杜仲MVA途径相关基因的鉴定及荧光定量PCR引物筛选

Related gene identification of MVA pathway and primers screening of real-time PCR in Eucommia ulmoides

杜仲富含萜类物质,MVA途径是萜类物质合成的重要途径之一。从杜仲果实转录组数据KEGG中杜仲MVA途径共被注释30条Unigene,根据NCBI中的BLAST比较,分别鉴定了乙酰COA酰基转移酶,羟甲基戊二酰辅酶a合成酶,羟甲基戊二酰辅酶a还原酶,甲羟戊酸激酶,磷酸甲羟戊酸激酶,二磷酸甲羟戊酸脱羧酶基因,被注释7条,3条,11条,2条,2条,5条。表达差异分析表明,以上基因中分别为5条,3条,4条,1条,5条为幼果和成熟果实差异表达基因。对差异表达基因序列进行软件设计、引物特异性分析和实验验证,最终筛选出10对适合杜仲MVA途径SYBR GreenI荧光定量PCR的引物,为MVA途径基因表达差异研究及萜类物质积累的分子机理研究提供重要依据。

Eucommia ulmoides Oliv. is a famous Chinese endemic medicinal tree being rich of terpenoids, and MVA pathway is one important way of terpenoids synthesis. From KEGG, the transcriptome data of eucommia fruit, the MVA pathway has been annotated 30 Unigene; according to BLAST comparison in NCBI, acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase were identified and respectively annotated 7, 3, 11, 2, 2, 5 genes. The differences analysis of gene expression showed that of the six enzymes＇ annotated genes, there respectively were 5, 3, 4, 1, 5 genes which were the differentially expressed genes of the young fruit and mature fruit. We conducted The software design for the sequences of differentially expressed genes, analysis for specific primers and experiment validation were conducted, and ultimately 10 suitable primers for SYBR GreenI real-time PCR in MVA pathway of E. ulmoides were selected. The results provide an important basis for researching the gene expression differences with MVA pathway and the molecular mechanism of terpenoid accumulation.