基于COⅠ基因对东北林蛙、中国林蛙等蛙属24种的鉴定  

Identification of 24 Rana Species Including Rana dybowskii and Rana chensinensis Based on COⅠ Sequences

作　　者：王孟虎 孙一帆 许亮[2] 康廷国[2] 张腾腾 左亚锋 朱丽婷 孟祥松 汤建 徐倩[4] WANG Meng-hu;SUN Yi-fan;XU Liang;KANG Ting-guo;ZHANG Teng-teng;ZUO Ya-feng;ZHU Li-ting;MENG Xiang-song;TANG Jian;XU Qian(Bozhou University,Bozhou 236800,China;Liaoning University of Traditional Chinese Medicine,Dalian 116600,China;Bozhou Vocational and Technical College,Bozhou 236800,China;The Second Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230061,China)

机构地区：[1]亳州学院,安徽亳州236800 [2]辽宁中医药大学,辽宁大连116600 [3]亳州职业技术学院,安徽亳州236800 [4]安徽中医药大学第二附属医院,合肥230061

出　　处：《中国实验方剂学杂志》2021年第16期150-158,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中央本级重大增减支项目(2060302);中药原料产品研发安徽普通高校重点实验室(亳州学院)建设项目(KLAHEI18032);中药学教学团队(亳州学院)(2019jxtd131);亳州市科技重大专项(BKJZD201801);亳州市第二批“首席专家工作室”(亳组2020[2]号);安徽省高等学校省级质量工程项目(2016sxzx027,2016sjjd054);亳州学院校级项目(BYZ2017C02)。

摘　　要：目的:利用DNA条形码线粒体细胞色素C氧化酶亚基Ⅰ基因(COⅠ)技术对东北林蛙等24种进行鉴别,构建邻接(NJ)系统发育树,对24种进行系统聚类分析,为东北林蛙等蛙类的鉴别,分类及新种的发现提供一定的依据。方法:收集东北林蛙、中国林蛙、黑龙江林蛙、徂崃林蛙、桓仁林蛙各10只,提取其DNA并进行聚合酶链式反应(PCR)扩增,将扩增成功的序列测序,共测得50条COⅠ基因序列;从GenBank数据库中获得蛙科蛙属24种163条COⅠ基因序列及蛙科侧褶蛙属,臭蛙属,琴蛙属,水蛙属,湍蛙属各一条COⅠ基因序列;利用MEGA X进行序列比对,分析各物种COⅠ基因序列简约性信息位点,计算种内,种间遗传距离,构建NJ树进行系统聚类分析。结果:东北林蛙等24种COⅠ基因序列长度为554 bp,共有210个简约性信息位点,各物种种内遗传距离均<2%;除桑植蛙与明全蛙种间遗传距离为0.004外,其余各物种种间遗传距离范围为0.024~0.228;桑植蛙与明全蛙聚为一支,部分东北林蛙与Rana uenoi聚为一支,中国林蛙有2个独立的分支,其余各物种均独立聚为一支。结论:DNA条形码COⅠ技术能够对东北林蛙等21种进行鉴定及鉴别,支持桑植蛙与明全蛙,韩国林蛙与昆嵛林蛙为同物异名,其中一支中国林蛙可能是蛙科蛙属未下载的4个种之一或新种。这表明DNA条形码COⅠ技术不仅可以鉴定及鉴别东北林蛙等24种蛙类,也可以为蛙科蛙属的分类,新种或亚种的发现提供一定的科学依据。Objective: To identify 24 Rana species such as Rana dybowskii by mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ)gene-based DNA barcoding and build the neighbour-joining(NJ)tree for hierarchical cluster analysis,so as to provide a basis for the identification and classification of Rana species as well as the discovery of new species. Method: R. dybowskii,R. chensinensis,R. amurensis,R. culaiensis,and R. huanrenesis,ten for each species,were collected for DNA extraction and polymerase chain reaction(PCR)amplification and sequencing. A total of 50 COⅠ gene sequences were obtained. Then 163 COⅠ gene sequences for 24 species of Rana and one COⅠ gene sequence for Pelophylax, Odorrana, Nidirana,Hylarana,and Amolops were harvested from GenBank. After sequence alignment by MEGA X,the parsimonyinformative sites of COⅠ gene sequences were analyzed and the intraspecific and interspecific genetic distances were calculated,followed by the built of NJ tree and hierarchical cluster analysis. Result: The COⅠ gene sequences of 24 Rana species including R. dybowskii were 554 bp in length and there were 210 parsimonyinformative sites in total. The intraspecific genetic distance of each species was smaller than 2%. Except that the interspecific genetic distance between R. sangzhiensis and R. zhengi was 0.004,the genetic distances between the other species ranged from 0.024 to 0.228. R. sangzhiensis and R. zhengi were clustered into one branch and some R. dybowskii and R. uenoi into one branch. There were two separate branches for R. chensinensis and the other species were all clustered independently. Conclusion: COⅠ-based DNA barcoding enabled the identification of24 species of Rana including R.dybowskii. The findings supported that R. sangzhiensis,R. zhengi,R. coreana,and R. kunyuensis were the same species. One branch of R. chensinensis might be one of the four undownloaded species in Ranidae or a new species. The results have demonstrated that COⅠ-based DNA barcoding allows not only the identification of 24 spe

关 键 词：东北林蛙 中国林蛙 蛙科 蛙属 DNA条形码 线粒体细胞色素C氧化酶亚基I基因(COⅠ) 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学]