奶牛性别鉴定的研究与应用

选取牛雄性性别决定基因SRY (sex region of Y chromosome),根据基因序列设计特异引物,应用PCR技术对5头荷斯坦奶牛DNA样品进行扩增,鉴定其性别;并对设计的引物灵敏度进行检测;对已有的公、母各20头荷斯坦奶牛DNA样品进行PCR盲检,获取奶牛高灵敏度特异性引物,用于奶牛性别鉴定。结果表明,4头公牛DNA样品可以扩增出目标条带(66 bp),1头母牛DNA样品无法扩增出条带,阴性对照扩增无条带;最佳引物灵敏度为1.6 pg/μL,可以很好地满足性别鉴定需要。40头个体中,20头个体DNA样品可以扩增出条带,其余20头个体DNA样品无法扩增出条带,检测结果与实际性别对比准确率为100%。试验结果表明,设计的引物灵敏度比较好,能够满足奶牛性别鉴定的需要。     

PCR法鉴定雏鸽性别的初步研究

雏鸽的雌雄外貌形态差别甚微,极难区别。为有效准确地鉴别雏鸽的性别,淘汰非目的性别,从而降低饲养成本,提高经济效益。本研究通过采用PCR方法,选定特殊的性别CHD1基因及鉴定引物,克隆出雏鸽特定性别基因序列鉴定雏鸽的雌雄。结果表明:在雏鸽雌性样本中扩增出1条带(777 bp),而雄性中未见扩增带,显示出CHD1基因的性别特异性,PCR扩增性别鉴定结果与样本解剖后对性腺观察的形态学鉴定结果也完全一致。在87份样品中,阳性克隆结果为25,阴性克隆结果为62,故87只雏鸽中雌鸽25只,雄鸽62只,其准确率达100%。     

牙釉质基因鉴定山羊性别的研究

根据牙釉质(amelogenin,AMEL)同源基因在山羊X、Y染色体上存在不同程度碱基缺失的特点,设计1对引物,对山羊血液基因组DNA混合样进行PCR扩增,将PCR产物纯化、克隆并测序;然后对46个山羊血液DNA样本(♀:22个,♂:24个)进行性别鉴定。结果显示,雌性山羊样品经扩增只产生一条非特异性条带,雄性山羊样品产生一条非特异性条带和一条特异性条带;非特异性片段长度为349 bp,特异性片段为289 bp;46个山羊血液DNA样本鉴定结果与实际性别对比,雌性准确率为100%(22/22),雄性准确率为100%(24/24)。     

鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

牙釉质基因鉴定麇鹿性别

性别鉴定是调查野生种群雌雄性比的重要方法,对野生动物种群管理具有重要意义。而牙釉质(AMEL)基因在性染色体上具有较高的保守性,在鉴定动物性别方面得到了应用,本次试验采用北京麋鹿生态实验中心的15头糜鹿组织样本,其中11个来自雄性麇鹿鹿茸,4个来自雌性糜鹿静脉血液。对所取样本分别进行基因组DNA提取、AMEL基因片段PCR扩增、纯化、测序。得到了AMEL基因鉴定和实际雌雄性别个数差异不显著(P>0.05)。结果表明:雌性麋鹿产生1条322 bpX带和1条N带,雄性麋鹿则产生322 bpX带和277 bpY带以及1条N带,雄鹿(10/10)和雌鹿(4/4)性别鉴定结果分别都与实际性别符合,所以,使用鹿茸角和血液样本进行AMEL.基因扩增电泳分析可以对糜鹿性别鉴定。     

Sex Identification of the Black Swan (Cygnus atratus) using the Locus-specific PCR and Implications for its Reproduction

Over the last 4-5 years the small captive population of black swans (Cygnus atratus) has consistently failed to reproduce at the Chengdu Research Base of Giant Panda Breeding. The probable cause was hypothesized to be an abnormal sex distribution of the population. The black swan is an example of a sexually monomorphic species. The locus-specific polymerase chain reaction (PCR) approach based on the chromo-helicase-DNA-binding 1 (CHD1) gene, was adopted for the sex determination of the black swans. For this purpose, F1, F2 and R primers were designed using the primerselect software for amplification of the CHD1 gene region. DNA agarose gel electrophoresis showed that the female control displayed two bands, whereas only a single band was found in the male control. Sequence analyses of all seven unknown sex black swans demonstrated the sex-specific DNA band for female. Therefore, it was inferred that all the individuals of the black swan population are females, which has resulted in unfertilized eggs and reproduction failure. This method can be extended to the sexing of other monomorphic avian species and will assist in the design of breeding projects.     

