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。

Paris fargesii is an important medicinal plant and its secondary metabolites, steroidal saponins, synthesized by the mevalonate pathway are the active ingredients. The study on the gene of the key enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductases (HMGR) in the mevalonate pathway is helpful for the regulation of steroidal saponins syntheses. Methods In the basis of degenerate primers designed according to mRNA homologous conserved regions of 11 HMGRs from 11 plant species published on NCBI and total RNA extracted from the seedling of P. fargesii, a 404 bp fragment of HMGR was amplified by reverse transcription PCR technology, which is a new cDNA as searching in the Genbank. Results Sequence analysis showed that the obtained fragment had 76%-81% identification compared with other seven plant species. The deduced amino acid seqence was predicted to have a wider and higher homology with other plants. Conclusion Protein characteristic zones, conserved regions, and phylogenetic analysis by Prosite and ClustalX could give preliminary evidence for the existence of HMGR in P. fargesi, which is the report of HMGR gene isolated from the plants in Liliaceae for the first time.

创新研究群体科学基金项目(2005DKA21404)