郁金香ACC氧化酶基因RNAi表达载体的构建

Construction of RNAi Expression Vector Targeting ACC Oxidase Gene in Tulipa Gesneriana L.

为了构建郁金香ACC氧化酶基因的RNAi表达载体.采用Trizol法提取了郁金香花瓣总RNA,根据郁金香ACC氧化酶基因编码区序列,设计了带有限制性内切酶位点的特异性引物,成功地扩增得到了郁金香ACO基因保守片段.将此片段以正向和反向插入到中间载体pBSin内含子的两端,构建成发卡结构的RNA片段,利用pCAMBIA35S-1301作为桥梁载体,构建了带发卡结构的RNAi表达载体pCAMBIA-ACO.

The total RNA was extracted from the petals of Tulipa gesneriana L. and reverse transcripted to first strand of eDNA. A pair of special primers was designed according to conservative region of 1-aminoeyclopropane-1-earboxylie acid oxidase （ACC oxidase） gene. The conserved sequence fragment of ACC oxidaso gene was amplified by RT-PCR. The sequence was inserted into the two sides of intron of middle expression vector pBSin in positive and negative orientation, so the Hairpin RNA structures were constructed. The RNAi expression vector pCAMBIA- ACC oxidase had been constructed by utilizing pCAMBIA-1301 vector.