【作者】 洪桂云；

【导师】 陈宏权； 章孝荣；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 本实验建立了用聚合酶链式反应（Polymerase Chain Reaction，PCR）技术体外扩增哺乳动物SRY特异序列鉴别哺乳动物胚胎性别的方法。依据牛、山羊、兔、猪等哺乳动物Sry基因高度同源性设计一对SRY引物，两条引物均为22个碱基，对公牛的SRY特异的163bp序列进行体外扩增。从公、母牛的组织或血液提取基因组DNA，用作PCR扩增基因组DNA的模板，同时在立体显微镜下收集羊-兔异种克隆胚胎，先用0.145mol／L NaCl冲洗2次，再移入20μL胚胎处理液中（10mmol／L Tris-HCl pH8.3，50mmol／L KCl，2mmol／L MgCl₂，0.45％NP-40，0.45％Tween-20，0.1μg／μL蛋白酶K），将羊-兔异种克隆胚胎及其处理液在55℃水浴作用60min，然后将此胚胎溶解物直接作为PCR扩增胚胎DNA的模板。按照析因设计，建立最适PCR扩增牛基因组DNA和羊-兔异种克隆胚胎DNA条件：反应体系中加入无MgCl₂的缓冲液和3×2×3因子组合浓度的SRY引物（每种引物浓度分别为：0.1μmol／L，0.3μmol／L，0.5μmol／L），dNTPs（浓度分别为：0.1mmol／L，0.2mmol／L）和MgCl₂（浓度分别为：0.75mmol／L，1.5mmol／L，2.25mmol／L）；模板为公牛基因组DNA（2μL约100ng）或羊-兔异种克隆胚胎溶解物20μL；2IU Taq DNA聚合酶。反应体系为50μL。反应在微机控制Flexigene型PCR仪上进行，反应参数为：95℃预变性5min后，按95℃变性1min，58℃退火1min，72℃延伸1min，进行30或35个循环后，72℃延伸9min，于4℃结束反应。扩增产物在2％琼脂糖凝胶上电泳，用PCR Marker作分子量标准，紫外灯下观察，最适PCR反应条件为扩增出最强的SRY产物带而无非特异性扩增带出现时的反应条件。结果表明：SRY引物扩增牛基因组DNA时，PCR最适条件为：0.1μmol／L SRY引物，0.1mmol／L dNTPs和1.5mmol／LMg²⁺，退火温度为58℃，循环次数30次；SRY引物扩增胚胎DNA时，PCR扩增最适条件为：0.5μmol／L SRY引物，0.1mmol／L dNTPs和1.5mmol／L Mg²⁺，退火温度为58℃，循环次数35次。 采用PCR扩增牛基因组DNA最适条件分别扩增了黑白花奶牛9头（6，3）、皖北黄牛11头（3，8）、波尔三羊3只（2，1）、兔4只（1，3）和猪7头（3，4）的基因组DNA。所有雄性样品中均出现清晰可见的特异DNA扩增带，而雌性样品和空白对照中无扩增片段出现。同时采用PCR扩增羊-兔异种克隆胚胎DNA最适条件扩增了15个羊-兔异种克隆胚胎DNA（4个胚胎的供体为公羊，11个胚胎的供体为母羊），所有以公羊为供体的羊-兔异种克隆胚胎样品均出现清晰可见的特异DNA扩增条带，而以母羊为供体的羊－兔异种克隆胚胎样品和空白对照中无扩增片段出现。结果表明PCR法可以用来鉴别哺乳动物的性别以及鉴别哺乳动物胚胎的性别，而且用此法鉴定哺乳动物早期胚胎性别具有相对简单、快速、费用低和准确率高等优点。 本实验还采用设计的性别鉴定引物，按照最优PCR扩增胚胎DNA条件配制了PCR性别鉴定试剂盒。经实验该性别鉴定试剂盒使用准确性高，可降低污染，方法简便易行，成本低，在生产实践中有很大的实用价值。更多还原

【Abstract】 A Polymerase Chain Reaction(PCR)-based method for sex determination of mammals embryos by amplification of mammals SRY-specific sequence was established. A pair of primers of 22 bases in length were designed based on regions of sequence homology among cattle, goats, rabbit and pigs to amplify a 163-bp region of cattle Sry. Genomic DNA extracted from male and female cattle, were directly used as template of PCR. The goat-rabbit interspecies clone embryos were collected washed twice with 0.145 mmol/L NaCl, then were transferred to 20uL lysate buffer(10 mmol/L Tris-HCl pH8.3, 50 mmol/L KC1, 2 mmol/L MgCl2, 0.45% NP-40, 0.45% Tween-20, 0.1μg/μL Proteinase K), and incubated for 60 min at 55℃. The lysates were directly used as embryos DNA template of PCR. Optimal PCR conditions developed using the factorial protocol for SRY primers using genomic cattle DNA and goat-rabbit interspecies clone embryos DNA were as follows: 100 ng of genomic DNA from a male cattle or 20 μL goat-rabbit interspecies clone embryos lysate were amplified, with 2 IU Taq DNA polymerase, supplied MgCb-free buffer and a 3×2×3 factorial combination of concentrations of SRY primers (0.1 μmol/L, 0.3 μmol/L, or 0.5μmol/L), dNTPs ( 0.1 mmol/L, or 0.2 mmol/L each), and MgCl2( 0.75 mmol/L, 1.5 mmol/L, or 2.25 mmol/L). The total volumes of PCR were 50μL. All reactions were carried out in Flexigene. After initial denaturation at 95℃ for 5 min, the amplification was performed by using 30 or 35 cycles of denaturation at 95℃ for 1 min, annealing at 58℃ for 1 min, and extension at 72℃ for 1 min. A final period of extension was carried out for 9 min and final holding at 4℃. The PCR products were resolved after electrophoresis in 2% agarose gel with ethidium bromide. The PCR products were visualized while the gel was exposed to ultraviolet light. Optimal conditions were defined as those that generated the strongest PCR products (thebrightest band) for SRY without non-specific banding. The results showed that optimal PCR condition for SRY primers using genomic cattle DNA as template were as following 0.1μmol/L SRY primers, 0.1 mmol/L dNTPs, 1.5 mmol/L MgCl2, annealing at 58℃ and 30 cycles; while optimal PCR conditions for SRY using embryo lysate as template were 0.5μmol/L SRY primers, 0.1 mmol/L dNTPs, 1.5 mmol/L MgCl2, annealing at 58℃ and 35 cycles.Using optimized genomic DNA PCR conditions for SRY primers, genomic DNA from nine black-white cattle, eleven Wuanbei yellow cattle, three Boer goats, four rabbit, and seven pigs were amplificated, The results showed that the sexing method was verified. All male samples had bands representing SRY-specific, whereas all female samples and control samples had no band. Using optimized embryo DNA PCR conditions for SRY primers, fifteen goat-rabbit interspecies clone embryos were amplificated, the sexing method was verified too. All male goat-rabbit interspecies clone embryos samples had bands representing SRY-specific, while female goat-rabbit interspecies clone embryos and control samples had no band. The result showed that PCR amplification of SRY-specific DNA sequences may be used to determine mammal’s sex and mammal embryonic sex. The advantages of PCR-based embryo sexing are relatively simple, rapid, and inexpensive, but maintaining high degrees of accuracy.On basis of the optimal embryo DNA PCR conditions for SRY primers, the PCR sex determination kit was made, which could be used in practice and has adventages of convenience, less cost, more clear, and accurate.更多还原