睾丸特异蛋白Y基因(TSPY)对奶牛早期胚胎性别的鉴定  

Sex Determination of Preimplantation Bovine Embryosusing Testis-specific ProteinY Gene (TSPY)

作　　者：黄金明[1] 王长法[1] 李建斌[1] 仲跻峰[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,济南250100

出　　处：《农业生物技术学报》2009年第3期433-438,共6页Journal of Agricultural Biotechnology

基　　金：山东省自然科学基金项目(No.Y2007D72);山东省农业良种工程项目(No.2006LZ10-04)资助

摘　　要：为方便性别鉴定方法在现场应用,利用睾丸特异蛋白基因(TSPY)建立了奶牛早期胚胎的非电泳性别鉴定方法。设计并合成了TSPY雄性特异和雌、雄共有基因引物,并利用已知性别的奶牛血液DNA为模板,初步建立了性别鉴定的PCR反应体系和非电泳的性别鉴定方法。灵敏度试验结果显示,雄性特异引物和共有基因引物对在模板10～60pg时,其性别鉴定的准确率为100%,提示TSPY基因具备鉴定胚胎性别的可能;同时采用非电泳法和环介导的等温扩增(LAMP)法鉴定6枚胚胎的性别,两者结果完全一致;用非电泳法检测TSPY基因鉴定了43枚胚胎的性别,对其中鉴定为雌性的21枚胚胎分别移植给自然发情后6～8天的受体,结果9头出生的犊牛均为母牛。结果表明,TSPY基因是一个很好的雄性特异标记,非电泳检测TSPY基因对奶牛早期胚胎性别的鉴定结果准确可靠。In order to determine bovine embryo sex under farm condition more feasible, the non-electrophoretic method to detect the testis-specific protein Y encoded （TSPY） gene for sex determination of bovine early embryos was established and examined.Primers were designed to amplify a portion of the TSPY gene and a male and female common gene was used as an internal control primer. PCR optimization was carried out using a DNA template （10-60 pg） from bovine whole blood. Sensitiveness test showed that the accuracy of sex determination was 100%. Furthermore, six embryo samples were diagnosed by the non-electrophoretic method and the sexing results were consistent with those of the loop-mediated isothermal amplification （LAMP） method. The result showed that TSPY was a sexing method as reliable as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant and all delivered female calves. The results showed that the sex predicted accuracy of this protocol was 100%. TSPY gene is a good male specific marker and indicates that a non-electrophoretic method is feasible and accurate to detect TSPY gene for sexing preimplantation bovine embryos.

关 键 词：奶牛胚胎 性别鉴定 睾丸特性蛋白Y基因(TSPY) PCR 

分 类 号：S188[农业科学—农业基础科学]