牙釉质基因巢式PCR鉴定猪ICSI胚胎性别  

Sex Identification of Porcine ICSI Embryo by Nested Amplification of Amelogenin Gene

作　　者：孙俊丽[1,2] 廖海洪 张冰 孙如玉 卢克焕 卢晟盛[1] SUN Jun-li;LIAO Hai-hong;ZHANG Bing;SUN Ru-yu;LU Ke-huan;LU Sheng-sheng(Guangxi University,College of Animal Science ＆ Technology,Guangxi Nanning 530004,China;Guangxi Institute of Animal Sci ence,Guangxi Key Laboratory for Livestock Genetic Improvement,Guangxi Nanning 530001,China)

机构地区：[1]广西大学动物科技学院,广西南宁530004 [2]广西畜牧研究所家畜遗传改良重点实验室,广西南宁530001

出　　处：《西南农业学报》2018年第10期2202-2207,共6页Southwest China Journal of Agricultural Sciences

基　　金：广西自然科学基金项目(2015jjAA301641);广西畜牧研究所基本业务费项目(桂牧研科2017-01)

摘　　要：【目的】建立可准确鉴定猪胚胎性别、可靠的巢式PCR反应体系及研究卵胞浆内单精子显微注射技术(Intracytoplasmic sperm injection,ICSI)对胚胎性别的影响。【方法】根据猪X染色体和Y染色体上牙釉质基因(Amelogenin gene,AMEL)第三内含子上9～10 bp的缺失设计了巢式PCR引物,并利用10个猪耳组织基因组DNA样品优化了PCR扩增条件,巢式PCR—荧光标记的半自动基因组扫描技术鉴定205个猪ICSI早期囊胚性别。【结果】特异性扩增了199个ICSI样品,6个ICSI胚胎扩增失败。经PCR产物片段扫描,只在178～179 bp处有峰的,判定为雌性胚胎,在169～171和178～179 bp 2处有峰则判定为雄性胚胎。共确定雄性胚胎71个,雌性胚胎128个,其中公母比例1∶1.8。【结论】基于牙釉质基因的巢式PCR方法可用于猪胚胎性别鉴定。性别比例偏离在一定程度上说明在ICSI囊胚中存在部分孤雌胚胎。【Objective】The objective of this study was to obtain an accurate and reliable method for determining the sex of porcine embryos through nested-PCR amplification of amelogenin（AMEL） gene,and evaluate the effect of ICSI microoperation on embryo gender.【Method】Based on the absence of 9-10 bp on the third intron of porcine AMELx and AMELy genes,the nested PCR primers were designed,and the PCR amplification condition was optimized by using 10 pig ear genome DNA samples.A total of 205 pigs ICSI early blastocysts were tested by nested PCR and sequence scanning to identify the gender.【Result】One hundred and ninety-nine ICSI samples were specifically amplified.Six ICSI embryos failed to amplify.After PCR product fragment scanning,the embryos of only one peak at 178-179 bp was determined as female,while that of two peaks at 169-171 and 178-179 bp was determined as male.71 male embryos and 128 female embryos were ensured.The ratio of male and female was 1∶1.8.【Conclusion】The nested-PCR method can be used for the gender identification of pig embryos.Gender imbalance partly indicates the existence of parthenogenetic embryos in ICSI blastocysts.

关 键 词：牙釉质基因 猪ICSI胚胎 巢式PCR 性别鉴定 

分 类 号：S828[农业科学—畜牧学]