X射线辐照处理甘草样品中CHS基因多态性及其功能解析

Analysis of CHS gene polymorphisms and function in X-ray irradiated Glycyrrhiza uralensis

查尔酮合酶(chalcone synthase,CHS)是催化甘草黄酮类化合物生物合成的第一个限速酶,具有重要调控作用。本文以X射线辐照处理的黄酮高含量甘草以及未经辐照处理的黄酮低含量甘草为实验材料,分析黄酮高/低含量组甘草的CHS基因多态性,筛选其主流单倍型,并解析其功能。6株样品共克隆得到109条长度均为1170 bp的CHS cDNA序列,存在220个变异位点(错义突变位点116个),可分为85种单倍型,编码65种氨基酸序列类型,存在96个变异位点。黄酮高含量组主流氨基酸序列类型为aa-20和aa-45,低含量组主流氨基酸序列类型为aa-11,结合靶点预测结果显示aa-20的383位点和aa-45的229位点位于结合位点区域,与底物结合相关,而aa-11的229位点和383位点均与底物结合无关,因此推测CHS的229位点和383位点对其功能具有重要影响。本文对丰富的CHS基因型进行序列分析,找到了影响其活性的重要位点,为解析其功能奠定了基础,也为进一步阐述甘草黄酮类化合物生物合成的分子调控机制提供了思路。

Chalcone synthase(CHS)is the rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis.It plays an important role in the regulation and control of flavonoids biosynthesis.In this study,X-ray irradiated G.uralensis samples with high or low content of flavonoids were studied.The CHS gene polymorphism in these samples were analyzed,the specific haplotypes were identified,and CHS function was parsed.109 CHS cDNA sequences with a length of 1170 bp were cloned,220 variable sites(116 missense mutation sites)were found and 85 haplotypes were identified,which encoded 65 amino acid sequences with 96 variable sites.aa-20 and aa-45 were the most common amino acid sequences in samples with high flavonoid content,while aa-11 was the most sequence in samples with low flavonoid content.Molecular docking results showed that the mutation sites at 383 in aa-20 and 229 in aa-45 were related to substrate binding,while the mutation sites at 383 and229 in aa-11 were not involved.Therefore,we speculate that the two mutation sites have significant influence on the function of CHS.We analyzed a large number of CHS cDNA sequences and identified the important functional sites,which will provide a basis for further functional studies.This paper will provide ideas for further research of the molecular regulation of the flavonoid biosynthetic pathway in G.uralensis.