute denaturation (denaturati〇n) 2 · 58 ° C 2 chaining (anneal ing) 3.72C 3 points extension (extension) 4.72 ° C 7 points are completed and 34 loops between "Step 1" and "Step 3" are extended. The PCR product was analyzed using 1% agarose electrophoresis with the presence of the N褥 gene. Example 6 Six-line resistance screening and analysis of the present invention: (1) Viral infection method: The above-mentioned translatability (WLm), non-translatability (pore „^ WLmts, wLmAs) and transcribed double-stranded RNA (WLmds) The transgenic material was inoculated with ffSMoV and other distantly quarantined P. variabilis virus in isolation greenhouse conditions. The virus-infected leaves were first ground in a 10 mM squash solution containing 1 〇 sodium sulfite (pH 7 Q). After the gold-sanded sand is applied to the leaves of the host plant, the solution is rubbed with the solution to achieve the purpose of virus infection. (2) Evaluation criteria for virus resistance · According to the tomato spotted wilt virus The status of infected tobacco was determined by the following group: (1) Infection (labeled as S): 4-7 days after infection, the same typical symptoms as 23 200826836 non-transgenic plants appeared on the leaves. (ii) Medium resistance (labeled MR): compared with non-艟A m α qy-non-褥 gene plants, the symptoms are delayed until 7 days. (Hi) Same resistance (marked as) ·· No performance Symptoms, resistant to viruses*(ιν) Marked as R): medium resistance (MR) and high resistance (HR) 2 are classified as resistant (R) groups. Therefore, in the resistant (R) group, the plants are resistant to viruses to a certain extent. (3) Vaccination with WSMoV: —+ The results of WTMoV, WLints, WLmAs and WLmds transgenic infections in WSMoV were summarized as follows: [Table 1] Transgenic test strain coefficient “LWsm〇vh^ dye

Ssstt A d fmmmmInf LLLLL resistant (R) plant height resistance coefficient (%) (HR) (%) ooooo 3 3 3 3 3 medium resistance (MR) (% i \ty )y )y \iy \J ^ 3 ο o 7 of 3 0 0 6 0 3 4 4 3 3j Γν ΓΧ rv rv 0 2 2 1-11 11 1x 11 \1/ \ly \ly \»y 7 3 3 7 0 6 3 3 6 0 4 6 6 6 7 /IV /IV Γν /IV rv 4 9 9 0 1 1A IX 1A OA~ )))))_ 13 3 3 0 0 1 :> 3 3 οΛ 112 2 3 4 1 /IV /IV rv Xc-, ▲ As can be seen from Table 1, the rna corresponding to WSMov L RNA depends on the rna potential shifting to the reserved area. It can be used with WLmt, WLmts, WLmAs & for its anti-blative and non-translating wsMqV, regardless of its article 24 200826836 Sense, antisense, or reversal repeats are more 艟f# From the inverted WLm, the WLm can be significantly improved (from & to 63.3% to 7〇〇%) 41* oblique "^ from! 7 l · 0 protection. 7 (K(U) for QV resistance, but for plants (4) Inoculation with other tomato spotted wilt virus: To assess the broad resistance of transgenic tobacco, from WLm, wLmt,

The transgenic WLmts WLmAs and WLmds were inoculated with γπν, GRSV, INSV and PCFV, and the results were summarized as follows: [Table 2]

Infected with GRSV coefficient of resistance (R) coefficient (%) Southern resistance (HR) (%) Medium resistance (MR) (°/〇) WLm 14 6 0 6 (42.9) (〇) (42.9 ) WLmt 19 10 4 6 (52.6) (21.1) (31.6) WLmts 19 11 7 4 (57.9) (36.8) (21.1) WLmAs 20 10 5 5 (50.0) (25.0) (25.0) WLmds 21 15 6 9 with resistance Number of Southern Resistance (R) Strains (HR) (%) (%) (71.4) (28.6) (42.9) Medium Resistance (MR) (%) 7 2 5 (50.0) (14.3) (35.7) 10 4 6 (52.6) (21.1) (31.6) 13 4 9 (68.4) (21.1) (47.4) 11 6 5 (55.0) (30.0) (25.0) 15 2 13 (71.4) (9.5) (61.9) (Continued from Table b) Transgenic infection with INSV infection with PCFV infection

