Study On Construction Of Expression Vector Of F3'H Gene From Eupatorium Adenophorum And Transformation To Tobacco

Eupatorium adenophorum is a kind of global exotic weeds and spreads extensively in south and westernsouth of China. They spread fast and have drawn the attention of the society. E. adenophorum have strong allelopathy, and can product the allelopathic substances to inhabit the peripheral plants' growth. During the metabolism pathway they can produce allelopathic substances, flavonoid 3'-hydroxylase (F3'H) can be induced by 9-Oxo-agerphorona, implying that F3'H gene is related to allelopathy of this weed. We could further explore the molecular mechanism about the allelopathy of E. adenophorum through research of F3'H gene that related to allelopathy and functional analysis, then study the function and role of the secondary metabolism pathway about F3'H gene, thus we could expect to provide the theoretical basis of control and management of E. adenophorum, and decline the economic loss and ecological destruction.The over-expression vector of F3'H gene was constructed and transformed into tobacco, then the physiological and molecular analysis was done in the research.The template is the plasmid DNA contained full-length F3'H gene which was conserved by our laboratory, the primers contained Xba I restriction endonuclease site, the PCR were performed. After electrophoresis, target PCR product was recovered, then linked with pMD18-T vector, named F3'H-T. Digestion by Xba I, the correct F3'H gene fragment was gotten from F3'H-T vector. At the same time, the PBI121 vector was digested by Xba I, linearized and dephosphorylated, then the F3'H gene fragment and PBI121 vector were linked by T4 DNA lingase. Next,the linked product was transformed into Escherichia coli competent cell(DH5α), the positive clones were picked out and identified.Finally,the F3'H gene and PBI121 vector were transformed into tabacoo.The result showed that the flower color of transformants expressing F3'H was the faintest among all the transformants and untransformants, flower color of transformants expressing PBI121 was deeper than that of expressing F3'H, but fainter than the untransformed control plants, and some expressing F3'H flowers had stripe between red and pink. It indicated that F3'H gene had close relationship with the formation of anthocyanins in the secondary metabolism pathway. Semi-quantitative PCR showed that F3'H gene could be transcripted and expressed in the transgenic tobacco, and had different accumulation.Real-time PCR suggested that expression accumulation in transgenic tobacco declined compared to the wild-type tobacco,but the varity extent were diferent.Therefore ,we presumed that expression of F3'H gene was affected by endogenous gene afer transformed into tobacco, which lead to the change of the content of anthocyanin in transgenic tobacco and the color slightly faint of flowers.Additionally, physiological detection about transformants and untransformants demonstrated that POD and PAL activity in the transformants were higher than that in the untransformants.The primers which contained attB1 (aaaaaagcaggct)and attB2(aagaaagctgggt) sequence were designed, the RNAi vector was successfully constructed by Gateway technique via BP recombianant reaction and LR recombianant reaction and transformed into tobacco in the paper too.