Studies On The Construction Of Two Chemical-inducible Expression Vectors And Transient Expression Analysis Of VGCfE: Gus In Tobacco

Rice serve as a major food crop in China, in order to effectively stabilize the sterility of sterile lines in the process of the two-line hybrid rice seeding at low temperatures, our group plans to construct the Chemical inducible RNA interference expression vector p3301VGCfE:Udtli and reporter gene Gus expression vector p3301 VGCfE:Gus, and then transfect corresponding plant materials to investigate the regulation carried out by the chemical inducible expression system in the rice sterile fertility conversion. The preliminary study has fulfilled the following goals:1. Construction of chemical inducible RNA interference expression vector p3301VGCffi:RNAiIn this study, the existence of the OsUdtl gene was confirmed according to the sequence of the rice Y58S chromosome VII on Genebank, and the 3'UTR sequence of the OsUdtl gene was then obtained by the blast results and the information submitted by Genebank. We designed gene specific primers and successfully amplified DNA which is about 450bp, taking genome of rice Y58S as template. The sequencing results of the acquired DNA segment proved to be correct according to the Blast results. Through ligation independent cloning, the two 3'UTR pieces were respectively linked to the two ends of the GA20 oxidase intron in opposite direction, hence the pUCC-LWri vector was constructed, After that,3'UTR-intron-3'UTR was inserted into the vector p3301 VGCfE through RF cloning due to limited restriction sites. Tested by PCR assay, the chemical inducible RNA interference expression vector proved to be successfully constructed.2. Construction of Chemical inducible reporter gene expression vector p3301VGCfE:GusAfter the synthesis of pUC57-4635S vector, two identical restriction sites of the p1381z vector and 4635S fragments were selected. The vector and fragment were digested, purified and connected using the T4 DNA ligase. The clones were identified by PCR and sequencing. We designed 4635S-Gus fragment specific primers to amplify the fragment based on the principle of LIC. Then the fragment was linked to the vector p3301 VGCfE through ligation-independent cloning. Tested by PCR assay the Chemical inducible reporter gene expression vector p3301 VGCfE:Gus proved to be successfully constructed.3. Agrobacterium-mediated transfection of tobacco by reporter gene expression vector p3301VGCfE:GusThe successfully constructed vector p3301VGCfE:Gus was transected into tobacco leaf mediated by Agrobacterium. After the induction of the inducer methoxyfenozide, the reporter gene Gus expression was observed by Gus staining. Compared with the control group in which no Gus expression was detected, leaves tranfected by p3301VGCfE:Gus expressed the Gus gene. This shows that the chemical inducible expression system is able to express the Gus gene after induction of the inducer.