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[摘要]
目的 从穿心莲中克隆穿心莲内酯合成途径中的香叶基香叶基焦磷酸合成酶(geranylgeranyl diphosphate synthase,GGPS)基因,并进行组织表达等特征研究。方法 采用CTAB-LiCl法从穿心莲茎和叶中提取总RNA,简并引物扩增获得保守区段,RACE法获得基因全长,ProtParam等软件分析基因特征,实时荧光PCR法检测不同发育时期穿心莲茎和叶中基因的表达。结果 获得了一个长1 047 bp的GGPS基因,编码一条由348个氨基酸组成的蛋白质序列,与长春花的GGPS蛋白亲缘关系较近,N端含有一个由47个氨基酸构成的质体信号肽。在穿心莲的茎和叶中,GGPS基因在花蕾期表达量较高,初花期降低;到了花果混合期表达量升高,青果期下降。表明穿心莲花蕾期和花果混合期相关代谢成分的合成可能更活跃。结论 从穿心莲中克隆了GGPS基因,为开展穿心莲内酯合成途径的调控奠定了基础。
[Key word]
[Abstract]
Objective Andrographolide is the main bioactive substance in Andrographis paniculata, and popularly used as active pharmaceutical ingredient (API). We have cloned the gene encoding geranylgeranyl diphosphate synthase from A. paniculata and characterized its tissue expression pattern. Methods Total RNA was extracted with CTAB-LiCl extraction method; Conserved fragment was amplified and cloned with degenerated primers, and a full length ORF encoding geranylgeranyl diphosphate synthase was obtained with RACE method and analyzed by bioinformatic softwares, e.g. ProtParam. Tissue expression pattern was predicted with real time PCR. Results We have cloned a 1 047 bp GGPS gene encoding a sequence with 348 amino acids. This amino acid sequence contained a plastid targeted N-terminal signal peptide and has high similarities with the GGPS protein from Catharanthus roseus. The GGPS gene has expressed in a dynamic state in stems and leaves of A. paniculata. The expression reached a high level at bud stage, then decreased at early flowering stage, increased at flowering and early seed setting stage again, and finally decreased at seed setting stage. Considering the above expression characteristics, biosynthesis of metabolites regulated by GGPS was deduced more active at bud stage and flowering and early seed setting stage. Conclusion GGPS is a key enzyme in biosynthesis of andrographolide. We have cloned the GGPS gene from A. paniculata, and provide a sharp tool in genetic engineering of andrographolide biosynthsis pathway.
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[基金项目]
基于遗传与环境的穿心莲品质保障技术示范研究(2012BAI29B02)