西藏小型猪胚胎成纤维细胞的分离培养及性别鉴定

目的探索和建立西藏小型猪胚胎成纤维细胞体外分离培养及性别鉴定的技术方法。方法取35d的西藏小型猪胚胎分离胚胎成纤维细胞,进行体外原代培养及传代培养,观察细胞成纤维细胞的形态和生长状况。根据猪Y染色体上性别决定基因SRY设计引物进行性别鉴定,同时以β珠蛋白作为内参基因,建立PCR反应体系鉴别胚胎的性别。结果西藏小型猪胚胎成纤维体外分离后,呈贴壁生长,快速增殖。PCR性别鉴定结果表明雄性胚胎细胞可扩增出一特异性SRY基因条带,而雌性则没有。该法可快速鉴定胚胎的性别,可用于体细胞克隆动物早期性别鉴定。结论研究结果表明利用胶原酶消化法所获得的西藏小型猪胚胎成纤维细胞可在体外稳定培养并传代,利用PCR鉴定猪胎儿性别具有简单、快速、准确的特点,可应用于克隆猪研究中体细胞系的早期性别鉴定。     

绵羊胚胎性别鉴定试剂盒的研究

根据绵羊Y-染色体的特异序列和常染色序列分别设计了确定公羊Y-染色体特异序列的3对特异性引物和内标基因的4对特异性引物。单重PCR扩增绵羊基因组DNA,筛选出了3对Y-染色体特异引物和3对羊DNA特异内标引物。将不同的绵羊Y-染色体特异引物与内标引物组合,利用多重PCR扩增绵羊基因组DNA,筛选出了1个可用于羊早期胚胎性别鉴定的PCR引物组合:A0/C1。按照最优PCR扩增DNA条件配制了绵羊PCR性别鉴定试剂盒并成功应用于绵羊血液、已知性别的绵羊成纤维细胞和胚胎,表明本研究建立的体系完全可用于绵羊早期的胚胎性别鉴定。     

CHD基因与非平胸鸟类性别鉴定

自1995年CHD基因被应用于鸟类性别的分子鉴定以来,非平胸类鸟类性别鉴定问题正逐步被解决,CHD基因已经成为非平胸鸟类性别鉴定最重要的分子标记。阐述和分析了CHD基因在扩增目标及引物设计、取样范围及辅助技术和所涉足的科研领域等方面的发展及现状。期望随着CHD基因研究的深入,建立起完善的基于CHD基因的鸟类性别鉴定体系,通过更广泛的应用,促使涉及鸟类性别鉴定的科学研究向着更深、更广的方向发展。     

奶牛ZFY、ZFX基因片段的克隆及测序

奶牛的早期胚胎性别鉴定是奶牛胚胎分割及移植技术产生较大经济效益的前提,用PCR技术进行早期胚胎性别鉴定具有准确、快速、灵敏度高的特点.利用人和鼠的性别分化相关的DNA序列ZFY、ZFX基因序列设计的引物对公牛和母牛的染色体DNA、PCR扩增,将扩增产物定向克隆到pUC118上,获得ZFY、ZFX转化子,并测定了ZFY、ZFX基因的序列,发现两者同源性达88.2%,以此为基础可设计引物和探针,以PCR方法进行高灵敏度的奶牛性别鉴定.     

两温度梯度多重PCR鉴别牛早期胚胎性别的技术

稳定、可靠和快速的牛胚胎性别鉴定方法在生产应用中具有重要意义.通过两温度梯度PCR方法对牛基因组、克隆胚胎、胚胎样品进行性别鉴别实验研究,建立了稳定、简便、快速的牛早期胚胎性别鉴别两温度梯度PCR方法,鉴定时间仅为57分钟.采用两温度梯度PCR方法对30枚奶牛胚胎进行了早期性别鉴别,并将鉴别的15枚胚胎(11枚为雌性,4枚为雄性)移植到同期处理的15头受体母牛体内.60天后妊娠检查,有7个受体成功受孕,5头受体怀孕晚期流产,流产犊牛全部为母犊.结果产下1公1母两头犊牛,流产个体与出生个体的性别与PCR鉴别结果完全相符.     

两温度梯度多重PCR鉴别牛早期胚胎性别的技术研究

稳定、可靠和快速的牛胚胎性别鉴定方法在生产应用中具有重要意义。通过两温度梯度PCR方法对牛基因组、克隆胚胎、胚胎样品进行性别鉴别实验研究,建立了稳定、简便、快速的牛早期胚胎性别鉴别两温度梯度PCR方法,鉴定时间仅为57分钟。采用两温度梯度PCR方法对30枚奶牛胚胎进行了早期性别鉴别,并将鉴别的15枚胚胎（11枚为雌性,4枚为雄性）移植到同期处理的15头受体母牛体内。60天后妊娠检查,有7个受体成功受孕,5头受体怀孕晚期流产,流产犊牛全部为母犊。结果产下1公1母两头犊牛,流产个体与出生个体的性别与PCR鉴别结果完全相符。      

哲罗鲑性别特异性标记筛选

研究通过比对哲罗鲑Hucho taimen (Pallas)基因组草图与虹鳟(Oncorhynchus mykiss)Y染色体序列, 获得哲罗鲑性别相关的候选序列, 并设计3对PCR扩增引物, 以此筛选哲罗鲑性别特异性标记。初步筛选结果显示, 在设计的3对引物中, 引物ST2在雌鱼中无扩增条带, 在雄鱼中有153 bp的扩增条带, 可作为哲罗鲑雄性特异性候选标记。为了消除样本降解及失误等因素导致的条带缺失, 研究以12S rRNA为参照, 采用双重PCR法, 在12S rRNA引物扩增出条带的前提下, 用ST2引物条带的有无来判断性别, 雌鱼为单带, 无ST2引物条带; 雄鱼为双带, 有ST2引物条带。同时为了验证本方法的可靠性, 对已知性别的哲罗鲑48尾雌、雄样本进行了检测, 结果显示该方法遗传性别鉴别准确率为100%。用此标记筛选哲罗鲑雌、雄鱼简单易行, 为哲罗鲑遗传学研究、单性养殖和性别控制育种等研究奠定了基础。     

