鹿类动物物种特异性遗传标记的研究
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。
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 traditional
methods 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.
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。
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 traditional
methods 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.
引文
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2 盛和林、曹克清、李文军等．中国鹿类动物，华东师范大学出版社．1992．
3 张荣祖．中国哺乳动物分布．中国林业出版社．北京．1997．
4 王玉玺．驼鹿繁殖研究．野生动物．1981．(2)：35-36．
5 王会志、盛和林．四川盆地西北缘林麝种群密度及保护与利用．兽类学报．1988，8(4)：241-249．
6 吴家炎、郑生武、韩亦平等．白唇鹿形态及生态地理分布的初步研究．华东师范大学学报哺乳动物生态学专辑．1990，71-78．
7 胡金元、袁西安．狍的繁殖．野生动物．1982，(4)：27-29．
8 胡江华、张恩迪、盛和林等．圈养林麝发情期行为的研究．华东师范大学学报哺乳动物生态学专辑．1990，8-7．
9 于孝忠．胜山猎场驼鹿食性研究(硕士论文)．1989，1-33．
10 陈化鹏、肖前柱．带岭林区马鹿冬季食性研究．兽类学报．1989，9(1)：8-15．
11 李继承、颜廷峰．林区野生马鹿食性与危害幼林的研究．动物学杂志．1989，(3)：34-36．
12 郑生武、皮南林．马麝的生态研究．动物学报．1979，23(2)：176-186．
13 卞祖福，段传风，袁丽华．三种驯鹿茸和梅花鹿茸化学成分比较测定(3)—性激素的测定特产科学实验．1987，(4)：39-40．
14 李文军．中国狍的生物学和管理．硕士研究生论文(1990)．
15 岳秉飞．鹿的外激素分泌腺及其功能．生物和特产．1986，(4)：29-30．
16 赵广英．大兴安岭驯鹿正常生理指标的测定和繁殖．动物学杂志．1976，(1)：37-38．
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