【作者】 王丹；

【导师】 金成汉；

【作者基本信息】 延边大学 ， 基础兽医学， 2003， 硕士

【摘要】 为选出更高效、实用的牛胚胎性别鉴定方法，本实验根据已经报道的牛性染色体上DNA序列，选择并合成了8对扩增性染色体上DNA序列的引物。采集晋南牛(中国黄牛品种)、荷斯坦牛(奶牛品种)血液样本，标记，提取基因组DNA并测得浓度。用PCR法应用8对引物分别对所有的成年牛血液提取的模板DNA进行扩增，使用电泳方法或非电泳法(引物DYZ-1)分析产物。经过对PCR条件的摸索和优化，非电泳法对血液模板DNA扩增的准确率达到了100％。为了把非电泳法应用于牛胚胎性别鉴定生产中，实验通过FSH法对雌晋南牛超数排卵，非手术法冲胚。胚胎评级后装管，使用乙二醇常规法冷冻后放入液氮罐中保存至使用。胚胎解冻后，使用玻璃针法取样，裂解。每一个取样胚胎的样本数为3-4份不等，取其中一份样本用SE引物扩增，如扩增有效，则其余的样本都用来做DYZ-1扩增；否则再取一份样本用SE扩增，直至扩增有效。以SE扩增结果为参照，验证DYZ-1的判定结果。血液模板DNA扩增结果与胚胎样本扩增结果都表明非电泳法在比SE引物电泳法节约1小时以上的同时，具有比SE更高的灵敏度，在模板浓度低至1-2拷贝时仍能达到90％的扩增有效率。本研究在我国首次成功地把非电泳PCR法应用到牛胚胎性别鉴定之中，减少了胚胎性别鉴定的操作步骤，并且与国外研究相比，进一步缩短了性别鉴定所需要的时间，从而使胚胎移植现场性别鉴定更加可行。在5对未见报道用于性别鉴定的微卫星DNA多态性引物中，筛选出2对具有性别特异性的引物，经过进一步的研究可能用于胚胎性别鉴定及品种鉴定。更多还原

【Abstract】 In order to develop a more efficient and applicable method of embryo sexing, 8 pairs of primers for sex chromosome DNA sequences were chosen and synthesized basing on some reported DNA sequences on XY chromosome. Blood samples of Jin-nan cattle and Hostem cattle were labeled to extract genome DNA, concentration of which was gauged. Both electrophoretic and nonelectrophoretic PCR sexing was used in the research and nonelectrophoretic PCR sexing both had reached an accuracy of 100% through a series of PCR condition system ameliorating.FSH was injected to cause superovulation of Jin-nan female cattle, followed by insemination procedure; then on day 7, embryos were flushed and collected for examining the applicability of nonelectrophoretic method in embryo sexing. After being graded, embryos were put in pipes for routine freezing in cryoprotectant with ethylene glycol. Pipes with embryos were stored in liquid nitrogen until been used. After thawing, embryos were sampled by needle and then there was a DNA releasing procedure right before PCR. Each embryo had 2-4 samples; one was amplified by SE first. In case of successful amplification, other samples were all amplified by DYZ-1; otherwise another sample was amplified until a successful amplification occurred. The result of DYZ-1 was compared with result of SE for the same embryo to confirm if it was right. Nonelectrophoretic PCR sexing seems more sensitive to low DNA concentration than SE, meanwhile it takes about 1 hour less than SE. It reached an efficiency of 90% even when the template concentration was only 1-2 copies.This study successfully applied nonelectrophoretic PCR to bovine embryo sexing for the very first time in our country, it also reduced a traditional step in embryo sexing. Furthermore, compared with abroad research, it further shortened the time for sexing, therefore made PCR sexing in field more applicable. In 5 pairs of microsatellite polymorphic DNA primers used in this research, 2 pairs being tested sex specific showed potential usability for bovine embryo sexing.更多还原