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作 者 ：苟君波, 李长福, 陈方方, 李兆波, 黎佳

期 刊 ：植物科学学报 2013年 31卷 4期 页码：398-405

关键词：野葛；异黄酮合酶；甘草素；大豆苷元；

Keywords:Pueraria lobata, Isoflavone synthase, Liquiritigenin, Daidzein,

摘 要 ：异黄酮是野葛(Pueraria lobata)中的主要活性成分,而异黄酮合酶(IFS)是催化异黄酮生物合成的第一步关键酶,尽管野葛的IFS基因已被分离,但其功能还未得到任何验证。本研究以中国安徽省郎溪县的野葛为材料,利用RT-PCR技术成功克隆到野葛IFS基因,命名为PlIFS,PlIFS开放阅读框大小为1566 bp,编码521个氨基酸,将该基因克隆到GAL1启动子控制下的酵母表达载体pESC-TRP上,得到重组质粒pESC-TRP-PlIFS,通过LiAc/ssDNA/PEG方法将其转化进酿酒酵母(Saccharomyces cerevisiae)WAT11中进行异源表达,并在酵母体内对其活性进行验证,结果显示PlIFS能催化甘草素生成大豆苷元,表现出异黄酮合酶活性特征。荧光定量PCR分析显示,PlIFS基因主要在野葛的根中表达,这与活性物质异黄酮主要在野葛根中的积累模式一致。

Abstract:Isoflavones are the main active component of kudzu(Pueraria lobata).The first committed step in the production of isoflavones is performed by a cytochrome P450 enzyme,IFS.While IFS genes have been isolated from kudzu,no biological functions have been identified to date.We cloned an IFS gene via RT-PCR,named PlIFS,using kudzu collected from Langxi(Anhui,China)as plant material.The PlIFS contained an open reading frame(ORF)encoding 521 amino acid residues.The amplified PlIFS gene was cloned into pESC-TRP vector,with yeast GAL1 divergent promoters as the control.The constructed recombinant plasmid,named pESC-TRP-PlIFS,was transformed into Saccharomyces cerevisiae strain WAT11 according to the LiAc/ssDNA/PEG method.The PlIFS expressed in yeast was able to catalyze daidzein formation from liquiritigenin,indicating that it had IFS activity.Gene expression pattern analysis revealed that PlIFS was expressed in roots at the highest level,which was consistent with isoflavones accumulation in kudzu plantlets.

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