块根特异启动的木薯SBE Ⅰ、SBEⅡ基因RNAi载体的构建及鉴定

摘要

利用基因重组技术,分别克隆木薯SBE Ⅰ基因ORF中的494 bp的片段和SBEⅡ基因ORF中的340bp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.%A 494 bp fragment named SI from the Open Reading Frame(ORF)of SBE I and a 340 bp fragment named S Ⅱ from the ORF of SBE Ⅱ were cloned from cassava and fused into a larger fragment named S Ⅲ with Overlap Extension PCR(SOE PCR). The S Ⅲ fragment was inserted into the plant expression vector pART27 RNAi in forward and reverse direction; Also the original 35S promoter in the vector was replaced with a root specific promoter Sporamin, and then a coding hairpin RNAi expression vector Sp-pRNAiS Ⅲ was constructed, which could, lay a foundation to breed new cassava varieties with high amylase content.