【作者】 赵建军；

【导师】 张兆旺；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 本试验根据兔SRY基因序列设计两对引物作为兔雄性特异性引物;根据兔APP基因序列设计3对常染色体引物作为内标引物。根据这些引物组合建立了兔早期胚胎性别鉴定的双重和巢式PCR反应体系。针对所建立的双重和巢式PCR体系进行了不同扩增条件的筛选和优化,并对巢式PCR结果进行了电泳法和非电泳法检测的比较。同时将兔SRY基因序列分别与人、牛、绵羊和小鼠SRY基因进行了同源性比较,并用本试验中所采用的SRY巢式引物分别对人、牛、绵羊和小鼠基因组DNA及冲卵液进行扩增,检测分析引物的特异性和分析胚胎性别鉴定过程中可能存在的污染因素。用24只公兔、9只母兔的血液和肝脏组织基因组DNA样品进行多重和巢式PCR扩增。结果表明,多重PCR扩增公兔基因组,可以扩增出282bp的SRY基因片断和173bp的APP基因片断,母兔只能扩增出173bp的APP基因片断,扩增灵敏度为100pg;巢式PCR两轮扩增后均进行电泳法和非电泳法检测扩增产物,结果表明公兔基因组DNA扩增出282bp的SRY基因片段(电泳法),在紫外分析仪下管内有明亮的荧光(非电泳法),母兔没有扩增产物也无荧光,扩增灵敏度为10pg。多重PCR扩增灵敏度明显低于巢式PCR扩增。但两种PCR扩增性别鉴定结果均与实际性别完全一致,准确率为100%。对12只母兔分别用进口FSH+hCG(8只)和PMSG+hCG(4只)进行超数排卵处理,FSH+hCG组(排卵点72.4±13.6,胚胎52.6±14.4)比PMSG+hCG组(排卵点32±6,胚胎21.8±4.2)获得了更好的超排效果(P<0.01)。将部分胚胎移植给4只同期化处理的母兔,怀孕2只,其中一只妊娠20天流产,另一只顺利产下8只小兔。部分超排胚胎用BIO-RAID显微操作仪和徒手切割法比较,结果表明,兔早期胚胎采用徒手切割法更灵活、简便,易于在生产现场进行操作,但此方法要达到精确控制取样细胞数量需要反复练习操作技巧。用本试验建立的PCR反应体系,对46枚超排胚胎性别鉴定结果表明,多重PCR只能对32细胞及以上胚胎细胞获得准确鉴定,不能作为兔胚胎PCR性别鉴定的应用;巢式PCR可以对8分胚(约4细胞)进行准确鉴定(同一胚胎扩增符合率100%),而且电泳法与非电泳法检测结果一致。兔SRY基因序列与其他几种哺乳动物(人、牛、绵羊、小鼠)同源性分析结果表明,兔SRY基因序列与牛、绵羊和小鼠SRY基因序列同源性在80%~82%之间,与人的为87%,且在本试验所设计的SRY基因引物,只对兔雄性特异,其他动物(人、牛、绵羊、小鼠)雄性DNA及冲卵液均不影响鉴定结果。本论文中建立的巢式PCR性别鉴定体系可以在实际生产中推广应用。更多还原

【Abstract】 In this study, two pairs of primers were designed according to the sex determining region of the Y (Sry) gene of rabbit as male specific primers, and three pairs of primers were designed according to the amyloid precursor protein gene (APP) of rabbit euchromosome as control primers. These primers were combined by twos in order to establish the multiplex PCR and nest PCR system used to sex rabbit embryo, and then, the system were chosen and optimized. The results of nest PCR were also compared using electrophoresis and non-electrophoresis. At the same time, the sex determining region of the Y (Sry) gene of rabbit was compared with the gene of human, bovine, sheep and mouse, and using the nest PCR system established in the study, the gene of human, bovine, sheep and mouse were amplified in order to detect the specific bands and the possible pollution during the sexing embryo.The genomic DNA from blood and liver of twenty-four male rabbits and nine female rabbits were used to multiplex PCR and nest PCR.The result shows that there are two specific bands—the 282bp of SRY gene and 173bp of APP gene when the male rabbits DNA were amplified using multiplex PCR, but there is only one specific band—173bp of APP gene when the female rabbits genomic DNA were amplified. The products of nest PCR were detected after reaction using electrophoresis and non-electrophoresis, the result shows that there are specific band about 282bp when the genomic DNA from male rabbits were used after electrophoresis and there is bright fluorescence in the PCR tubes when they were put in the ultraviolet radiation analyzer and the sensitivity is 10pg, but nothing can be seen when the genomic DNA of female were used. The sensitivity of multiplex PCR is obviously low than nest PCR, but the results of embryo sexing of these two kinds of PCR are consistent with practical sex and the accuracy was 100%.Twelve rabbits were supervulated using imported FSH+hCG(8) and PMSG+hCG(4),and the result shows that the ovulation points and embryos of FSH+hCG are 72.4±13.6 and 52.6±14.4 respectively.It’s remarkably better than PMSG+hCG—32±6 and 21.8±4.2 respectively. A part of embryos were transfered into four rabbits that were synchronizated, as a result, two rabbits gestated and one of them aborted after twenty days,the other layed eight small rabbits.The rest were splitted by BIO-RAID Micromanipulator or hand. The result is using hand cut the embryo is more flexible and simple, and it’s also easy to be used on the field, but the disadvantage is the number of cells that get from embryo aren’t exact.更多还原