论文部分内容阅读
目的克隆北柴胡皂苷生物合成途径关键酶异戊烯基焦磷酸异构酶(EC 5.3.3.2,isopentenyl diphosphate isomerase,IPPI)的全长cDNA,为研究柴胡皂苷的生物合成与基因调控奠定基础。方法 PCR矩阵法快速筛选北柴胡全长cDNA文库。结果获得了北柴胡IPPI的全长cDNA(GenBank No.gq433719),其核苷酸序列长1 117bp,编码319个氨基酸的蛋白。NCBI Blastx结果显示与胡萝卜Daucus carotasubsp.sativus(abb52064)的IPPI氨基酸序列相似性最高,一致性为92%,相似度为97%。保守结构域搜索显示含有IPPI共有的催化活性位点、金属结合位点及Nudix基序。TargetP1.1和SignalP3.0分析表明北柴胡IPPI N端含有长26 bp的叶绿体信号肽。结论首次克隆了北柴胡IPPI的全长cDNA,将促进后续北柴胡IPPI基因表达特性及其在柴胡皂苷合成代谢中功能的研究。
Objective To clone the full-length cDNA of isopentenyl diphosphate isomerase (IPPI), a key enzyme in the biosynthesis pathway of saikosaponin, and lay a foundation for studying the biosynthesis and gene regulation of saikosaponin . Methods The full length cDNA library of Bupleurum chinense was rapidly screened by PCR matrix method. Results The full length cDNA of IPPB (GenBank No. gq433719) was obtained. The nucleotide sequence of it was 1117bp and encoded a 319 amino acid protein. The results of NCBI Blastx showed the highest similarity to the IPPI amino acid sequence of carrot Daucus carotasubsp. Sativus (abb52064), with a consistency of 92% and a similarity of 97%. Conserved domain searches revealed the catalytic site, the metal binding site and the Nudix motif shared by IPPI. Analysis of TargetP1.1 and SignalP3.0 showed that the NIPP of N. canadensis contained a 26 bp long chloroplast signal peptide. Conclusions The full length cDNA of IPPB from N. bidentis was cloned for the first time, which will promote the subsequent IPPi gene expression and its function in the anabolic process of saikosaponin.