四个警用犬种嗅认行为相关OR基因SNP筛查及分子机制研究

Dogs have a greater olfactory ability which is crucial to themselves. As for the shortcomings of methods in testing dog’s smelling such as ethology methods, odor detection of police-dogs is in question. Therefore clarifying the mechanism of the olfactory differences will endow the study with scientific value and practical significance. The former study showed that there was a relationship between SNP of OR genes and smelling behavior of dogs and an interaction between SNP and the kind of odor. Firstly, single base extension, capillary electrophoresis, behavior test, SAS 9.2 PROC-GLM and Logistic Regression Analysis will be applied to find the correlative SNP of OR genes and its number in the four breeds of German Shepherd Dog, Labrador Retriever, English Springer Spaniel and Belgian Malinois. Secondly, a plasmid pIRES-OR is constructed and transfected into IE6 cells while stimulated by odorant solutions. Finally, fluorescent-probe assay is utilized to examine the concentration of Ca2+ while ELISA assay is used to test the concentration of SEAP and Dual Glo luciferase is used to test the concentration of cAMP, which will exploit the mechanism of SNP in IP3 and cAMP signaling pathways. The research will provide the theoretical bases and trail supports for the genetic differences of dogs’ olfactory sense at the molecular, cellular and organic levels.

犬嗅觉系统具有卓越的气味识别能力并对犬生命活动有重要意义。犬嗅认行为选育方法的缺憾与警犬气味识别结论被质疑的现状使得阐明犬嗅觉差异遗传机制具有重要科学价值和实际意义。前期实验结果显示警用德国牧羊犬嗅认行为与OR基因SNP具有相关性，且SNP与气味种类交互作用。为发现犬嗅认行为相关OR基因SNP及其数量，本研究拟通过单碱基延伸和毛细管电泳技术检测OR基因型、测评犬嗅认行为、SAS 9.2 PROC-GLM、Logistic 回归分析，筛选德国牧羊犬、拉布拉多犬、史宾格犬、马里努阿犬嗅认行为相关SNP及其数量；为分析SNP在IP3和cAMP信号通路中的作用机制，课题组构建OR基因表达载体、转染IE6细胞，在适宜气味刺激下，利用荧光探针标记示踪法检测Ca2+浓度、ELISA检测SEAP浓度、双荧光素酶报告基因检测cAMP浓度。本研究从分子、细胞、整体水平为犬嗅认行为遗传差异提供实验和理论依据。

德国牧羊犬、史宾格犬、拉布拉多犬和马里努阿犬，是被世界各国警察频繁用于气味识别作业的四个犬种。但是，许多国家的法庭质疑并不予采纳警方提供的犬气味判定结果。本项目①依据警犬和导盲犬工作能力标准测评犬嗅认行为：受试犬选自公安部警犬技术学校、大连医科大学导盲犬培训中心和河北李浩犬类养殖有限公司；②毛细管电泳技术检测OR基因SNP位点69个；③PROC-GLM和Logistic回归分析基因型效应；④细胞学方法研究嗅觉机制：构建敏嗅与非敏嗅cOR2K2-like:c.518AA/GG、cOR08A09:c.118CC/GG和cOR16C11:c.368CC/AA表达载体3对，Gaolf共转染HEK-293细胞，香草醛、a-Damascone和乙酸丁香酚酯气味刺激，双荧光素酶报告基因检测cAMP浓度。研究结果显示①嗅认行为相关SNP：被检测的德国牧羊犬7个SNP位点均与嗅认行为不相关.因此，目前发现的德国牧羊犬嗅认行为相关SNP仍为前期研究的5个SNPs包括cOR2K2-like:c.518G>A等；拉布拉多犬2个即cOR16C11:c.368 C>A 和OR7215:c.251C>T；马里努阿犬2个即cOR08A09:c.118G>C、cOR0527:c.847G>A；史宾格犬1个即cOR3045:c.206C>T。②嗅觉机制： OR重组质粒转染Hek-293细胞，在分别受到3种气味物质刺激后，OR均表现出识别活力、细胞内cAMP含量增加且敏嗅型>非敏嗅型即cOR2K2:c.518GG>AA、cOR08A09:c.118GG>CC和cOR16C11:c.368CC>AA。项目研究筛查出四个警用犬种嗅认行为相关SNP、从分子层面解释犬嗅认行为差异的遗传机制，为分子标记辅助警用犬种选育提供理论基础和生产实践可能。