利用Gateway技术构建黄瓜HPL基因的RNA干扰载体

Construction of RNAi Vectors for HPL Gene of Cucumber Using Gateway Technology

为探讨黄瓜脂氢过氧化物裂解酶基因(HPL)下调表达对黄瓜果实挥发性醛类物质合成的影响,设计1对特异引物,以黄瓜pDONR201-HPL质粒为模板,通过PCR扩增出323bp的片段。利用基于同源重组原理的Gateway技术,将扩增的片段通过BP反应连接到入门载体pDONR201中。对扩增出的323bp的片段进行测序,测序结果显示该序列与原序列同源性为100%。在此基础上,通过LR反应将目的片段插入过表达的植物RNA干涉载体pHellsgate8,构建黄瓜HPL基因的RNAi表达载体pHellsgate8-HPLi,分别经限制性内切酶XhoⅠ、XbaⅠ酶切后获得323bp的片段,与预期结果一致。采用热击法将pHellsgate8-HPLi转化农杆菌GV3101,经菌液PCR扩增获得323bp的目的条带,表明RNAi表达载体pHellsgate8-HPLi已成功转入农杆菌中。

In order to probe the effects of down-regulated expression of HPL gene on biosynthesis of volatile aldehydes in cucumber, a 323 bp interference fragment was obtained by PCR amplification with the specific primers and using cucumber pDONR201-HPL plasmid as a template. The fragment was introduced into the pDONR201 vector via BP reaction based on the homologous recombination of Gateway technology. It showed that the sequence of interference fragment was totally identified with that of HPL gene. Then the interference fragment was introduced into the destination vector pHells gate8 via LR reaction to form RNAi expression vector pHellsgate 8-HPLi, which was detected by re- striction enzyme Xho I and Xba I and it was consistent with the expectation. The plasmid of pHells- gate8-HPLi was transformed into Agrobacterium strain GV3101 by heat shock and the 323 bp frag- ment was amplified using the bacteria liquid PCR.