mRNA差异显示法筛选黄花石蒜中加兰他敏合成相关基因

Screening of the gene related to galantamine biosynthesis of Lycoris aurea Herb. by mRNA differential display

目的筛选和克隆黄花石蒜加兰他敏相关基因。方法采用mRNA差异显示法(mRNA differential displayPCR,DD-PCR),对7月份黄花石蒜产加兰他敏含量最高的部位花蕊和含量最低的部位花葶进行基因表达差异的研究,筛选其差异片段,再对差异片段进行回收、克隆、测序及序列分析和同源性比较。结果黄花石蒜花蕊和花葶中存在明显的基因表达差异,发现差异条带44条,经序列分析和同源性比较,确认其中2个条带与黄花石蒜加兰他敏合成相关,另一条可能与加兰他敏运输代谢调控有关。结论通过DD-PCR得到的3个同源序列为研究黄花石蒜中加兰他敏的合成途径提供了有力依据。

Objective To isolate and clone the gene related to galantamine biosynthesis of Lycoris aurea Herb. Methods The specially expression cDNA fragments of the gene related to galantamine biosynthesis were screened by differential display polymerase chain reaction（DD-PCR） after extract- ing the total RNA from the pistils tissues and the scape tissues of the Lycoris aurea Herb cropped in July which had differential contents of the galantamine. Then these fragments were separately cloned to plasmid PMD19-T and subsequently underwent sequence analysis and homogenous comparison, Results 44 differential display bands were found in gene expression between the pistils tissues and the scape tissues of the Lycoris aurea Herb. According to the se- quenee analysis and homologenous comparison, two of them were confirmed to related with galantamine biosynthesis, one of them was possible related with galantamine transport regula tion. Conclusion The three homologous sequences of Lycoris aurea screened by DD-PCR provide strong basis of galantamine synthetic pathways.