土鳖虫药材基原的DNA条形码鉴定及隐存多样性研究

The Tubiechong is a traditional Chinese medicine with large demand in the Chinese market. According to the Pharmacopoeia of the People's Republic of China (2010 Edition), the origin of Tubiechong should be Eupolyphaga sinensis or Steleophaga plancyi. At the germplasm resources level, the Tubiechong has numerous breeding varieties, and the wild Tubiechong also has large genetic or morphological diversity. Our preliminary work has supported the existence of cryptic species within Eupolyphaga sinensis collected from different localities. At the market level, it is very hard to distinguish between Tubiechong and its adulterant when the crude drug has been processed into powder. The Tubiechong from different sources, as well as its relative species, habitually used species and adulterant, will be taken as research object in this project. The DNA barcoding candidates COI gene and ITS2 region of these specimens will be amplified and sequenced. It will be the first systematic research of Tubiechong resources on the molecular level. By the means of genetic distance and phylogenetic analysis, we will discover the cryptic diversity within Eupolyphaga sinensis or Steleophaga plancyi, study the relationship among cryptic species, and judge the authenticity of so called "hybrid Tubiechong". The DNA barcoding identification method of Tubiechong will be established, and the feasibility of this method on processed medicinal materials will be tested. This research will provide important evidences for the germplasm resources protection and quality control of Tubiechong.

土鳖虫为市场需求量较大的传统中药材，2010 年版《中国药典》规定其基原为地鳖或冀地鳖。种质资源层面，土鳖虫养殖品种名目繁多，野生资源的遗传、性状差异较大。本研究前期工作支持不同产地地鳖中存在隐存种。市场流通层面，经炮制后成粉末状的土鳖虫与混伪品难以区分鉴别。本研究拟以涵盖正品、正品近缘种、习用品和常见混伪品的大量不同来源土鳖虫为研究对象，扩增DNA条形码COI 基因及辅助序列ITS2。首次在分子水平进行土鳖虫药源梳理，从野生及养殖种群（或品种）中，通过遗传距离、系统发育分析，进一步揭示地鳖或冀地鳖内部的隐存多样性，并分析隐存种种间亲缘关系，人工"杂交土元"的真伪及起源。确立土鳖虫药材的DNA条形码基原鉴定方法，验证该方法对炮制处理后药材鉴定的可行性。研究结果将为土鳖虫资源保护及药材质量控制提供重要依据。

相对于植物资源，针对药用动物的中药资源学研究开展较少，特别是隐存资源的发现研究。本研究收集了土鳖虫样品68份并利用DNA条形码技术发现隐存资源5种。应用线粒体基因组技术以及比较形态学方法对部分隐存资源进行分析。最后，我们尝试利用宏条形码技术从大黄蛰虫丸中鉴定土鳖虫成分，并在中成药中发现了不同隐存资源混合使用的情况。