Screening Of Virus Disease Related Genes In N.tomentosiformis And Resistance Identification To Tobacco Mosaic Virus

Virus diseases is one of the most serious diseases in tobacco. Exploring and application virusreisistance related genes become especially important while breeding resistant cultivar is the mosteffective method to against virus diseases. Early most resistance related genes are obtained by cloingtechnology and examinated function by dentification. Altogether, there are282disease resistance geneswhich containing NBS specific structure were found from Nicotiana tomentosiformis.In this research, atotal of8partial sequence of virus resistance related genes were amplified by PCR, meanwhile its RNAiexpression vectors were constructed successfully through Gateway technology. For the prupose ofidentificate R genes function and breeding resistant cultivar tobacco,4RNAi expression vectors weretransformed into tobacco Honghua Dajinyuan by agrobacterium mediated transformation, at the sametime several important factors that influencing transformation efficiency most were also optimized. Themain results are as follows:1. A total of8virus resistance related genes RNAi expression vectors were constructed successfully.The partial sequence of R genes with specific banding site attB were amplified by PCR technology,which primers were designed according to the sequences obtained from Nicotiana tomentosiformissequencing. Using GATEWAY technology, the RNAi expression vector of R gene was constructedsuccessfully by insered the partial sequence of R genes into the vector of pH7GWIWG2(I).2. Establishment of transformation systems in tobacco Honghua Dajinyuan with4virus resistancerelated genes. In this research, several factors such as sterilization time of75%alcohol and20%Naclo,concentration of hygromycin during leaf regenetation and rooting progress as well as infection timeof agrobacterium and its concentration that influencing transformation efficiency most were optimized.3. Research on transgenic tobacco resistance for TMV. After inoculated with TMV, the diseaseindex and TMV RNA relative content were tested.The DI and TMV relative amount in some transgenictobacco are significantly higher than CK. It is further verified that the virus disease resisitance relatedgenes play an active role in system of tobacco resistance to virus.