Studies On NPR1 Gene Cloning In Capsicum Annuum L., Vector Construction And Tobacco Transformation

The research of the molecular mechanism of plant defense reaction is the basis of clarifying the mechanism of plant disease-resistance, and the effective genetic modification. It showed that the R gene played the critical role in the the disease-resistant reaction, but the disease-resistant mechanisms mediated by the R gene in diffent plants has not been clarified so far. Local ETI can activate systematic disease-resistance SAR (Systemic acquired resistance),NPR is the important regulate protein of the SAR, the analysis of the structure and function of NPR and its relation with other proteins ,which will help to clarify the disease-resistant mechanisms mediated by the R gene. To get the transgenic mutants of the over expression and RNA interference of NPR for the research of the impact that the NPR gene has on the other relative genes, this study screened a NPR1 cDNA from the cDNA library of Capsicum annuum L, constructed the over expression vector and RNA interference vector of NPR1 cDNA, transformed into tobaccos respectively to obtain transgenic tobaccos. The main conclusions are:1. By PCR with special primers ,the fragment of NPR1 cDNA was isolated from the cDNA library of the L-11 leaves of Capsicum annuum L. that treated by UV, whose length is 1.8kbp including a complete ORF encoding 582 amino acids which containes four conserved domains (BTB, DUF, ANK, NPR1-like).2. Constructed the overexpression vector and RNA interference vector of NPR1 by Gateway vector technology, using pDONR201, pk7WG2 and pHELLSGATE12. After sequencing these vectors, transformed tobaccos with Agrobacterium-mediated method and obtained T0 generation plants of transgenic overexpression and RNA interference.3. Extracted DNA from transgenic tobaccos, detecting with PCR obtained 39 plants of T0 generation of transgenic overexpression, and 7 plants of RNAi. Extracted RNA from positive plants with Trizol method, and transcribed RNA into cDNA . Identifying the transgenic tobaccos with RNA inferred that the overexpression and RNAi effect of NPR1 in tobacco K326.