High number of strains MR line C resistance R Η medium resistance R Μ high number of resistant strains MR line with resistance to middle % HR% R Μ 25 200826836 WLm 5 2 3 10 4 6 (35.7) (14.3) ( 21.4) (71.4) (28.6) (42.9) WLmt 11 5 6 14 7 7 (57.9) (26.3) (31.6) (73.7) (36.8) (36.8) WLmts 13 7 6 19 9 10 (68.4) (36.8) ( 31.6) (100.0) (47.4) (52.6) WLmAs 11 2 9 15 5 10 (55.0) (10.0) (45.0) (75.0) (25.0) (50.0) WLmds 16 9 7 16 6 10 __(76.2) (42.9) (33.3) (76.2) (28.6) (47.6) Above the WLm and Lu' except that the number WLm-16 can only fight against wSMoV, all other tested strains showed varying degrees of extensive resistance. In particular, the number WLm-11 and the number WLm-30 can simultaneously express resistance against WSMoV, TSWV, GRSV, INSV and PCFV; the number WLm U represents resistance among WSMoV, TSWV, GRSV and PCFV (MR) , and high resistance (HR) for INSV; number WLm_3〇 shows high resistance (HR) for WSMoV, GRSV and PCFV, and medium resistance (MR) for Tswv and nsv. In the case of WLmt, 6 strains, numbered WLmt-4, 5, 7, 9, 13 and 17, exhibited varying degrees of resistance to the aforementioned five viruses. In particular, two of the lines: Zhuangzi_4 and WLmt_13e are shown in Figure 2, and WLmt-13 is immune to the aforementioned five viruses. There are different strains for the WLmts, and the number of the 7 lines is: L6:. 24; its "UtS_7 shows a strain that is immune to all five viruses. For WLmAs, the numbers WLmAs-7, 8, 19 exhibit varying degrees of resistance against the aforementioned 5 20 and 21 viruses. Among them 26 200826836 WLmAs-7 showed resistance to immunity against all five viruses. Further, among the plants of the WLmdS strain, the numbers WLmds-2, 4, 10, 15, 16, 22, and 24 have different degrees of resistance, respectively, however, none of them can completely immunize all five viruses. (5) Conclusions on resistance screening and analysis: WLmAs and WLmds are not only able to show a wide range of resistance to wSM〇V by WLm, WLmt, WLmts, and transgenic plants. Viruses such as TSWV, GRSV, Guan and PCFV also showed varying degrees of resistance. Therefore, it can be seen that the transgenic plant produced by the present invention has broad resistance and can effectively (iv) at least a five-cyan and closely related genus Fusarium genus virus. The broadly resistant strains can be self-pollinated to obtain progeny' for genetic separation and resistance analysis of progeny. Example 7 In the seventh embodiment of the present invention, the transcript expression of the transgenic tobacco plants of the resistant strains was analyzed: (1) Northern blotting: the selected transgenic tobacco was subjected to northern hybridization. Analysis to confirm the amount of transgene expression in the transgenic plants. The total RNA of non-transgenic and transgenic tobacco was extracted and quantified and analyzed by electrophoresis using a 12% colloid containing formaldehyde (f〇rmaldehyde). Then, after electrophoresis, the colloid is transferred to the leaf "with the state 27 200826836 nylon film (Hybond-N nylon is responsible for the high-retention regional reaction of the replicase, the results are shown in the third figure, measured 0 membrane), to - Because the film # is also used as a probe for heterozygous Kodak BioMax film exposure detection from the above-mentioned northern heterozygous analysis, compared to the non-detection signal = transgenic grass plant, and two (礼.4 and ― -13) shows the infected transgenic lines in the retrovirus. In the plants with extensive resistance of the present invention, the transgenes all exhibit low transcription. Therefore, it is known that the broad resistance comes from RNA silencing. Example 8 The eighth embodiment of the present invention analyzes the number of transgenic copies carried by plants with resistant strains: (1) Southern blotting: screening for resistant transgenic tobacco Southern heterozygous analysis to confirm the number of transgenes in the transgenic plants (c〇py number): Extract the total DNA of non-transgenic and transgenic tobacco, quantify, cut with restriction enzymes & pi, and at 12 〇 Electrophoresis analysis was carried out with 〇.8% colloid. After electrophoresis, the colloid was transferred to Hyb〇nd-N nylon membrane, and the gene fragment of high-retention region of replicase was labeled with a-2P-dATP as a probe. The heterozygous reaction was carried out, and the results were detected by K〇dakBi〇Max negative film as shown in the fourth figure. (2) It can be seen from the above-mentioned southern heterozygous analysis that non-transgenic plants as negative control cannot be measured. Signals, and Table 28 200826836 Transgenic plants that are now widely resistant include one or more copies of the transgene. [Conclusions] (1) The method of the present invention does provide effective and extensive control of a variety of tomato spotted wilt viruses. Strategy: The present invention successfully utilizes a highly reproducible region of a replicase of Watermelon silver 卯ίί/e ηγμ (WSMoV, a species of tomato spotted wilt virus) to successfully produce transgenic tobacco with broad resistance, which can effectively simultaneously Against five different and closely related tomato spotted wilt viruses, these five viruses include WSMoV itself, tomato spotted wither virus (Tomato spotted ιν!it Virus, ritual ± round spot Sick Qin

