人参多向耐药性转运蛋白基因保守区序列的克隆及分析

Cloning and analysis on pleiotropic drug resistance transporter gene conserved fragments from roots of Panax ginseng

目的对人参多向耐药性(PDR)转运蛋白基因进行克隆及序列分析。方法利用其他植物PDR基因的保守区域设计简并引物,以人参根总RNA为模板,采用RT-PCR方法扩增人参PDR基因片段并连接至pGEM-T Easy载体上,阳性克隆经PCR检测后测序。结果得到一段长693 bp的基因序列,序列分析表明,该片段编码231个氨基酸,与植物PDR基因核苷酸和氨基酸序列同源性分别在76%和80%以上。分析表明该序列属于PDR转运蛋白的核苷酸结合域,具有PDR转运蛋白的C端Walker A、ABC标签模体和C端walker B 3个保守功能域。结论首次从人参中克隆出PDR转运蛋白基因,为研究人参次生代谢产物的转运和积累机制奠定了基础。

Objectlve To clone and analyze the sequence of pleiotropic drug resistance （PDR） transporter gene from the roots of Panax ginseng. Methods Degenerate primers were designed based on the conserved sequences of the PDR genes from other plants. Total RNA form the roots of P. ginseng was used as template. PDR gene fi-agment was obtained by reverse transcription polymerase chain reaction （RT-PCR） and PCR products are sub-cloned into pGEM-T Easy vector. The positive clone identified by PCR was sequenced. Results A 693 bp gene fragment was obtained, encoding 231 amino acids. Sequence analysis suggested that the nucleotide sequence and the translated amino acid sequence shared over 76% and 80% of homology respectively with PDR transporter gene sequences from other plants. The predicted amino acid sequence was nucleotide-binding domain （NBD） and shared C-terminal Walker A, Walker B, and ATP-binding cassette （ABC） signature conserved functional domains with other plant PDR transporter gene. Conclusion It is the fwst report that PDR transporter gene is cloned from P ginseng. This work provides a foundation for investigation on the transportation and accumulation mechanism of secondary metabolites in P ginseng.