【作者】 梁文明；

【导师】 吴海龙；

【作者基本信息】 安徽师范大学 ， 动物学， 2010， 硕士

【摘要】 黑麂(Muntiacus crinifrons)是我国特有的濒危鹿科动物,具有很高的学术研究价值。由于分布范围狭窄,加之传统狩猎习俗和经济价值等因素,该物种长期遭受非法捕猎的生存压力,野生种群数量已十分稀少,因而,黑麂被列为国家一级重点保护野生动物。尽管如此,野外盗猎现象仍时有发生,出售在市场上的黑麂被去皮后,执法人员几乎无法利用形态学特征等宏观手段进行鉴定。为了切实保护黑麂,为相关执法部门提供一种便捷、可靠、高效的工具来鉴定黑麂组织样品,本研究基于AFLP方法开发了黑麂种特异性SCAR探针。首先,利用引物Ep7和Mp8获得了长度为707bp的黑麂AFLP特异性条带,对该特异性DNA片段进行纯化、克隆、测序。根据所测DNA片段序列信息,利用Primer Premier 5.0和Oligo 6.0软件设计了黑麂特异引物(P-F/P1-R),并获得了长度为298bp的SCAR。为了检测特异引物的可靠性,对引物进行了群体扩增检测。同时,利用哺乳动物cyt b通用引物L1和H1对所有待检样品DNA的质量进行了检测。结果显示,在全部9种43个个体样品中,25个黑麂样品全部扩增出2条目标条带,即长度298bp的黑麂特异条带和长度约为466bp的cyt b基因片段;而代表其它物种的18个个体均仅扩增出单一的cyt b基因片段。所有个体都能被哺乳动物cyt b通用引物扩增,说明待检样品DNA的质量全部符合PCR扩增要求,引物可靠性检测结果达到对目标动物进行法医学鉴定的目的。综上所述:本研究已成功地将黑麂种特异性AFLP标记转化为SCAR标记,并得到了具有高度物种特异性的SCAR探针。利用本研究开发的SCAR特异探针,只要根据黑麂特异条带的有或无,就能快速、准确地鉴别出黑麂。因此,该探针可作为执法部门鉴定黑麂组织样品的有效工具,以加强对偷猎行为的执法力度。此外,利用该探针可以基于无损伤取样法(如粪便)对特定区域黑麂野生种群动态进行监测。更多还原

【Abstract】 Black Muntjac (Muntiacus crinifrons), a rare and endangered deer endemic to China, holds a high value in academic research. The species is currently regarded as the state key protected wild animal due to its limited distribution range and small population in the wild. However, because of the economic value of the meat, antlers and skin, the species has been hunted as the main targeted animal in traditional hunting practices and is still suffered from poaching at the present time. Usually, in order to escape from punishment, the poached black muntjac was peeled and sold together with carcasses of other animals, such as Chinese muntjac or livestocks. To provide a convenient, reliable and effective tool for authentication of tissue specimen such as, meat, skin and other tissues of the species, in this study, a Sequence Characterized Amplified Region (SCAR) derived from the black muntjac species-specific Amplification Fragment Length Polymorphism (AFLP) marker has been developed.Initially, a 707-bp species specific DNA fragment of black muntjac was detected by an AFLP primer pair (Ep7/Mp8). Then, the DNA fragment was purified, cloned and sequenced. Subsequently, based on the specific AFLP fragment sequence, a black muntjac-specific primer pair (P-F/P1-R) was designed by using Primer Premier 5.0 and Oligo 6.0, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified across all the samples, PCR amplification with the cyt b universal primers as positive control. As expected, out of the all forty-three DNA samples representing nine different species, all the twenty-five black muntjac samples presented two bands (about 298bp for SCAR and 466bp for cyt b). But the others failed to produce the SCAR with merely showing one band, the 466-bp cyt b fragment. The results indicated that the black muntjac-specific AFLP marker has been converted into a SCAR marker successfully and the SCAR primers were highly species-specific. Just based on the presence and absence of the SCAR band, we could discriminate black muntjac from other species rapidly and accurately. Therefore, the SCAR primers developed in this study will provide an effective tool for forensic authentication of tissues from black muntjac and strengthen the detection of poaching. And also, the SCAR probe may be useful for population survey in the wild through noninvasive sampling method.更多还原