基于EST-SSR的金银花分子鉴别方法研究

利用简单有效的方法鉴定药用植物种及其变种一直是中药材鉴定首先要解决的问题。金银花是临床常用大宗中药材之一,源自忍冬科植物忍冬(Lonicera japonica Thunb.)的花蕾。为了有效利用金银花EST序列开发功能性分子标记,本文通过生物信息学手段对本实验室保存的忍冬及其变种红白忍冬(L.japonica var.chinensis Thunb.)EST序列进行分析,共获得3 705条忍冬EST-SSRs,且平均每4.05 kb检出一个SSR(simple sequence repeat);2 818条红白忍冬EST-SSRs,平均每7.49 kb检出一个SSR。分析结果表明金银花及其变种红白忍冬主要SSR重复为二核苷酸,其次为三核苷酸,主要重复基序类型为AG/TC和GAG/TCT。通过同源比对,在忍冬及其变种中共筛选出87对重复次数具有差异的EST-SSR。选出15对碱基数差异大于6的EST-SSR进行验证,结果表明其中的13对引物在52份金银花类药用植物中具有有效扩增产物,且引物jp.ssr4、jp.ssr64及jp.ssr65可用于忍冬及其变种、山银花原植物的鉴别。

EST-SSR identification of Lonicera japonica Thunb

Simple and effective methods are needed for the identification of Chinese medicinal material species and their variety. Lonicera japonica Thunb. is one of Chinese herbal medicines widely demanded. A total of 3 705 EST-SSRs of L. japonica and 2 818 EST-SSRs of L. japonica var. chinensis Thunb. were identified from EST database in our lab. In average, there was one EST-SSR per 4.05 kb in L. japonica ESTs and per 7.49 kb in L. japonica var. chinensis ESTs, separately. The identified SSRs in L. japonica were consisted of 51.98% dinucleotide and 34.61% trinucleotide repeats, while SSRs in L. japonica var. chinensis had 57.45% dinucleotide and 30.09% trinucleotide. The results reviewed that the classes AG/TC and GAG/TCT were predominant in the dinucleotide motifs and the trinucleotide motifs, respectively. Total 87 EST-SSRs were identified of significant difference between L. japonica and L. japonica var. chinensis. PCR products were obtained from 52 L. japonica samples in 13 out of 15 SSR markers tested. The polymorphism in L. japonica, L. japonica var. chinensis and other honeysuckles could be distinguished by three markers (jp.ssr4, jp.ssr64 and jp.ssr65) tested.