甘草CHS基因多态性与甘草苷含量的相关性分析

Analysis on the correlation between chalcone synthase gene polymorphism and content of liquiritin in licorice

2015版《中华人民共和国药典》明确规定,甘草药材中甘草苷含量不得低于0.5%,然而目前市售栽培甘草大量存在甘草苷含量不达标的现象。由于功能基因多态性对于次生代谢产物的积累至关重要,因此本文试图对甘草苷生物合成途径中的关键酶——查尔酮合酶(chalcone synthase,CHS)进行基因多态性分析,筛选出甘草苷高/低含量样品对应的特异基因型,为进一步解析甘草苷生物合成的分子机制提供参考。本文利用HPLC法测得60株甘草样品中4种主要黄酮类化合物(甘草苷、异甘草苷、甘草素、异甘草素)的含量,应用Spearman相关性分析及χ2检验进行统计学分析,发现4种黄酮类化合物含量间具有相关关系且不同基原间含量差异显著,乌拉尔甘草>光果甘草>胀果甘草。因此筛选出含量最高的5株乌拉尔甘草及含量最低的5株胀果甘草分为黄酮高、低含量组,对其CHS基因进行克隆及序列分析。10株样品共获得了336条长度为1 175 bp的CHS c DNA序列,存在249个变异位点,其中错义突变位点141个,可分为137种单倍型;编码389个氨基酸残基,存在130个变异位点,可分为102种氨基酸序列类型;主流CHS氨基酸序列类型为AA-3,黄酮高含量组甘草样品特有的主流氨基酸序列类型为AA-35,黄酮低含量组甘草样品特有主流氨基酸序列类型为AA-36,AA-35与AA-36在193位与229位处存在I/V、V/T突变。采用Discovery Studio 2.5对蛋白三维结构进行分析,结果表明AA-35的229位的缬氨酸为底物丙二酰辅酶A的结合位点。MEGA 5.0同源性分析表明甘草CHS亲缘关系良好,与其他物种区分度良好。本文筛选获得了黄酮高/低含量组甘草样品的特异CHS基因型,对于指导优质甘草的分子育种具有重要意义。

Chinese pharmacopoeia stipulates that the content of liquiritin in licorice slices should be no less than 0.5%. However, there are lots of unqualified licorice slices in the herbal medicine markets. Due to the important role of functional gene polymorphism in secondary metabolism, this study attempts to analyze the influence of chalcone synthase （CHS） gene polymorphism on liquiritin biosynthesis and find out the unique haplotypes in licorice samples with high or low content of liquiritin, and to provide a basis for further analysis of molecular mechanism in flavonoid biosynthetic pathway. The contents of the 4 main flavonoids （liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin） in 60 licorice samples were assayed by HPLC and the results were analyzed by Spearman and χ^2 tests. The contents of the 4 main flavonoids were related to each other and obviously different in different original plants. They were highest in Glycyrrhiza uralensis samples and lowest in Glycyrriza inflate samples. Five G. uralensis samples with the highest liquiritin contents and five G. inflate samples with the lowest liquiritin contents were selected to clone the CHS cDNA sequences. 336 CHS cDNA sequences with a full length of 1 175 bp were obtained, 249 variable sites （141 missense mutation sites） were found, and 137 haplotypes were determined. 130 variable sites were found in the 336 CHS amino acid sequences and 102 types were determined. AA-3 is the major type of CHS in licorice, AA-35 is the special major type of CHS in the group with high flavonoids contents and AA-36 is the special major type of CHS in the group with low flavonoids contents. The mutation sites between AA-35 and AA-36 are I/V at 193 and V/T at 229. Discovery Studio 2.5 analysis of the three-dimensional structure of the CHS protein shows that the valine at site 229 of AA-35 is combined with malonyl-CoA. Homology analysis indicates that the homology of CHS among different species is low. This study is significant for identification of the unique haplotypes in licorices with high or low content of liquiritin and guiding the further molecular breeding of high-quantity licorice.