不同地理居群破布叶的psbA-trnH和ITS2序列及其聚类分析  

Cluster analysis on psb A-trnH and ITS2 sequences of Microcos paniculata from different geographical populations

作　　者：林爽[1] 吴海燕[2] 张宏意[1] 李坤平[2] 

机构地区：[1]广东药科大学中药学院,广东广州510006 [2]广东药科大学药学院,广东广州510006

出　　处：《中草药》2017年第7期1403-1408,共6页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(31300273);广东省科技计划项目(2015A030302082)

摘　　要：目的探讨破布叶Microcos paniculata不同地理居群间的DNA序列变异与地理分布的关系,为其种质资源评价和基原植物的分子鉴定提供参考。方法对破布叶14个居群14份个体样品以改良的3×CTAB法提取基因组DNA,选择特异的引物扩增ITS2和psb A-trnH序列;PCR扩增产物直接双向测序分析。DNAMAN 8.0软件处理测序数据,MEGA6.0软件计算K2P遗传距离,运用NJ法构建系统聚类树。结果破布叶14个居群的ITS2序列均为462 bp,共检测到14个变异位点,居群间遗传距离0.000 0~0.019 8。除云南景洪居群样本的psb A-trnH序列存在8 bp缺失外,其他13个居群样本的psb A-trnH序列均长387 bp,无变异,居群间遗传距离为0.000 0。结论不同地理居群破布叶的psb A-trnH较ITS2更为保守,二者PCR扩增引物特异性好,扩增效率和测序成功率高,对其药材及基原植物的分子鉴定可采用psb A-trnH为主,ITS2序列为辅的DNA条形码技术。Objective To investigate the relationship between variations of chloroplast DNA psbA-tmH and nuclear ribosomal DNA ITS2 sequences and the geographical origins of Microcos paniculata, and seek for some evidence for its germplasm resources evaluation and molecular identification. Methods Total genomic DNA was extracted from M. paniculata and the psbA-tmH and ITS2 sequences were amplified by PCR and directly sequenced. The raw data were dealt with DNAMAN 8.0 and the pairwise distance was calculated based on Kimura 2-parameter model using MEGA 6.0. The phylogenic tree was constructed by Neighbor-Joining method. Results The ITS2 sequences of M. paniculata from different populations were 462 bp with 14 variable sites. The nucleotide divergence between ITS2 sequences in pairwise comparison was calculated and the result showed that the distances between M. paniculata populations is about 0.000 0 -- 0.019 8. The sequences length of psbA-tmH was 387 bp, except for one isolated from Jinghong, Yunnan province which was 379 bp. Conclusion The psbA-trnH sequences ofM. paniculata are more conservative than the ITS2 sequences, which can effectively identify M. paniculata from its adulterants and the closely relate species. The ITS2 sequence is so sensitive with the geographical variation that it can show the different characteristics of different populations in molecular level.

关 键 词：破布叶 PSB A-trnH ITS2 DNA条形码 分子鉴定 

分 类 号：R282.12[医药卫生—中药学]