Construction And Utilization Of RNAi Vectors For The Functional Candidate Genes In Rice

RNA interference is the process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences. The RNAi technology is an effective strategy to be widely used to identify the function of genes in plants. Compared to other technologies, RNAi is sequence specific and silencing genes with high efficiency. In this paper, Gateway technology is used to replace the previously conventional cloning method to construct RNAi vectors for the functional candidate genes in rice and the RNAi constructs were applied to transformation analysis.Gateway technology is a high throughput cloning and expression platform that allows rapid and highly efficient cloning of targeted gene or any DNA segment across multiple vectors. It is based on the site-specific recombination reaction mediated by phageλ. DNA fragments or targeted genes flanked by recombination sites can be transferred into vectors that contain compatible recombination sites.Several PCD related candidate genes, such as OsCDC5, OsDAD1 and OsPDCD5, were used as target genes for the construction of RNAi vectors. Firstly the fragments for RNA interference were choosed for each gene after bioinformatics search, then attB PCR primers were designed and applied to amplify the attB-PCR products, entry clones were created using this attB-PCR products by BP recombination reaction, and LR recombination reaction were processed to construct expression clones for each target gene.The RNAi expression vectors were then transformed into IR64 by agrobacterium mediated transformation. After co-culture, selection, pre-regenetation, regenetation, root growing, the transgenic plants were obtained. Several important factors influencing transformation efficiency of indica rice were also optimized.