油菜RNA干扰(RNAi)文库的构建

The Construction of Oilseed Rape RNA Interference(RNAi) Library

为了通过构建高通量RNAi(RNA干扰)双元载体来得到油菜RNAi文库,对PCR引物设计时,在目的片段5′端和3′端分别引入合适的酶切位点序列,在3种中间载体的基础上构建高通量RNAi双元载体pHBMT4。该载体Bsu36Ⅰ酶切位点间的片段可被油菜的任意一种功能片段取代。载体pHBMT4含有用以检测载体功效的gus报告基因,原多克隆位点处被一个插入目标序列表达框取代,该框含有的反向重复nos终止序列可自然形成发夹环结构引发转录后水平的基因沉默(PTGS)。通过抽提的油菜总DNA与载体pHBMT4相连转化获得了油菜RNAi文库,为以后研究油菜的基因组功能打下良好的基础。

In order to construct RNAi（RNA interference） library of oilseed rape by a high-throughput RNAi vector,the PCR primers were added with proper adapter when designed to get various complementary sticky ends.The plasmid pHBMT4 was constructed based on three kinds of intermediate vectors.The sequence among Bsu36 Ⅰ could be displaced by any genomic fragment of oil seed rape.There were two kinds of different selectable marker genes combined for the plasmid pHBMT4;Kanr gene was used to select the recombinant plasmid in E.coli and Hyr gene was used to select the transgene plant.The gus reporter gene used to test the efficiency of the plasmid,was also combined for the pHBMT4.The multiple cloning sites（MCS） was displaced by an insert sequence reading frame consisting of the inverted repeated nos sequence that could spontaneously form hairpin loop to induce posttranscriptional gene silencing（PTGS）.When the insert sequence was displaced by the genomic sequence of oilseed rape,the RNAi library of oilseed rape was successfully obtained,which was helpful to study the genomic function of oilseed rape.