非损伤性方法的建立及其在亚洲黑熊分子遗传学研究中的应用

哺乳动物野生群体和濒危物种的分子遗传学研究长期以来受到组织样品来源的限制。因为传统的分析方法，如蛋白质电泳、ＤＮＡ限制性片段长度多态及ＤＮＡ序列分析等，需要采集诸如血液、肌肉、肝脏、心脏、肾脏和脾脏等组织材料以提取足量的蛋白质和ＤＮＡ。而这又常常涉及捕捉、损伤甚至杀死动物。本工作探索建立一种非损伤性的途径以解决这一困难。我们采用Ｂｉｏ—Ｒａｄ公司“ＩｎｓｔａＧｅｎｅＰｕｒｉｆｉｃａｔｉｏｎＭａｔｒｉｘ”从遗落于地上的亚洲黑熊单根毛发样品中提取出基因组ＤＮＡ。这种方法简便、快速，仅需一个步骤──在该试剂中以１００℃裂解细胞。不必再经酚和氯仿抽提。我们用ＰＣＲ技术从这种毛发ＤＮＡ中扩增了线粒体细胞色素ｂ基因片段，并测定了该片段的ＤＮＡ序列。我们的工作为开展亚洲黑熊及其他哺乳动物野生群体的分子遗传学研究奠定了基础。

A NON-INVASIVE APPROACH TO THE GENETIC ANALYSIS OF ASIATIC BLACK BEAR

Genetic analysis of mammalian free-ranging populations and endangered species utilizing molecular .methods, such as protein electrophoresis, DNA RFLP and DNA sequencing, has long been hampered by tissue-sampling problems. We established anon-invasive approach to help to overcome the problems. The genomic DNA of the Asiaticblack bear was extracted from single hair shed on the ground using instaGene Purification Matrix (Bio-rad) with a simple cell lysis stop by boiling in the presence of the matrix. Our protocols allow simple, easy and fast DNA preparation by eliminating labor intensive phenol / chloroform extraction. Mitochondrial cytochrome b gene fragment was amplified from hair DNA using PCR technique. Double stranded PCR product was directly sequenced by the dideoxy method using heat denaturation. Our success on determination of mitochondrial cytochrome b genesequence from hair could be considered as a first step in the population genetic study of thefree-ranging Asiatic black bears.