PCR扩增妊娠奶牛牙釉蛋白基因进行胚胎性别鉴定的研究

牙釉蛋白（amelogenin,简写为AML）基因是牙齿发育过程中丰富表达的多拷贝基因,AML基因的同源基因分别定位在XY染色体上。本试验利用x—Y同源的牙釉蛋白基因序列设计一对特异性引物（牛AML基因序列的扩增片段长度：雌性为只有467bp的特异性扩增片段：雄性为同时具有341bp和467bp的两条特异性扩增片段）,应用PCR技术同时扩增X和Y染色体上的特异性片段,扩增产物用PAGE电泳分离技术,经硝酸银溶液染色及扫描分析进行妊娠奶牛早期胚胎的性别鉴定。结果显示,从X染色体上扩增出467bp的片段．从Y染色体上扩增出341bp的特异性片段。由此可知,PCR扩增妊娠奶牛牙釉蛋白基因可以进行胚胎的性别鉴定。     

应用Sry-PCR扩增鉴定狍(Capreolus capreolus)的性别

根据人的SRY基因核心序列设计合成1对引物C1、C2，应用PCR技术对野生狍(Capreolus capreolus)Sry基因(哺乳动物Y染色体DNA雄性特异区)进行扩增．结果在野生狍雄性样本扩增出1条带(221bp)．而在雌性样本未见扩增带．显示了Sry基因的性别特异性。应用这1对引物对42个未知性别狍肌肉组织样本进行了性别鉴定．结果雄性22个，雌性20个。对Sry-PCR产物克隆测序，得到185bp的Sry基因部分核苷酸序列。本试验为狍种群性别比率及其种群动态变化机制研究提供了资料。     

Development of Y‐chromosome‐Specific SCAR Markers Conserved in Taurine,Zebu and Bubaline Cattle

Sex pre‐selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR‐specific primers are derived from the few single‐copy Y‐chromosome‐specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male‐specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male‐specific markers (OPC16₃₂₃ and OPF10₁₁₆₈) by means of RAPD. These markers were successfully converted into SCARs (OPC16₇₂₆ and OPF10₉₈₄) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16₃₂₃ marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16₃₂₃ is also highly similar to a bubaline Y‐chromosome‐specific sequence. The primers derived from the two Y‐chromosome‐specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.     

牛早期胚胎性别鉴定PCR反应体系优化的研究

根据牛SRY基因序列设计合成2对巢式PCR引物作为性别鉴定引物,根据牛-珠蛋白基因序列设计了一对引物作为内标引物建立了牛胚胎性别鉴定的PCR反应体系。公牛可以扩增出187bp的SRY基因片段和255 bp的-珠蛋白基因片段,母牛只能扩增出255 bp的-珠蛋白基因片段。由于巢式PCR只需3～8个细胞就可以在紫外灯下看到扩增结果,而常规PCR则需要较多的细胞,所以胚胎性别鉴定时使用巢式PCR效果更好。     

秦岭羚牛性别鉴定的初步研究

根据牛的Sry基因核心序列设计合成1对引物F1、R1。应用PCR技术对羚牛(TAKIN)Sry基因进行扩增,结果在羚牛雄性样本扩增出1条带(230 bp),而在雌性样本未见扩增带,显示了Sry基因的性别特异性,应用这1对引物对60只未知性别的羚牛组织样本进行了性别鉴定,结果雄性14个,雌性46个。该试验为羚牛种群性别比率及其种群动态变化机制研究提供了资料。     

Sry基因和Y染色体重复序列在牛早期胚胎性别鉴定中应用效果的比较

本试验以牛Sty基因和Y染色体重复序列作为雄性特异性基因,分别设计引物,建立多重巢式PCR体系,比较二者在牛早期胚胎性别鉴定中的应用效果。试验结果表明,当扩增体系中的模板量为一个胚胎细胞的DNA量时,以Y染色体重复序列构建的扩增体系比Sry基因具有更高的灵敏度和稳定性,更适合用于牛早期胚胎性别鉴定。     

昆明小鼠不同性别早期胚胎体外培养发育潜力观察

自主设计2对引物,提取已知性别小鼠肝细胞DNA,扩增雄性小鼠特有的Sry基因及雌、雄小鼠共有的ZFX基因,建立双重PCR性别鉴定方法.提取小鼠早期8细胞胚胎单卵裂球、双卵裂球DNA,分别进行双重PCR性别鉴定,选择出早期8细胞胚胎的适宜模板量;将已测性别的8细胞胚胎按照雌、雄性别分开培养,观察统计雌、雄8细胞胚胎发育至囊胚的比率.试验结果显示,与单个卵裂球DNA模板量相比,双卵裂球的模板量检出率高,两者之间差异显著(75.00%Vs 93.33%,P<0.05);经过性别鉴定的雄性8细胞胚胎比雌性8细胞胚胎发育至囊胚的比率高,两者之间差异显著(53.33%Vs 33.33%,P<0.05).结果表明,采用双重PCR性别鉴定方法,以双卵裂球的DNA量为模板能够有效的对小鼠早期8细胞胚胎进行性别鉴定;雄性胚胎在体外培养条件下比雌性胚胎具有更好的发育潜力.     