Wroundnut hngspot virus, GRSV, impatiens necrotic spot virus (INSY), and peanut chlor〇tic fan — sp〇t virus (pcFv). It is known from the above that the method of the present invention can confirm that the transgenic tobacco plants can produce good resistance to five closely related tomato spots under greenhouse conditions, and the establishment of a broad transgenic resistance, a good tomato control Verticillium virus is a viral disease strategy. (2) The present invention is applied to other plants: In addition to the transgenic tobacco disclosed in the above embodiments, the constructed transgene can also be used in crops such as pangas and melon which are seriously endangered by the picrovirus and have important economic value. . Transgenic hens with broad resistance 29 200826836 You and melon crops are also covered for the other main purpose of the invention. The above description is merely a preferred embodiment of the present invention. It is not intended to limit the scope of the present invention. Any person having ordinary skill in the art can use the present invention without departing from the technical features of the present invention. The present invention is intended to disclose equivalent embodiments of the various changes or modifications made in the art, and without departing from the technical features of the present invention, it is still within the scope of the technical features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the construction of each transgene in the second embodiment of the present invention. Fig. 2 is an appearance view of the plants of the sixth embodiment of the present invention, numbered WLmt-13 and WLmt-29, infected with a virus. Fig. 3 is a diagram showing the N〇rthern blotting of the seventh embodiment of the present invention. Fig. 4 is a diagram showing the s〇uthern blotting of the eighth embodiment of the present invention. [Explanation of main component symbols] (1) WSMoV: Watermelon silver mottle virus, NC-003832 (accession code, the same below) (3) TSWV: Tomato spotted wilt virus, NC_002052 (4) INSV: Impatiens necrotic Spot virus, NC-003625 (5) GRSV: Groundnut ringspot virus 200826836 (6) PCFV · Peanut chlorotic fan-spot virus 31

Claims (1)

