Generation Of Transgenic Nicotiana.Benthamiana Expressing Sw-5b Resistance Gene To Control TSWV

Tomato spotted wilt disease is a kind of widely distributed disease,which cau-sing severe symptoms in many plants.The disease caused by Tomato Spotted Wilt Virus(TSWV),the Bunyaviridae Tospovirus typical members.Sw-5b is a R gene wit-h CC-(NB-ARC)-LRR structure,obtained from the Peru tomato,the R gene can effectively limit the spread of TSWV in tomato,only cause slight allergic reaction.In this study,we transfer the tomato R gene Sw-5b into N.benthamiana with transg enic technology.Plasmid p2300-Sw-5b was constructed containing tomato R gene Sw-5 b,and transfer to N.benthamiana with leaf disk method via agrobacterium,we obtani ng transgenic N.benthamiana after tissue culture,then we do some preliminary moleula r detection and disease resistance identification,the results are show follows:The construction of Sw-5b agrobacterium expression vector:in this stud-y,we designed specific primers and cloned the 3.7kb Sw-5b gene from resistant toma-to.We insert the gene into the engeered agrobacterium expression vector p2300(rep-laced 35S promoter to Sw-5b native promoter)via double enzyme digestion in Sm-al I and Pst I sites,the construct is verified by PCR and transfer into agrobacterium GV3101,the agrobacterium containing p2300-Nsm(TSWV NSm protein)co-infiltrated N.benthamiana,can cause a hypersensitive reaction(Hypersensitive Response,HR)sh-owed that the vector was constructed successfully.Study on Sw-5b transgenic N.benthamiana:we use leaf disk method to transfe-r p2300-Sw-5b expression vector into N.benthamiana,we determined the transgenic plants were homozygous by trait segregation ratio of T1 generation.The Sw-5b transgeni-c N.benthamiana inoculate with TSWV.HR was happened on inoculated leaf,7dpi,t-he transgenic N.benthamiana growth normally,The system leaves was used for Dot-b-lot detection and extract total RNA and detected by RT-PCR,nether detected TSWV i-nfection,prove that the transgenic N.benthamiana can effectively resist TSWV infecti on.This study successfully obtained Sw-5b trensgenic N.benthamiana resistant to TS-WV,the specific resistance mechanisms need further research.