黑麂Y染色体的鉴别和Sry基因的克隆及定位

The Study of the Sry Gene of Muntiacus crinifrons

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针, 应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的 SRY（sex determining region of the Y chromosome ） 基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样，与人SRY基因的同源性均为83%。

The single Y chromosome of Muntiacus reevesi an d Y 1,Y 2 ,X+4，1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the meta phase karyotype of Muntiacus crinifrons were painted( by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y 2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly(the product of the DOP-PCR of Y 1，Y 2，X+4，1 chromosome of Muntiacus crinifrons were used as the template s of the next amplifica tion using the special primer devised according to the human SRY gene .One band was obtained only from Y 2 chromosome , then it was cloned to the T -vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y 2 ch romosome of the Muntiacus crinifrons.