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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-LiC1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。
Objective To clone the gene of geranylgeranyl diphosphate synthase (GGPS) in andrographolide synthesis pathway from Andrographis paniculata (Andrographis paniculata). Methods CTAB-LiC1 method was used to extract total RNA from the stem and leaves of Andrographis paniculata. The conserved regions were amplified by degenerate primers. The full-length cDNA was obtained by RACE and analyzed by ProtParam software. The real- And leaf gene expression. The results showed that a GGPS gene of 1 047 bp encoded a 348 amino acid protein sequence with a close genetic relationship with the GGPS protein of Catharanthus roseus and a 47 amino acid plastid signal peptide at the N terminus. In the stems and leaves of Andrographis paniculata, the expression level of GGPS gene was higher at budding stage and decreased at first flowering stage. The expression of GGPS increased at the mixed stage of flower and fruit, and decreased at green fruit stage. The results indicated that the synthesis of the metabolic components related to the mixed bud of Bud and Lotus could be more active. Conclusion GGPS gene was cloned from Andrographis paniculata, which laid the foundation for the regulation of andrographolide synthesis pathway.