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【目的】筛选和克隆何首乌中二苯乙烯苷代谢相关酶基因。【方法】采用mRNA差异显示技术,对何首乌中二苯乙烯苷含量差异悬殊的不同组织(根、茎、叶)进行差异表达基因的筛选,将得到的差异基因连接pMD19-T载体进行测序后,通过数据库比对预测其基因功能。【结果】发现差异片段51条,选取其中9条进行半定量PCR验证,获得3条阳性差异片段。其中1条阳性片段为何首乌茎和叶特异表达,与甘氨酸脱氢酶高度同源,另外2条阳性片段为何首乌块根特异表达,分别与烯酰辅酶A水合酶、氨基肽酶N高度同源。【结论】通过mRNA差异显示技术得到的3个同源序列可为进一步研究何首乌中二苯乙烯苷生物合成途径提供帮助。
【Objective】 The aim of this study was to screen and clone the enzyme gene related to stilbene glycoside metabolism in fleece-flower root. 【Method】 The differential expression genes were screened in different tissues (roots, stems and leaves) with different stilbene glycoside contents in Polygonum multiflorum Thunb.ensis by mRNA differential display technique. After the differentially expressed genes were ligated to pMD19-T vector and sequenced, Predict the gene function through database comparison. 【Result】 Fifty-one differential fragments were found and nine of them were selected for semi-quantitative PCR validation. Three positive differential fragments were obtained. One of the positive fragments of Polygonum multiflorum stem and leaf specifically expressed highly homologous with glycine dehydrogenase, the other two positive fragments of Radix Polygonum specific expression, respectively, and enoyl-CoA hydratase, aminopeptidase N highly homologous. 【Conclusion】 The three homologous sequences obtained by mRNA differential display technique can be used to further study the biosynthesis pathway of stilbene in Polygonum multiflorum.