应用于多个沉香属物种鉴定的DNA条形码序列筛选  

Screening of DNA Barcoding Sequences for Identification of Multiple Species of Aquilaria L.

作　　者：康勇 刘杨 杨云 冯剑 郑希龙 魏建和 KANG Yong;LIU Yang;YANG Yun;FENG Jian;ZHENG Xi-long;WEI Jian-he(Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine&Key Laboratory of State Administration of Traditional Chinese Medicine for Agarwood Sustainable Utilization,Hainan Branch of Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Haikou 570311,China;Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine,Ministry of Education&National Engineering Laboratory for Breeding of Endangered Medicinal Materials,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China)

机构地区：[1]中国医学科学院北京协和医院药用植物研究所海南分所,海南省南药资源保护与开发重点实验室,国家中医药管理局沉香可持续利用重点研究室,海口570311 [2]中国医学科学院北京协和医院药用植物研究所,中草药物质基础与资源利用教育部重点实验室,濒危药材繁育国家工程实验室,北京100193

出　　处：《中国药学杂志》2019年第23期1926-1932,共7页Chinese Pharmaceutical Journal

基　　金：国家重点研发计划项目资助(2018YFC1706400);中组部“万人计划”项目资助(99950534);国家自然科学基金项目资助(81703660);国家中药材产业技术体系资助(CARS-21);海南省自然科学基金项目资助(319QN343);中国医学科学院医学与健康科技创新工程资助(2016-I2M-2-003)

摘　　要：目的本实验旨在从常用DNA条形码序列中筛选出有效的序列,从而实现沉香属内常见物种的快速鉴别。方法本实验以8个不同国家5种常见的沉香属物种为材料,对其核基因ITS2,叶绿体基因mat K、rbc L和trn H-psbA的序列特征进行评估,并通过构建系统进化树分析不同序列及组合对沉香属物种的鉴别效率。结果4种序列均具有较优的PCR扩增成功率及测序成功率,其中mat K序列变异位点和SNPs最多,trn H-psbA最少,Barcoding gap检验结果表明,鉴定效果最优序列依次是mat K>ITS2>rbc L>trn H-psbA,mat K片段具有明显的遗传变异间隔区域;通过计算平均节点支持率及同质性检验,mat K+ITS2+rbc L组合构建进化树可将多个沉香属物种分别聚为一支,且具有最高的节点支持率;基于Taxon DNA的BBA方法验证,mat K序列与其他序列组合的鉴定效果最好。结论使用DNA条形码技术鉴定沉香属物种,单一片段中鉴别成功率最高的为mat K序列,应用mat K+ITS2+rbc L的序列组合可以实现沉香属不同物种的准确鉴别。OBJECTIVE To screen out efficient sequences from DNA barcoding sequences in order to rapidly identify multiple species of Aquilaria.METHODS Five species of Aquilaria were collected from eight countries as test materials.The ITS2 sequence in nuclear genome as well as matK,rbcL and trnH-psbA sequences from chloroplast genome were analyzed and evaluated.Phylogenetic trees based on different sequence combinations were built to evaluate the identification efficiency of Aquilaria species.RESULTS All four kinds of sequences exhibited relatively high success rate of PCR amplification and sequencing.matK had the most variation sites and SNPs while trnH-psbA had the fewest.Barcoding gap test demonstrated that the identification effect of sequences from high to low was matK>ITS2>rbcL>trnH-psbA.According to the homogeneity test and average node supporting rate,phylogenetic trees based on the combination of matK+ITS2+rbcL could clearly divide different species into separated clusters with high node supporting rate.The matK showed the best identification effect when combined with other sequences as shown by the BBA method of Taxon DNA.CONCLUSION When using DNA barcoding technique to identify Aquilaria species,matK has the highest identification rate among single barcoding candidate sequences.And the combination of matK+ITS2+rbcL can precisely distinguish multiple Aquilaria species.

关 键 词：DNA条形码 沉香属 进化树 物种鉴别 

分 类 号：R284[医药卫生—中药学]