中国大鲵非损害性DNA取样及提取方法的比较研究  

Comparative Study on the Methods of Non-Disruptive DNA Sampling and Extraction in Chinese Giant Salamander(Andrias davidianus)

作　　者：王金秀 兰香英 罗庆华[1,2] 邓克国[1] 梁志强[3] 蒋万胜 WANG Jin-Xiu;LAN Xiang-Ying;LUO Qing-Hua;DENG Ke-Guo;LIANG Zhi-Qiang;JIANG Wan-Sheng(Hunan Engineering Laboratory for Chinese Giant Salamander’s Resource Protection and Comprehensive Utilization,and Key Laboratory of Hunan Forest Products and Chemical Industry Engineering,Jishou University,Zhangjiajie 427000;College of Biology and Environmental Sciences,Jishou University,Jishou 416000;Fisheries Research Institute of Hunan Province,Changsha 410153,China)

机构地区：[1]吉首大学大鲵资源保护与综合利用湖南省工程实验室,林产化工工程湖南省重点实验室,张家界427000 [2]吉首大学生物资源与环境科学学院,吉首416000 [3]湖南省水产科学研究所,长沙410153

出　　处：《动物学杂志》2022年第5期641-652,共12页Chinese Journal of Zoology

基　　金：国家自然科学基金项目(No.32060128,32060238);湖南省创新平台与人才计划项目(2020RC3057);质兰基金会项目(No.2020040371B);吉首大学生态学双一流学科建设经费及研究生校级课题(No.Jdy20086,DNGC2012,DNGC2021)。

摘　　要：基于DNA分子的研究方法在物种分类及系统学、生态遗传学、保护生物学等领域被广泛运用,但针对动物DNA取样方法的比较研究整体上较为缺乏。动物DNA取样要在对实验对象造成最小化影响和获得满足研究需要的DNA之间保持平衡,这需要不断探索取样方法的优缺点和适用性。本研究以人工繁育的水生有尾两栖动物中国大鲵(Andrias davidianus)为研究对象,比较口腔拭子、皮肤拭子、皮肤脱落物和尾静脉采血4种取样方式对DNA质量的影响。另外,基于其中皮肤拭子样品,进一步比较试剂盒法、高盐法、苯酚氯仿法和磁珠法对DNA提取效果的影响。结果表明,4种非损害性取样方法均能获取目标物种的DNA,且均未对取样对象的行为或适合度产生明显影响,但在DNA质量和浓度方面存在一定差异,其中,相对最好的为尾静脉采血,其次是口腔拭子和皮肤拭子,最差的为皮肤脱落物样品。而基于皮肤拭子样品采用4种不同DNA提取方法所获得的DNA效果整体差异不大。本研究通过以中国大鲵作为水生和非皮毛类动物的代表类群,研究并总结了不同DNA取样方法和提取方式的优缺点及注意事项,可为未来中国大鲵或其他珍稀濒危动物的非损害性取样方法及相关分子生态学研究提供借鉴。[Objectives]DNA-based research methods have been widely applied in the study of species taxonomy and phylogeny,ecological genetics,and conservation biology,however,the comparative studies on DNA samplings are generally scarce.In this study,we aimed to compare the performance and applicability of different DNA sampling and extraction methods by using the captive-bred Chinese giant salamander(Andrias davidianus)as study objects.[Methods]DNA samples were obtained by four sampling methods including buccal swabbing,skin swabbing,shed skin sampling and tail venous blood sampling(Fig.2).DNA was extracted by four different methods including Kit method,high salt method,phenol-chloroform method and magnetic bead method based on the most accessible skin swabbing samples.All the DNA qualities and concentrations were detected by gel electrophoresis and nucleic acid protein analyzer,and each of the sampling DNA was identified by PCR and sequencing of the mitochondrial COI fragments.In addition,the daily and feeding behaviors of the experimental animals were observed after samplings.[Results]Our results showed that the daily and feeding behaviors of A.davidianus didn’t change visibly,thus the four sampling methods could belong to the category of non-disruptive DNA sampling that has minimal impact on the fitness,behavior or welfare of the experimental animals.Among the four different sampling methods,the tail venous blood sampling performs optimally,followed by buccal,skin swabbing,and then shed skin sampling in terms of DNA quality and concentration(Fig.3a,b).However,the DNA obtained by four different extraction methods based on skin swabbing samples were generally similar(Fig.3c,d).Although different sampling methods performed differences in obtaining DNA,all the extracted DNA could be amplified of the target COI gene fragments(Fig.3e,f).[Conclusion]By summarizing the advantages,disadvantages and precautions of different DNA sampling and extraction methods(Table 1),we suggest that the most appropriate sampling method should

关 键 词：非损害性取样 非损伤性取样 DNA提取方法 中国大鲵 水生动物 

分 类 号：Q958[生物学—动物学]