Construction Of Transgenic Tobaccos Carrying CP And RdRp Genes Of Tomato Spotted Wilt Virus And Rice Black Streaked Dwarf Virus

Rice black-steaked dwarf virus(RBSDV)can infect rice,corn,wheat and other gramineous crops,causing rice black dwarf disease,corn roughing disease and other diseases.Its genome consists of ten dsRNA segments,which includes S1 encoding RNA dependent RNA polymerase(RdRp)and S10 encoding coat protein(CP).Tomato spotted wilt virus(TSWV)can infect more than 900 species of 70 families,and the disease is distributed around the world.Its genome consists of three single-stranded RNA segments(L,M and S).RNA L is a negative-stranded RNA,whose complementary strand encodes RNA-dependent RNA polymerase(RdRp).RNA S is an ambisense RNA and its complementary strand encodes coat protein(N).Both viruses can cause serious production hazards and economic losses.In order to further study these two RNA viruses at the molecular biology level,we constructed the transgenic tobaccoes containing CP and/or RdRp gene of RBSDV and TSWV in this study,which will be used to build up the reverse genetics system on RBSDV and TSWV.Using pROKⅡ as basic binary vector,8 transgenic vectors(pROKⅡ-TS-CP、pROKⅡ-TS-CP-8H、pROKⅡ-RB-CP、pROKⅡ-RB-CP-8H、pROKⅡ-TS-RdRp、pROKⅡ-TS-RdRp-8H、pROKⅡ-RB-RdRp、pROKⅡ-RB-RdRp-8H)containing CP and RdRp of RBSDV and TSWV with or without His-tag were constructed.These 8 vectors were respectively transformed into the tobacco(N.tabacum,NC89)using Agrobacterium tumefaciens-mediated leaf culture.After pre-culture,co-culture and selective culture,the resistant callus were screened out and the adventitious buds were transferred into rooting medium to obtain complete regenerated plants.The callus was induced by pre-differentiation,co-culture and selective culture,and then transferred into rooting medium and culture tank,and finally moved into soil.Then,we can respectively obtain the monovalent and bivalent transgenic tobaccos expressing CP and RdRp of TSWV and RBSDV stably through the tissueculture technique.Based on the detection of PCR、RT-PCR and Western blot,tobaccos containing RBSDV CP(with His-tag)or TSWV CP(with His-tag)were achieved,and other monovalent and bivalent transgenic tobaccos are performing.