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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。
Objective To clone and sequence the ginseng multidrug resistance (PDR) transporter gene. Methods Degenerate primers were designed based on the conserved regions of other plant PDR genes. The total RNA of ginseng root was used as a template. The PDR gene fragment of ginseng was amplified by RT-PCR and ligated into pGEM-T Easy vector. The positive clones were sequenced by PCR . A 693 bp gene sequence was obtained. Sequence analysis showed that the fragment encoded 231 amino acids, which shared 76% and 80% identity with the plant PDR nucleotide and amino acid sequences, respectively. The analysis showed that the sequence belonged to the nucleotide binding domain of PDR transporter and had three conserved domains of C terminal Walker A, ABC tag motif and C terminal walker B of PDR transporter. Conclusion The PDR transporter gene was cloned from ginseng for the first time, which laid the foundation for the study of the mechanism of transport and accumulation of secondary metabolites in ginseng.