基于ITS 2序列的15种淫羊藿属药用植物分类鉴定研究
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摘要
为探讨内转录间隔区2(ITS 2)序列及其二级结构在15种淫羊藿属(Epimedium)中的鉴定效率。利用试剂盒提取叶片总DNA,对ITS 2序列进行PCR扩展与测序,计算K 2 P遗传距离,构建系统发育树,预测其二级结构。结果表明,ITS 2序列在15种淫羊藿属植物中的通用性好,扩增成功率与测序成功率均为100%。从K2P种间和种内遗传距离看,ITS 2在淫羊藿属中的种间变异较小,部分种类种内遗传距离大于种间遗传距离,鉴定成功率约为60%。采用比对法(Blast)分析,ITS 2鉴定效率为75%。由ITS 2系统进化树可知,淫羊藿属与鬼臼属各为一支,易于分开。除薄叶淫羊藿、竹山淫羊藿、巫山淫羊藿外,其余种类均能较好的进行聚类;除粗毛淫羊藿、保靖淫羊藿外其余均不能与NCBI下载到的序列进行较好聚类。研究表明,15种淫羊藿属植物ITS 2序列在ML系统进化树中具较好的鉴别能力,但淫羊藿属各样本种内与种间遗传较为混乱,遗传距离复杂,ITS 2二级结构形态结构亦较为相似,将各种间进行区分存在一定难度,不适合单独作为该属条形码使用。
In order to explore the efficiency of identification for the appraisal of Epimedium genera of medicinal plants in the internal transcribed spacer(ITS 2) 2 sequence and secondary structure in 15 Epimedium species.The Extraction Kit was used to extract the total DNA,PCR expansion and sequencing of ITS 2 s,calculate K 2 P distance,building phylogenetic trees,predict its secondary structure.The results showed that ITS 2 had good generality in 15 species of Epimedium,the success rate of amplification and sequencing were 100%.From K2P interspecific and intraspecific genetic distance show that,the interspecific variation of ITS 2 in Epimedium is small,some species in genetic distance was greater than the interspecific genetic distance,appraisal success rate was about 60%.By using comparison method(Blast) analysis,ITS 2 appraisal efficiency was 75%.According to the evolution tree of ITS 2 system,Epimedium genera and ghost mortar respectively,easy to divide.All the other species could cluster well except the E. membranaceum, E. zhushanense and E. wushanense. Except for E. acuminatum and E. baojingense,all the others could not cluster well with the sequences downloaded from NCBI.The research showed that the ITS 2 s of 15 species of Epimedium had good identification ability in the ML phylogenetic tree,but the intra-and inter-species inheritance of each sample of Epimedium was relatively chaotic,the genetic distance was complex,and the morphology structure of ITS 2 secondary structure was also relatively similar.Therefore,it was difficult to distinguish all kinds of ITS 2,which was not suitable for the DNAbarcoding of Epimedium alone.
In order to explore the efficiency of identification for the appraisal of Epimedium genera of medicinal plants in the internal transcribed spacer(ITS 2) 2 sequence and secondary structure in 15 Epimedium species.The Extraction Kit was used to extract the total DNA,PCR expansion and sequencing of ITS 2 s,calculate K 2 P distance,building phylogenetic trees,predict its secondary structure.The results showed that ITS 2 had good generality in 15 species of Epimedium,the success rate of amplification and sequencing were 100%.From K2P interspecific and intraspecific genetic distance show that,the interspecific variation of ITS 2 in Epimedium is small,some species in genetic distance was greater than the interspecific genetic distance,appraisal success rate was about 60%.By using comparison method(Blast) analysis,ITS 2 appraisal efficiency was 75%.According to the evolution tree of ITS 2 system,Epimedium genera and ghost mortar respectively,easy to divide.All the other species could cluster well except the E. membranaceum, E. zhushanense and E. wushanense. Except for E. acuminatum and E. baojingense,all the others could not cluster well with the sequences downloaded from NCBI.The research showed that the ITS 2 s of 15 species of Epimedium had good identification ability in the ML phylogenetic tree,but the intra-and inter-species inheritance of each sample of Epimedium was relatively chaotic,the genetic distance was complex,and the morphology structure of ITS 2 secondary structure was also relatively similar.Therefore,it was difficult to distinguish all kinds of ITS 2,which was not suitable for the DNAbarcoding of Epimedium alone.
引文
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[2]国家药典委员会.中国药典2015版一部[S].北京:中国医药科技出版社,2015:327-328.
[3]何顺志.中国淫羊藿属植物彩色图鉴[M].贵阳:贵州科技出版社,2014.06.
[4]柴士伟,刘芳.淫羊藿化学成分分离鉴定[J].天津中医药,2016(2):114-117.
[5]孟宁,孔凯,李师翁.淫羊藿属植物化学成分及药理活性研究进展[J].西北植物学报,2010,30(5):1063-1073.
[6]严福林,何顺志,徐文芬,等.中国淫羊藿属药用植物引种后性状变异及分类[J].贵州农业科学,2015,43(9):153-156.
[7]郭宝林.淫羊藿属的分子系统学研究[D].北京:中国医学科学院药用植物研究所,1999.
[8]王悦云,孙庆文,杨雯,等.中国淫羊藿属药用植物花粉特征及其分类学意义[J].西北植物学报,2015,35(7):1367-1377.
[9]王霖娇,田静,盛茂银.10种淫羊藿属植物核型分析及其分类学的意义[J].四川农业大学学报,2017,35(03):389-395.
[10]盛茂银.中国西南地区淫羊藿属植物遗传与进化研究[D].南京大学,2011.
[11]蒋昱.四川淫羊藿属植物DNA条形码筛选与鉴定研究[D].四川农业大学,2012.
[12]Zhang Y J,Du L,Ao L,et al.The Complete Chloroplast Genome Sequences of Five Epimedium Species:Lights into Phylogenetic and Taxonomic Analyses[J].Frontiers in Plant Science,2016(7):306.
[13]Yanjun Zhang,Lulu Yang,Jianjun Chen,et al.Taxonomic and phylogenetic analysis of Epimedium L.based on amplified fragment length polymorphisms[J].Scientia Horticulturae,2014(2):25.
[14]Yousaf Z,Hu W,Zhang Y,et al.Systematic validation of medicinally important genus Epimedium species based on microsatel lite markers[J].Pakistan Journal of Botany,2015,44(2):477-484.
[15]任保青,陈之端.植物DNA条形码技术[J].植物学报,2010(01):1-12.
[16]陈士林,庞晓慧,姚辉,等.中药DNA条形码鉴定体系及研究方向[J].世界科学技术(中医药现代化),2011(05):747-754.
[17]陈士林,宋经元,姚辉,等.药用植物DNA条形码鉴定策略及关键技术分析[J].中国天然药物,2009(05):322-327.