The Study Of Geminivirus Coat Protein And Viral Binding Protein Gene-mediated Virus Resistance In Plant

Geminiviruses occurred worldwide, and cause severe diseases in several economically important crops. In recent years, with bio-invasion of B biotye Whitefly Tomato Yellow Leaf Curl Virus (TYLCV) has outbreaked in a large area of the tomato-producing areas, and serious threats had been posed to agricultural production. Whitefly is the transmission vector of TYLCV, and the GroEL chaperone protein, which produced by the symbiotic bacteria of vector, can protect the circulation of the virus in the insect. Once the disease occurs, it is difficult to control. Recently the development of RNA silencing technique in the plant may be an effective way to solve this problem.In this study, a comparison has been made between TYLCV Y10 coat protein and other isolates. Different sequences of two 150bp segments and about 3 different length coat protein gene segments of TYLCV are selected to construct dsRNA plant expression vector respectively, then they are transformed into tobacco and tomato, on the purpose of obtaining resistance plants with strong and wide resistance to TYLCV, furthermore, to find the optimal length of inducing RNA-mediated virus resistance. Furthermore, to explore the use of RNAi to cut off the transmission of the virus, dsRNA plant expression vector is also constructed using virus transfer-mediated or GroEL protein gene segments in Whitefly. These studies provided a basis for further construction of dual-gene, infection and epidemic are cut off in two ways, which are virus infection and virus-transferring mediator, thus laying the foundation work on establishing the technical system of molecular mechanism for geminivirus. Study results and main conclusions are as follows:1 RNA Mediated Transgenic Resistance to TYLCCNV by transforming N. benthamiana and tomato with the CP gene segments dsRNA1.1 Studies on different homologous in amino acid sequence of CP gene dsRNA-mediated virus resistanceThe homology of 94.94% of the 159bp segment and 81.32% of the 158bp segment of the CP were selectec for construnction of plant expression vector based on the comparision of mino acid homology of most CP gene sequence of TYLCV and TYLCCNV, CP segment is successfully constructed dsRNA expression vector pBIN19-2HCP plant and pBIN19-2LCP, And introduced into N. benthamiana and tomato via Agrobacterium tumefaciens-mediated transformation. After tissue culture we got 56 and 43 transgenic N. benthamiana,61and 56 transgenic tomato. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant.The resistance ratio of transgenic N. benthamiana of TO progeny to TYLCCNV is 47.1%and 72.7%, respectively.1.2 Studies on CP gene segment of different length dsRNA-mediated virus resistanceAbout 150bp,300bp and 450bp length of the CP segment are selected, and The CP segment is successfully constructed dsRNA expression vector pBIN19-2CP159(pBIN19-2HCP), pBIN19-2LCP302 and pBIN19-2CP457. These vectors were further introduced into N. benthamiana and tomato via Agrobacterium tumefaciens-mediated transformation.56,57 and 38 transgenic N. benthamiana,59, 77 and 54 tomato plants were achieved. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant. The resistance ratio of transgenic N. benthamiana of TO progeny to TYLCCNV is 72.7,80.7%and 89.6%, respectively.Tl progeny from resistance plants are used to analyze the resistance, and it revealed that the resistant strain ratio of T1 progeny is 12.5%,22.5%,10%and 17.5%, respectively.2 RNAi interfere Whitefly transmission capacity by transforming N. benthamiana and tomato with the GroEL gene segments dsRNAAbout 300bp and 500bp of the GroEL gene segment are selected, GroEL gene segment is successfully constructed dsRNA expression vector pBIN19-2G502 and pBIN19-2G306, And introduced into N. benthamiana and tomato.via Agrobacterium tumefaciens-mediated transformation. After tissue culture we got 46 and 73 transgenic N. benthamiana,66 and 91 transgenic tomato. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant. No resistant type and higher rates of disease tolerance have been found in transgenic N. benthamiana of TO progeny, which has been transferred two different length of GroEL gene segment dsRNA, and contemporary, the ratio of the virus-tolerance plants is different in two inoculation method. Feed the whitefly two days with the infected in TO generation of transgenic tobacco first and then non-transgenic plants, and PCR examination showed that transgenic plants with transferred GroEL gene have a certain interference to whitefly's transmission capacity.3. The preliminary analysis of the resistance in the transient expression of dsRNA TYLCCNV CP gene in N. benthamianaIn order to initially ensure whether TYLCCNV CP gene dsRNA can induce viral resistance, and prevent blindness of trans-gene. We were constructed successfully plant expression vector pBIN19-GFP and pBIN19-CP-GFP. These genes transiently expressed after agro-infiltration in N. benthamiana, GFP fluorescence was observed in N. benthamiana leaves. We first infiltrated A.tumefaciens with pBIN19-2CP457in N. benthamiana leaves, followed by infiltration of pBIN19-CP-GFP, the result was a shutdown of GFP expression; or followed, after 16 days, by inoculation of TYLCCNV, the result was immunity to TYLCCNV.