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Improvement of the Resistance of Nicotiana Tabaccum to Virus by RNAi and Initial Establishment of Ge

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Tutor: LiuDiQiu
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: CMV,RNAi,genetic transformation,antiviral,L.regale
CLC: S435.72
Type: Master's thesis
Year:  2011
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Abstract:
Virus disease is the second biggest disease after fungus diseases in agricultural production. Enormous economic loss in agriculture is caused by virus disease. The traditional prevention and control measures of virus disease are as flowing:(1) agricultural comprehensive prevention and control method, (2) mild strain cross protection, (3) the use of chemical pesticides. But these methods have some limitations and disadvantages, and they can not fundamentally solve the problems of virus disease. Recently, with the development of plant gene engineering technology, gene silencing has been developed quickly and has been become the important measure of controlling the plant virus disease. RNAi exists in a wide variety of eukaryotic organisms, and it is one of the natural plant defence mechanisms against virus infection. RNA interference (RNAi) is sequence-specific gene silencing induced by double-stranded RNA, and it can adjust and close the expression of the special genes in order to control advanced life action of all sorts of cells. The sence and antisense sequence of a gene can be cloned behind the same promoter and are separated by an intron. After the transcription, the RNA should match and form a hairpin RNA (hpRNA) structure. The hairpin RNA can form a stable dsRNA structure and can induce gene silencing. Cucumber mosaic virus (CMV) is a kind of plant virus with a tripartite sense single-stranded RNA. CMV is one of the most important plant virus and is of great academic value.Firstly, lily leaves with symptoms of CMV disease were collected from Chenggong country of Yunnan province in this study. The RT-PCR method were used to amplify the gene fragment of CMV RNA1 and RNA2, and the PCR products were sequenced. 1a gene fragment was in size of 344bp, and 2a gene fragment was in size of 308bp. Sequence alignment result showed that the cloning genes were highly homologous with the known with CMV 1a and 2a respectively. In present study, we adopted a high-level expression vector of pHellsgate2. The gene fragment of CMV 1a and 2a were respectively amplified by PCR using a pair of primers contained attB sequences, then the products were ligated into the pHellsgate2 vector by BP recombination reaction. The RNAi expression vectors were transferred into agrobacterium tumefaciens by electroporation. Then the RNAi vectors were introduced into tobacco by Agrobacterium-mediated method. Among the 83 lines of CMV 1a transgenic tobacco,31 were positive according to PCR analysis. And among the 212 lines of CMV 2a transgenic tobacco,132 were positive. In order to further study the resistance of transgenic plants to CMV. The positive striains were inoculated with CMV. Experimental results showed that the resistance of CMV was stronger in transgenic tobacco lines with RNAi vectors of CMV 1a and 2a than that in the wild type. The wild type lines inoculated with CMV showed twisted, lusterless, blocky and yellow-green mosaic symptoms. On the contrary, just individual transgenic tobacco lines revealed slight mosaic symptoms, and most were not infected by CMV. Among the 20 transgenic tobacco lines,12 strains had resistance to CMV, and among the 40 transgenic tobacco lines,21 lines had resistance to CMV. These results showed that double-stranded RNA of CMV 1a and 2a were expressed in tobacco, and the transgenic lines had different degrees of resistance to CMV infection.Lilium regale(L.regale) is a king of Chinese endemic species. L.regale has a very good ornamental value, and is good materials for genetic and breed because of its strong stress. In the study, the genetic transformation system of L.regale had been developed preliminarily. The Agrobacterium-mediated genetic transformation system L.regale was optimized by GUS gene transient expression in this experiment. Finally, the optimal parameter of L.regale were as followed:Agrobacterium concentration OD600 1.0, infection time of 25-30 minutes, co-culture temperature of 23℃, co-cuture time of 48h, and activation medium with50mg/L AS and without NH4NO3. And on the basis of the result of transient expression, we studied the stable expression of GUS gene in L.regale. As a result, among 36 transgenic plants of L.regale,31 lines were positive.
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