奶牛精子SRY基因原位杂交检测方法的建立

Establishment of an Effective Approach to Sperm Fluorescent in Situ Hybridization for Sry Gene in Dairy Cow

Sry基因是Y染色体性别决定基因,采用primer premier 6对SRY基因序列设计引物,以公牛基因组DNA为模板扩增,高效DNA地高辛标记和检测试剂盒制备探针,对X精子(Y精子纯度7%)、Y精子(Y精子纯度93%)和常规精子(Y精子纯度50%)DTT去凝集、蛋白酶K去除鱼精蛋白,杂交。结果表明:在荧光显微镜下可见标本背景清晰,精子头部成蓝色,边界清楚,X精子4.67%(n=600)有荧光信号,Y精子86.33%(n=600)有荧光信号,常规精子46.17%有荧光信号。实验证明了所选取Sry基因序列为Y精子携带,建立了精子原位杂交方法。

SRY gene is the special gene to determine the sex of Dairy Cow. SRY gene primers were designed by primer premier 6 And PCR applicatied with the Bulls genomic DNA was used astemplate. DNA labeled with high Digoxin and detection kit were used to produce SRY gene probes. X sperm （7% Y - sperm）, Y （93% Y - sperm） and conventional sperm （50% Y -sperm purity） were agglutinated with DTF, the protamine of X, Y and conventional sperm was removed with protease K, and X, Y and conventional sperm were hybridized, respectively. Results showed that： FISH images observed under fluorescence microscope for X, Y and conventional sperm with significantbackground, blue sperm head and clear boundary. The 4.67% sperm cells were found with fluorescent signal in X -sperm （n = 600） , 86.33% in Y -sperm （n = 600） , and 46.17% in conventional sperm. The experiment proved that Sry gene sequences were only carried by Y - sperm, rather than X - sperm. The method was established for sperm fluorescent in situ hybridiza- tion in dairy cow.