【作者】 袁巍；

【导师】 马建章； 孙中武；

【作者基本信息】 东北林业大学 ， 动物学， 2002， 硕士

【摘要】 随着对野生动物保护力度的加强和养鹿业的迅速发展，越来越多的刑事案件和民事案件涉及了鹿类动物种、亚种或品种的鉴别。对于鹿类动物骨骼、皮张、被毛、肌肉、内脏等组织、茸片、血液、胎儿及这些材料制备的各种产品的种属鉴定，传统的方法难以满足识别鉴定的需要，本文针对这一问题，从角蛋白和DNA水平上设计和建立了鹿类动物物种识别方法体系，几乎可以用于任何鹿类初级产品的鉴别。 1．成功地从鹿类动物毛干中提取角蛋白和DNA，在分子水平上建立了对鹿类动物无损伤性的研究方法，至此，本文的方法几乎可以适用于任何鹿类动物的初级产品的鉴定。 2．毛角蛋白SDS-PAGE谱带具有物种特异性，可以作为物种的蛋白指纹。 3．毛角蛋白多态性与取样部位有关，同种不同个体间，如果被毛来自身体的相同部位，角蛋白电泳结果一致，若被毛采自身体的不同部位，蛋白谱带存在差异。因此用角蛋白指纹识别物种时要强调毛样品的来源部位。 4．SDS-PAGE电泳方法分辨率和灵敏度有限，可能有一些角蛋白组份不能被检出，故SDS-PAGE获得的角蛋白指纹不宜用于分析物种之间系统关系，应当用来进行物种识别和鉴定。 5．经过对来自GenBank中的15种鹿类动物的Cyto b基因序列的比较，用通用引物L15774和HSF21作为模板的质量控制，设计了特异性引物DF／DR和CF／CR来鉴定马鹿、梅花鹿、狍；设计了麝类特异性引物MF／MR，用限制性内切酶Rsa Ⅰ酶切扩增产物来区分原麝和林麝。本文建立的5种鹿类动物识别方法体系达到了稳定、高效、灵敏、重复性好的水平。更多还原

【Abstract】 With the increasing development of wildlife conservation and foster deer enterprise,there are more and more criminal cases and civil cases that relate to the identification of deer species,subspecies or breeds. For the identification of deer species with body parts like bone,skin,hair,muscle,visceral organ,antler,blood,embryo,and their various products,the traditionalmethods are not likely powerful enough to discriminate these involved samples. For establishing an effective identification methodology,keratin and mtDNA polymorphisms were studied and taken as genetic markers of deer species. The methods established here were proved effective for any deer primary products.1. Keratin and DNA were successfully extracted from hair shaft of deer species,and hereby a non-invasive research method about deer species was set up. Thereafter,almost all deer primary product could be adopted in deer species identification.2 . Hair keratin polymorphisms generated through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed species specific patterns. The keratin patems could be regarded as species specific fingerprints.3. The hair keratin polymorphisms differ with the sampling sit on the body. For different individuals of the same species,an identical keratin fingerprint will be obtained if the hairs come from the same part of body,and vis versa. So,the sampling sit on the body must be clearly emphasized when applying keratin fingerprints to the identification of species.4. It was observed that some keratin components would be not detected through SDS-PAGE due to its low resolution and sensitivity. Therefore,the SDS-PAGE keratin fingerprints should be applicable to the species identification,but not to the phylogenetic analysis.5. Based upon the comparison of Cyto b gene sequences in 15 deer species downloaded from GenBank,a universal primer set L15774/HSF21 was used as positive control of the template quality,at the meantime,two species specific primer sets DF/DR and CF/CR were desgined to identify red deer (Cervus elaphus),sika deer (Cervus nippori) and roe deer (Capreolus capreolus) from other species. A musk deer (Moschus) specific primer set WF/MR was designed,Siberian musk deer(Afoschus moschiferns) and forest musk desr(Moschus berezovskii) could be discriminated when restriction endonuclease Rsa I was used to cut the PCR products. The species identification methodology of 5 deer species established in this paper achieved reliable,effective,sensitive and reproducible.更多还原