Structure Of RNAi Universal Vector System, Plant Transformation And Expression Verification

RNAi is the phenomenon of double-stranded RNA induced gene silencing of homologous sequences m RNA degradation in cells.It has great potential value in forest pest control.The key to realize RNAi technology is to build the RNAi vector which can express stably in the plant.In order to improve the efficiency of RNAi vector construction and promote the wide application of RNAi technology in forestry,this research created a set of general vector system about RNAi plant transformation,with the existing plasmid vector.On this basis,the two conservative sequences of chitinase gene Chi and molting hormone receptor gene EcR were selected from cotton bollworm which the gene sequences have a highly homologous with a large number of forest pests.The test built four RNAi plant transformation vectors successfully,and verified the feasibility of RNAi plant transformation vector system.There are two of RNAi plant transformation vectors(G0 and G3)were leaded to the Agrobacterium tumefaciehs,then transformed into the tobacco using the Agrobacterium-mediated leaf disc method,so the transgenic tobacco were obtained successfully,then the vector function was verified through feeding cotton bollworm larvae.On the same time,transforming Poplar deltocdes‘55/56’×P.deltocdes‘2KEN8’to obtain the transgenic lines by PCR preliminary detection.The main results were as follows:1.Creating and construction of the RNAi plant transformation vector system.Using the existing cloning vector pUCm-T and plasmid pHSN401,received the vector pGr8 which carries Xcm I enzyme loci,the vector can form T vector after a single enzyme,the T vector can form "A-T" connection with the target gene sequence which added A tail by the conventional PCR;Using the arabidopsis thaliana genome DNA and vector plasmid p ET28 a as template,obtain the vector pGr39 by enzyme digestion connection that contain the “stem ring” structure.It can take advantage of the principle that after the connection between the same tail enzyme,the original enzyme sites will disappeared,then to accepted the different target gene fragment in many times,which has relevant tail enzyme loci that come from pGr8.Then obtain the target genes of positive and negative sequence and the “stem ring” structure and constituted the important structure of RNAi vector to produce dsRNA.Finally connecting this structure and vector p BI121,making up complete RNAi plant transformation universal vector.Taking advantage of this vector system can be connected to the target gene with the plant transformation vector by the conventional PCR reaction,"A–T" cloning and the ordinary "enzyme-links",at last,obtain the RNAi transformation universal vector.Cloning the full-length sequence of chitinase gene Chi and ecdysone receptor gene EcR from Helicoverpa armigera genome,and selecting a conserved sequence as target sequences,then using the vector system constructed 4 RNAi plant transformation vectors,pGr320(GFP),pGr326(Chi),pGr402(EcR)and pGr408(Chi +EcR),successfully.2.Genetic transformation and expression detection of tobacco by RNAi plant transformation vector.Transformating the vectors pGr320(G0)and pGr326(G3)into tobacco by agrobacterium-mediated,the transgenic lines were obtained after Kan rooting screening and PCR detection.There were 7 strains expressing GFP gene and 10 strains expressing Chi gene.The dsRNA of tager genes were expressed at the 17 transgenic lines through fluorescence quantitative PCR detection,and there were some difference among these lines.The feeding-insect test showed that feeding the transgenic lines which expression Chi gene dsRNA of G3 vector,had suppressed the target gene expression in the body of 3instars Helicoverpa armigera,To some extent,the gene silencing realized.However,feeding the transgenic lines of expressing GFP gene dsRNA and wild type cannot changes the target gene expression in insects’ body.The electrophoresis figure of the accumulation of si RNA showed that feeding the transgenic lines of expression no-target gene dsRNA G0 vector and wild type tobacco,insect had no obvious phenomenon of mi RNA accumulation in the body,but there was a large number of si RNA of feeding the transgenic lines of expression target gene dsRNA.The determination results of plant height,leaf area,chlorophyll content,photosynthetic parameters,chlorophyll fluorescence parameters and biomass in the transgenic lines and control showed that genetic transformation of the RNAi vector had no inhibition or distortion effects on the plants.3.Genetic transformation and expression detection of Poplar deltocdes‘55/56’×P.deltocdes‘2KEN8’ by RNAi plant transformation vector.Transformation the G3 vector into Poplar deltocdes‘55/56’×P.deltocdes‘2KEN8’by agrobacterium-mediated,the complete plants were obtained by screening of Kan and Cef which can root on the selective medium.Finally there were two lines can be obtained by PCR detection,successfully.