甜瓜N-乙酰氨基葡萄糖基转移酶基因(CmGnT)RNA干扰载体的构建

Construction of RNA Interference Vector of CmGnT Gene in Melon

为探讨甜瓜N-乙酰氨基葡萄糖基转移酶基因(CmGnT)的功能,选取甜瓜CmGnT基因保守序列,利用特异性引物PCR扩增得到一段238 bp的干扰片段。利用Swa I单酶切将正向片段亚克隆到质粒载体p FGC1008的35S启动子与GUS之间,利用Spe I单酶切将反向片段亚克隆到质粒载体p FGC1008的GUS与终止子之间,构建了RNAi表达载体p FGC1008-CmGnT。通过冻融法将其转入根癌农杆菌C58中,菌液PCR验证表明RNAi表达载体p FGC1008-CmGnT构建成功并成功转入农杆菌。为进一步研究甜瓜CmGnT基因功能及甜瓜分子育种奠定重要基础。

In order to explore the functions of CmGnT gene,the paper selected conserved sequence ofCmGnT gene in melon,and amplified the interference fragment(238bp)by PCR with the specific primers.The forward fragment was subcloned into the plasmid vector pFGC1008between the35S promoter and GUS bySwa I single digestion,and the reverse fragment was subcloned into the plasmid vector pFGC1008between theGUS and the terminator by Spe I single digestion,and the RNAi expression vector pFGC1008-CmGnT wasconstructed.The recombinant plasmid pFGC1008-CmGnT was transformed into Agrobacterium tumefaciens(C58)by freeze-thaw method,and the results of PCR confirmed that the vector(pFGC1008-CmGnT)wassuccessfully constructed and transferred to C58.This study has laied an important basis for further exploring thefunction of CmGnT gene and molecular breeding of melon.