摘要
本试验建立了用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）体外扩增牛Ｙ染色体特异ＤＮＡ序列鉴别牛胚胎性别的方法。选择３头成年健康西门塔尔母牛为供体，在其自然发情后第９～１４ｄ，用ＦＳＨ超排处理，人工授精，发情后的第７ｄ（发情日为Ｏｄ），用非手术法采集胚胎，共获得１１枚Ａ、Ｂ级胚胎。在立体显微镜下分别对胚胎进行分割，各获得２个半胚。一个半胚在２０μＬ含１０ｍｍｏｌ／ＬＴｒｉｓ－ＨＣＩ（ｐＨ８．３）、５０ｍｍｏ１／ＬＫＣＩ、２ｍｍｏｌ／ＬＭｇＣｌ２、０．４５％ＮＰ－４０、０．４５％Ｔｗｅｅｎ－２０、０．１ｍｇ／ｍＬ蛋白酶Ｋ的处理液中５５℃水浴作用６０ｍｉｎ。该处理液直接作为ＰＣＲ扩增的模板，加入引物、底物、ＴａｑＤＮＡ聚合酶及相应的缓冲液进行ＰＣＲ。ＰＣＲ反应温度参数为９２．５℃变性，５５℃退火，７２℃延伸，共３５个循环。扩增产物在２％琼脂糖凝胶上电泳，出现Ｙ染色体特异条带判断胚胎为雄性，否则判断为雌性。切割得到的另一半胚分别移入１１头同期发情的黄牛或黑白花奶牛体内，最后产出４头西门塔尔牛犊，２头公牛，２头母牛，与ＰＣＲ方法鉴别的结果完全一致，表明所建立的方法可用于牛胚胎性别的鉴别。
A polymerase chain reaction(PCR)-based method for accurate sex deter-mination of preimplantation bovine embryos was established.11 Simmental cattle em- brvos were recovered from three female cattle. The embryos were divided into two parts under binocular microscope。 One of the semi-embryos was transferred to 20 μL lysate buffer(10 mmol/L Tris-HCI pH 8.3, 50 mmol/L KCI, 2 mmol/L MgCI2 ,0.45%NP- 40,0.45% Tween-20,0.1mg/mL Proteinase K ) and incubated for 60 min at 55℃. The lysates were directly used as templet of PCR, The amplification was performed by using 35 cycles of denaturation at 92.5℃, annealing at 55℃ and extention at 72℃ .Am- plified DNA products were resolved by 2%agarose gel electrophoresis. The embryos in whose lysates a bovine Y chromosome-specific amplification fragment appeared were i- dentificated as male, otherwise they were identificated as female。The other semi-em- bryos were transferred tO the uterus of pseudopregnant females。 2 males and 2 females were born from 11 implanted embrvos whose sexes were same as the results identificated by PCR。
出处
《中国兽医学报》
CAS
CSCD
1995年第2期112-115,共4页
Chinese Journal of Veterinary Science
基金
吉林省科委青年基金
关键词
牛
胚胎
性别
PCR
聚合酶链反应
bovine
embryo
PCR
sex Biological and Technical Centre of Jilin Agricultural Academy
