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目的研究球药隔重楼次生代谢产物甾体皂苷甲羟戊酸合成途径的一个关键酶即3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的基因,有助于对活性物质的合成进行调控,从而提高产量。方法比较NCBI上公布的11种植物11条HMGR基因mRNA的同源保守区域,设计简并引物,利用RT-PCR技术,以球药隔重楼新鲜茎总RNA反转录的cDNA为模板成功扩增出404 bp的保守区基因片段。结果序列分析表明,球药隔重楼HMGR基因片段与其他7种植物的一致性达到76%～81%,球药隔重楼HMGR氨基酸片段与其他植物有较高的同源性,经Genbank查询为一新的cDNA。结论通过蛋白质特征区、保守区以及进化树分析,初步确定该基因为HMGR基因,这是首次从药用植物球药隔重楼中克隆得到甲羟戊酸途径关键酶HMGR的基因片段,也是百合科植物首次获得的HMGR基因。
Objective To study the gene of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme involved in the biosynthesis of secondary metabolites of steroidal saponins mevalonate, The synthesis of active substances is regulated so as to increase the yield. Methods The homologous conserved regions of 11 HMGR mRNAs published on NCBI were compared. The degenerate primers were designed. RT-PCR was used to amplify the reverse transcribed cDNA of fresh stem total RNA of S. aureus. Add a 404 bp conserved region gene fragment. Results The sequence analysis showed that the homology of the HMGR gene fragment with other seven plant species was 76% -81%, and the amino acid sequence of the HMGR amino acid fragment of the spacer was high homologous with other plants. After Genbank query A new cDNA. Conclusion The HMGR gene was preliminarily identified by analyzing the characteristic regions of proteins, conserved regions and phylogenetic tree. This is the first time that a gene fragment of HMGR, a key enzyme of mevalonate pathway, was cloned from the spacer of medicinal plants, The first access to plants HMGR gene.