人成骨蛋白成熟肽基因在毕赤酵母中的克隆及其序列分析

采用PCR方法,根据献报道的人成骨蛋白-1（OP-1）成熟肽基因序列,设计并合一对引物,从含人成骨蛋白基因的质粒中扩增获得大小的420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,继之以pPIC3.5k为表达载体构建重组表达质粒,并经PCR及酶切鉴定。     

戴冕鹤性别的分子鉴定

戴冕鹤Grus nigricllis为单态性鸟,很难通过外观和形态区分性别.本文采用非损伤性采样羽毛提取DNA,利用P2-P8引物对CHD基因进行特异性扩增,分别用HaeIII和Asp700I处理PCR产物,然后经过限制性片段长度多态性分析,HaeIII处理后的PCR产物,雄性出现2条带,雌性出现3条带;Asp700I处理后的PCR产物,雄性出现1条带,雌性出现3条带,表明可以准确鉴定戴冕鹤的性别.本文建立了从非损伤采样的羽毛提取DNA,利用PCR和限制性片段长度多态性准确鉴定戴冕鹤性别的方法,为人工繁殖戴冕鹤时合理配置雌雄比例、提高繁殖率奠定了基础.     

鱼类性别异形和性别决定的遗传基础及其生物技术操控

鱼类养殖对世界食品特别是动物蛋白的持续供给做出了至关重要的贡献.鱼类生殖对策的多样性,特别是单性雌核生殖方式的利用已开创了鱼类遗传育种的典型范例.不少鱼类在生长和个体大小等重要经济性状上表现出显著的性别异形.性别特异分子标记的开发和性别控制生物技术的发展为增加鱼产量及其经济价值提供了重要的技术途径.随着基因组学和分子遗传学技术的迅速发展,鱼类性别异形的遗传基础逐步被揭示,鱼类性别决定机制及其性别决定相关基因的鉴定已经取得了重大进展.本文对此进行了概述,以期为该领域的深入研究提供一些方向性和目标性思考.     

哺乳动物性别决定和性反转

目前已知SRY仅是涉及性别决定过程的基因之一.近年来又发现和克隆了许多可能参与性腺分化与发育的基因,如副中肾抑制基因MIS,也称抗副中肾激素基因AMH;SRY相关基因SOX9;编码甾类因子的基因SFI;X-连锁的DAX基因;Wilm′s肿瘤抑制基因WTI;以及X-连锁的剂量敏感基因DSS等,并新建立了性别决定的Z-基因模型,DSS-基因模型和Jimenez等的模型,较合理地解释了哺乳动物性别决定的分子机理和以前难以解释的各种奇特的性反转现象,使性别决定的研究取得了长足的进展,但仍有一些悬而未决的问题有待于进一步探索.     

Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

家禽的性别鉴定方法

现代家禽生产,祖代鸡通过释肛鉴定性别,父母代鸡以羽速（快慢羽）判定公母,而商品代鸡则以羽色自别雌雄。常规性别鉴定方法耗资费力,且慢羽基因（K）与内源病毒基因（ev-21）紧密连锁,导致慢羽鸡群免疫反应降低,成活率和产蛋性能下降。以鉴定W染色体上性连锁DNA序列或基因为基础,从分子水平上鉴定家禽性别的研究有望填补家禽性别鉴定空白。     

番木瓜性别决定及其鉴定研究新进展

番木瓜有3种基本性别类型,性别遗传较为复杂.就其植株的多型性表现、性别决定及其鉴定研究、连锁遗传图的构建、分子标记辅助选择技术和花器的发育等方面的研究进展进行了综述,并对番木瓜性别鉴定的应用前景做了展望.     

Sex identification by alternative polymerase chain reaction methods in falconiformes

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.     

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.     

Sex Determination by Sex Chromosomes in Dioecious Plants

Abstract: Sex chromosomes have been reported in several dioecious plants. The most general system of sex determination with sex chromosomes is the XY system, in which males are the heterogametic sex and females are homogametic. Genetic systems in sex determination are divided into two classes including an X chromosome counting system and an active Y chromosome system. Dioecious plants have unisexual flowers, which have stamens or pistils. The development of unisexual flowers is caused by the suppression of opposite sex primordia. The expression of floral organ identity genes is different between male and female flower primordia. However, these floral organ identity genes show no evidence of sex chromosome linkage. The Y chromosome of Rumex acetosa contains Y chromosome-specific repetitive sequences, whereas the Y chromosome of Silene latifolia has not accumulated chromosome-specific repetitive sequences. The different degree of Y chromosome degeneration may reflect on evolutionary time since the origination of dioecy. The Y chromosome of S. latifolia functions in suppression of female development and initiation and completion of anther development. Analyses of mutants suggested that female suppressor and stamen promoter genes are localized on the Y chromosome. Recently, some sex chromosome-linked genes were isolated from flower buds of S. latifolia.     

Species and sex identification of Formosa landlocked salmon using loop-mediated isothermal amplification

Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts.     

Sex determining of cat embryo and some feline species

Sex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, beta-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.