Study on Quick Sex Identification Method in Pigeons by CHD Gene

In this experiment, the primer pairs were designed, and polymerase chain reaction (PCR) technique to amplify the segments of the partial sex linked gene chromo-helicase-DNA-binding (CHD) to identificate pigeon's sex using feather pulps and blood.We found that two bands were demonstrated in all female egg-laying pigeons, and the sizes of them were 400 and 700 bp, respectively;In all male egg-laying pigeons, only one band appeared with length of 700 bp.100 unknown sex samples were successfully identified (52 males and 48 females).Based on the checking results between molecular electrophoresis of pigeons and the truly sex of maturity pigeons, we detected CHD gene in 2 928 early age pigeons using PCR amplification and electrophoresis in agarose gel.The PCR amplification results showed 1 517 males and 1 411 females.     

通过CHD基因快速鉴定鸽子性别方法的研究

本试验采用PCR方法,依据性别基因连锁理论,通过羽毛和血液,不提取鸽子基因组DNA,直接利用特异性引物对其CHD基因的特异性片段进行扩增,琼脂糖凝胶电泳检测,结果显示雄鸽仅有1条带,在700bp左右,雌鸽有2条带,分别在400与700bp左右;利用该检测方法对100羽性别未知的雏鸽进行了性别鉴定,结果为雌鸽48羽、雄鸽52羽,并通过剖解雄鸽的生殖器官进行验证,准确率达到100%。用建立的方法测定了2928羽雏鸽用于蛋鸽留种试验,结果表明,雌鸽为1411羽、雄鸽为1517羽。     

Isolation of DNA from embryo and chorio-allantoic membranes and sexing by PCR in Japanese quail

1. The aim of this study was to evaluate the amount and quality of genomic DNA isolated from embryos and their chorioallantoic membranes (CAM) and to investigate the utility of different PCR methods for identifying the sex of Japanese quail embryos.

2. Fertilised eggs were incubated at 37°C for 120?h and DNA was isolated from samples of embryos and CAM. Target regions of the CHD-W gene or XhoI repeat sequence were amplified by PCR and examined on agarose gels or by using a capillary electrophoresis system.

3. DNA samples from embryos had significantly higher OD260 values than those from CAM, while OD260/280 values were not significantly different between embryos and CAM.

4. Gender identification was not possible by PCR amplification of the CHD gene region or XhoI repeat sequences examined on agarose gels, whereas males and females of Japanese quail were distinguishable when PCR products of the CHD gene were separated by capillary electrophoresis.

5. The results showed that high molecular weight DNA could be isolated from both embryo and CAM of Japanese quail. DNA isolated from CAM could be used for molecular genetic studies where embryos would be used for other purposes, such as in situ hybridisation. A capillary electrophoresis system could be used for identifying the gender of Japanese quail embryos.     

狍Sry基因PCR扩增的初步研究

为研究狍性别决定机制,采用聚合酶链式反应(PCR)技术对野生狍(Capreoluscapreolus)(n♀=2,n♂=2)Sry基因(哺乳动物Y染色体DNA雄性特异区)进行扩增。根据人的SRY基因核心序列设计合成了1对引物1,2。结果在野生狍雄性个体中扩增出1条带,大小约为220bp,而在雌性个体中未见扩增带,表明了Sry基因的性别特异性,为探讨野生狍的性别决定机制提供了分子资料。     

扩增SRY基因鉴定牛、羊胚胎性别的初步研究

本研究利用本试验室设计的SRY基因3对常规PCR引物扩增牛SRY基因和羊的SRY基因,结果表明:第2对引物扩增出公牛和公羊基因组的DNA 750 bp左右清晰条带,而母牛、母羊基因组DNA无此扩增条带,说明第2对引物可以应用于牛和羊的早期胚胎性别鉴定;     

兔早期胚胎PCR性别鉴定体系的建立

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1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。     

肠炎沙门菌LAMP检测方法的建立

为建立一种能快速检测肠炎沙门菌(SE)的方法,本研究根据基因库中SE种特异性基因(sdfⅠ)的保守序列,设计一套特异性环介导等温扩增(LAMP)引物,建立了SE的LAMP可视化检测方法。该方法的敏感性可达100fg DNA,高于常规PCR方法100倍;全部反应可在1h内完成;可通过肉眼观察颜色直接判定结果;对其他常见病原体的检测结果均为阴性。结果表明建立的LAMP方法简便、快速、灵敏、特异,可用于SE感染的快速检测。