200826836 X. Patent application scope: 1. A method for using the RNA replicase of the genus Panax sphaeroides (TbspoF/ri/s1) to regenerate into a plant and having a virus against the tomato spotted wilt virus. The method comprises the following steps: (a) Cloning an RNA against the tomato spotted wilt virus by a selection means to obtain at least one gene fragment according to a high-retention region of the RNA replicating enzyme; (b) the at least one gene The fragment is subcultured (subcl〇ne) to abandon "select" to the appropriate vector to form at least one transgene; (c) to transfer the at least one transgene into a tomato spotted wilt virus a plant host of the virus to form a transgenic plant; (d) inoculated with a tomato spotted wilt virus for resistance screening; (e) obtaining a transgenic plant having resistance. 2. The method of claim 1, wherein the tomato spotted virus belongs to the WSMoV. 3. The method of claim 2, wherein the RNA is dependent on nucleotides 3975 to 4928 of the viral complementary strand of the RNA replicase and the reserving region WSM〇V L RNA. 4. The method according to Item 3 of the scope of the patent application, the colonization method is carried out by RT-PCR, using SEQ ID Ν〇: 1 and SEQ ID N〇: 2 as primers for selection. A gene fragment. 5. The method of claim 3, wherein the method of colonization is carried out by RT-PCR using SEQ ID NO: 3 and SEQ ID NO: 2 as primers to obtain a Gene fragment. 32 200826836 6·If the patent application scope is based on the method described in item RT of RT-PCR, the method of selection is used as a primer, and SEQidn〇.:4 and seqidno..2 are used as primers. The cockroach is selected to obtain a gene fragment. .·2 7· If the scope of the patent application is 3, the method of RT-PCR is adopted, and the method of selection is used as a primer for colonization by 4"丨Α Ν〇.:5 and SEQ ID NO."喔 侍 侍 侍 侍 侍 侍 侍 侍 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 N0. :8 is a gene fragment for the selection of the primer. It has a ligation sequence (1 inker 9. If the patent application scope is 4 to the re-selection (4) includes: $Receive method, (a PGEM-7zf(+) vector for re-sequencing the gene fragment; glutamic acid (GUS) (b) the gene fragment and the fish enzyme gene are re-selected into V as the leader sequence of glucosinolate The method includes the method described in the eighth item, wherein the re-selector (4) combines the reverse gene fragment with a porcine gene having a ligation sequence and a -PBI121 binary vector: the transgene. U·If the application mentioned in item 1 of the scope of patent application utilizes the farmer’s date, it’s a matter of The 51 method converts the transgene into the plant host of the tomato Μ Λ Λ 。 。 33 33 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 Small to obtain a resistance to grass transgenic plants: Caesar 2 3 disease: the method described in Item 1 or 11, the plant host of the genus ... 'to obtain the method described in the article , the method of obtaining the resistant item, in order to obtain a plant host melon melon transgenic plant having resistance 14 such as the patent application range 1 or u. 15. The invention relates to a plant host of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Patent application number 17 In the method, the at least one of the genus of the genus T. genus further comprises wsM 〇 v. 19. An application of the method described in the scope of the application of the patent application, which comprises providing the transgenic plant obtained by the method The method of antiviral. The application of the transgenic plant line is as described in claim 19. 34 200826836 21. The transgenic plant is a tomato according to the application of claim 19. The application described in claim 19 of the patent scope, the transgenic plant is melon. 23. The transgenic plant is a watermelon as claimed in claim 19. 24. A primer pair for the selection of a highly reproducible region of an RNA-replicating enzyme against a tomato spotted wilt virus rna comprising Sf:Q jd NO· :2, SEQ ID NO: :5 or SEQ ID NO· : 7. . 25. The primer pair according to claim 24, further comprising SEQ II) N0·:1 for use in combination with SEQ ID NO: 2. 26. The primer pair according to claim 24, further comprising SEQ II) N0·:3 for use in combination with SEQ ID NO.: 2. 27. A primer pair as described in claim 24, further comprising SEQ ID NO.: 4 in combination with SEQ ID NO.: 2. 28. A primer pair as described in claim 24, further comprising SEQ ID NO.:6 in combination with SEQ ID NO.:5. 29. A primer pair as described in claim 24, further comprising SEQ ID NO.:8 in combination with SEQ ID NO.:7. XI. Schema: as the next page 35