马蔺重金属ATP酶基因HMA 2的RNAi载体构建及其遗传转化

Construction of RNAi expression vector of heavy metal ATPases gene HMA2 and genetic transformation in Iris lactea

重金属ATP酶基因HMA2介导的Cd长距离运输对植物体内Cd的分配及解毒起重要作用。然而,有关马蔺(Iris lactea)体内Cd长距离运输的分子机制仍不清楚。本研究以马蔺根中总RNA为模板,采用RT-PCR方法获得了重金属ATP酶基因片段,以中间载体pHANNIBAL和植物表达载体pART27为基础,采用酶切和连接的方法,构建了CaMV35S启动子驱动的含有IlHMA2基因片段反向重复序列的RNAi植物表达载体pARTG1G2,用冻融法将此重组质粒导入农杆菌GV3101,并转入到马蔺幼苗体内获得了IlHMA2-RNAi转化株系。这将为阐明IlHMA2介导的Cd长距离运输在马蔺适应重金属镉污染环境中的作用机制奠定基础。

Heavy metal ATPases HMA2 mediated Cd long-distance transport plays important roles in the distri- bution and detoxification of Cd2＋ in plants. However, the molecular mechanism underlying Cd2＋ long-distance transport in Iris lactea remains unknown. Total RNA was extracted {rom the roots of I. lactea to obtain a fragment of IIHMA2 by RT-PCR. Then RNAi expression vector pARTG1G2 with inverted repeats of HMA2 driven by the promoter CaMV35S was constructed based on the intermediate vector pHANNIBAL and the plant expression vector pART27 by restriction enzyme digestion and ligation. Moreover, the recombinant plasmid was transformed into agrobacterium GV3101 by freezing and thawing method, and then was transformed into I. lactea to obtain IIHMA2-RNAi lines. This would be a good basis for clarifying the IIHMA2 mediated Cd2＋ long distance transport in the adaptation of I. lactea to Cd2＋ contaminated environments.