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利用基因重组技术,分别克隆木薯SBEⅠ基因ORF中的494 bp的片段和SBEⅡ基因ORF中的340 bp的片段;并通过重叠延伸PCR方法,扩增融合SBEⅠ、SBEⅡ基因片段的大片段SⅢ,将其正向、反向插入植物RNAi表达载体pART27中,同时克隆块根特异性启动子取代原来载体上自有的35S启动子,构建块根特异启动的可编码发夹结构的RNAi表达载体Sp-pRNAiSⅢ,为后续培育高直链淀粉含量的木薯新品种等实验打下基础。
The 494 bp fragment of SBEⅠ gene ORF and 340 bp of ORF of SBEⅡ gene were cloned by gene recombination technique respectively. The large fragment SⅢ fused with the SBEⅠ and SBEⅡ gene fragments was amplified by overlap extension PCR Forward and reverse insertion into the plant RNAi expression vector pART27, cloning the root-specific promoter of the original 35S promoter to replace the original vector to construct a root-specific RNAi expression vector Sp-pRNAiS Ⅲ encoding hairpin structure is Subsequent cultivation of high amylose content of cassava new varieties and other experiments lay the foundation.