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

• 作 者: 张瑞腾 付秋实 马太光 郭秀霞 李灵芝 郭仰东 王怀松 李海平
• 机 构: 山西农业大学园艺学院,中国农业科学院蔬菜花卉研究所,中国农业大学园艺学院,
• 出 处: 中国蔬菜
• 发表日期: 2017年08期
• 关键词: CmGnT)RNA干扰 基因沉默 载体构建 乙酰氨基 葡萄糖基转移酶 单酶切 质粒载体 干扰载体 RNA 表达载体

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 investigate the function of CmGnT gene in melon, a conserved sequence of CmGnT gene from melon was selected and a 238 bp fragment was amplified by PCR using specific primers. The forward fragment was subcloned into the plasmid vector pFGC1008 between the 35S promoter and GUS by single digestion with Swa I and the reverse fragment was subcloned into the plasmid vector pFGC1008 between GUS and the terminator by Spe I digestion The RNAi expression vector p FGC1008-CmGnT was constructed. The recombinant plasmid was transformed into Agrobacterium tumefaciens C58 by freeze-thaw method. PCR analysis showed that RNAi expression vector p FGC1008-CmGnT was successfully constructed and transformed into Agrobacterium. It laid an important foundation for further study of CmGnT gene function and melon molecular breeding.