The Isolation And Identification Of Tomato Spotted Wilt Virus And Study On The Resistance Of Transgenic Tobacco Expressed DsRNA

The genus Tospovirus is the only phytopathogenic group in the family Bunyaviridae. Tomato spotted wilt virus (TSWV), a member of Tospovirus, is widely spread in the world, which can infect more than100genus including more than1000species of plants and cause serious losses. But it is seldom reported in China. For the past few years, with the spreading of western flower thrips (Frankliniella occidentalis), one of the main transmission vectors of TSWV, the distribution of TSWV is enlarging, the specie of its host is increasing. Since it’s difficult to control, TSWV has posed very serious threat to agricultural production. In order to explore the spread trend and the control methods of TSWV, establish the detection method of TSWV, eighteen samples were collected from tomato, pepper, tobacco, cowpea, and so on in Yunnan province. Some of TSWV isolates were isolated and identified, and their phylogenetic relationship were analysed. Using the theory of RNAi, inverted-repeat dsRNA vectors with different length segments were constructed and transformed into Nicotiana tobacum cv. Samsun NN. Transgenic tobacco was achieved, and the resistance of transgenic tobacco expressed dsRNA of TSWV has been studied and analysed.The results and main conclusions are as follows:1. The isolation and identification of TSWVIn this study, through isolating and identifying suspected TSWV samples from different areas, a TSWV strain was isolated from Iris tectorum collected in Yunnan Agricultural University. We identified it as TSWV based on the results of the host’s symptoms, ELISA and molecular detection, and named it as YN-2. The complete sequences of N protein and GN/GC glycoprotein were cloned, respectively. The results showed that the complete sequences of N protein were777nt and encoded258aa (accession number:KC294570), while the GN/GC glycoprotein sequences had3408nt and1135aa (accession number:KC294569). Their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins were analyzed. The phylogenetic relationship results showed that N protein and GN/GC glycoprotein of YN-2exhibited high homology with YN01in China:99.74%and99.53%, respectively. Through analysing their phylogenetic relationships, we found that the phylogenetic comparison of N protein and GN/GC glycoprotein was in different groups. We assumed that the genome reassortment may happen between M segment that encodes GN/GC glycoprotein and S segment that encodes N protein.Besides, eight TSWV stains were isolated from tomato, pepper and cowpea which were collected in different areas in Yunnan province, and the complete sequences of N protein and GN/GC glycoprotein of the eight TSWV stains were cloned, respectively. The complete sequences of N protein of the eight TSWV stains were777nt and encoded258aa, GN/GC glycoprotein were3408bp and encoded1135aa. And their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins in China were analyzed. Their phylogenetic relationship results showed that N protein and GN/GC glycoprotein of eight TSWV pathogens exhibited high homology with other TSWVs:99.34%and99.38%, respectively. Through analysing the phylogenetic relationships, we found that eight TSWV pathogens have the same origin, although they were isolated from different host plants.To our knowledge, this is the first time to report that TSWV is isolated from Iris tectorum and cowpea.2. Study on TSWV N gene dsRNA vectors being transformed Nicotiana tobacum cv. Samsun NN, and the resistance of the transgensic tobaccoTSWV N gene segment of different length dsRNA vectors (±313-1304and±536-1304,313and536for short, respectively) had been constructed using RNAi in2010, and the dsRNA vectors had been introduced into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation, respectively. After tissue culture, sixty and seventy-nine transgenic tobaccos were achieved. Through observing the phenotype of T0and T1progeny after TSWV inoculation, we found that the resistance effects of T0and T1progeny of transformed536was better than those of transformed313; and the resistance effects of To was better than its of T1in all of transgenic tobaccos.3. Study on the construction of dsRNA vectors with TSWV different length of GN/GC gene segment, and their expression in Nicotiana tobacum cv. Samsun NNUsing RNAi, four inverted-repeat dsRNA vectors with different length segments and different regions of TSWV GN/GC gene were constructed. According to TSWV GN/GC complete gene sequences, restriction enzyme site (Ascl, Swal, BamHl, Spel) were introducted when designing primers to amplied297bp,500bp,328bp and503bp segment, respectively, to construct inverted-repeat dsRNA expression vectors. The mature dsRNA vector, pFGC1008, was used as the inverted-repeat dsRNA vector, and335bp sequences of GUS gene in pFGC1008was used as the intron, pCAMBIA1304was used as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid was consistent with expected results by PCR analysis, restriction enzyme digestion and DNA sequencing.The inverted-repeat dsRNA plant expression vectors were transformed into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation. After tissue culture, twenty transgenic tobaccos transformed G13-1304were achieved, twenty transformed G15-1304, fifteen transformed G23-1304and sixteen transformed G25-1304were also achieved. The resistance of the transgenic tobacco needs to be further analyzed.