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 encoding isopentenyl diphosphate isomerase gene(IPPI) associated with saikosaponin biosynthesis pathway in Bupleurum chinense and to provide the basis for further studies on biosynthesis and regulation of saikosaponin.Methods Matrix-based PCR method was used to rapidly screen a B.chinense full-length cDNA library.Results The full-length cDNA of B.chinense IPPI was obtained(GenBank No. gq433719).The length of nucleotide sequence was 1 117 bp and an ORF with 319 amino acids was deduced.Blastx search showed it had the highest similarity to the corresponding protein from Daucus carota subsp.sativus(abb52064) with 92% identity and 97% similarity.Common conserved domains of IPPI were found in B.chinense IPPI including active site,metal binding site,and Nudix motif.A 26 bp long chloroplast signal peptide on the N terminal of the deduced amino acid sequence was predicted by Target P 1.1 and Signal P 3.0.Conclusion The full-length cDNA of B.chinense IPPI is cloned and reported here for the first time.This work will promote the studies on the gene expression pattern and regulatory functions of B.chinense IPPI in saikosaponin biosynthesis.

国家科技支撑计划重点项目(2006BAI09B01);中央级院所长科研基金项目(YZ-1-10)