早期胚胎玻璃化冷冻保存技术研究进展

本综述阐明了胚胎玻璃化冷冻原理，比较了玻璃化冷冻技术各种方法及其优缺点，分析了胚胎玻璃化冷冻的影响因素，展望了玻璃化冷冻技术的应用前景。     

蒙古牛线粒体DNA全基因组遗传多样性与起源研究

［目的］通过对蒙古牛线粒体DNA(mtDNA)的基因组进行测序,探究蒙古牛的mtDNA基因组遗传多样性与母系起源。［方法］采用DNA提取、三代测序及生物信息学方法。［结果］在36头蒙古牛mtDNA全基因组序列中,共检测到22种不同的单倍型,平均单倍型多样度(Hd)为0.970,平均核苷酸多样度(Pi)为0.00845,表明蒙古牛有丰富的母系遗传多样性。构建的IQ系统发育树发现,蒙古牛具有瘤牛和普通牛两个母系支系。［结论］蒙古牛有丰富的母系遗传多样性,拥有普通牛和瘤牛两个母系起源,以普通牛起源为主。     

The Impact of Reproductive Technologies on Stallion Mitochondrial Function

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen–thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.     

新型基因组编辑技术研究进展

基因组编辑技术是一种能精确靶向修饰生物基因组,实现对基因定点敲除和外源基因定点整合的技术。新出现的锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALEN)和规律性重复短回文序列簇与Cas9蛋白(CRISPRs/Cas9)系统3种新型的基因组编辑技术通过特异性结构识别靶位点,核酸酶发挥切割作用对靶位点进行定点编辑。3种新型基因编辑技术因具有高效准确、制作简单、耗时短等特点而在生命科学研究中得到广泛应用。论文对目前三种新型的基因组定点编辑技术的特点、结构原理、构建方法以及在传统生物模型、功能基因筛选、人类遗传病基因治疗等方面中的应用做一综述。     

Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

猪核线粒体假基因的分布特征研究

核线粒体假基因(NUMT)是线粒体DNA插入到核基因组中的DNA片段。目前,已经发现越来越多的真核生物基因组存在线粒体假基因现象。猪线粒体假基因在核基因组中的分布尚未见报道。研究通过猪线粒体基因组全序列与核基因组全序列进行比对,由BLAST程序鉴别出132个NUMT。结果表明:这些NUMT的大小在37～4 453bp,其中90%的NUMT长度处于40～1 000 bp的范围内,NUMT与相应的线粒体DNA片段之间的同源性数值范围为66%～100%。此外,鉴定出的NUMT序列几乎涵盖了包括线粒体DNA控制区在内的全部线粒体基因组,包括30个完整的线粒体基因,分布于猪的1、4、5、7、11、13、14、15、17号染色体上,近50%的NUMT定位在14号染色体上。     

小尾寒羊线粒体基因组遗传变异分析

本试验旨在利用基因组扫描策略和DNA混池测序技术分析小尾寒羊线粒体基因组遗传变异.结果表明,小尾寒羊线粒体基因组编码区共有95个变异位点,多肽编码区除ATPase8基因无突变外,其余12个多肽编码基因均有突变位点,变异位点总数为83个,错义突变为19个,分别为:T3544A、T4209C、C7501A、A8040G、G8265C、A9376G、C9975T、G10119A、G10938A、G11046A、G12571C、G13041A、C13576T、T13588C、C13777T、C13789T、T13837C、T13855C、A13876G.12S rRNA区域有3个变异位点,分别为:T281C、C291T、A538G;16S rRNA区域有5个变异位点,分别为:A1099T、T1112C、T2200C、C2444T、T2635C;tRNA区域有4个突变位点,分别为:tRNA-Tyr (G5295A)、tRNA-Lys (T7720G)、tRNA-His (C11607T)、tRNA-Ser (G11669A).由此可知,小尾寒羊线粒体基因组编码区多态性较丰富,该试验结果为核外遗传效应研究提供了分子生物学基础.     

A History of Equine Embryo Transfer and Related Technologies

The first successful equine embryo transfer was reported in 1972, 21 years after the first reported embryo transfer in cattle. Adaptations of embryo transfer and the various related technologies have generally been more rapid in the equine than in cattle, with the exceptions of superovulation, in vitro fertilization and cryopreservation. Recent progress has been achieved in all three of these areas. This paper presents a time line of various events in the history of equine embryo transfer and related technologies. Approximately 25,000 equine embryo transfers are being conducted per year, worldwide.     

秦川牛mtDNA全基因组遗传多样性与母系起源研究

[目的]从32头秦川牛全基因组中提取其mtDNA全基因组序列进行分析,以揭示秦川牛mtDNA基因组的遗传多样性与母系起源。[方法]采用mtDNA全基因组序列比对及生物信息学方法。[结果]在32头秦川牛mtDNA全基因组序列中,共检测到30种不同的单倍型,其平均单倍型多样度（Hd±SD）为0.996±0.009,其平均核苷酸多样度（π±SD）为0.0067±0.0010,表明秦川牛具有丰富的母系遗传多样性。构建的mtDNA全基因组系统发育树与单倍型网络图表明,32头秦川牛的mtDNA全基因组序列包括T2、T3、T4、I1共4个不同的支系,其中T2支系占21.88%,T3支系占40.625%,T4支系占9.375%,I1支系占28.125%。说明秦川牛具有普通牛和瘤牛两个支系,其中普通牛支系占71.88%,瘤牛支系占28.12%。[结论]秦川牛具有丰富的母系遗传多样性,有普通牛和瘤牛两个母系起源,但以普通牛起源为主。     

Genetic Diversity and Phylogenetic Relationships of the Complete Mitochondrial Genome in Chinese Indigenous Pig Breeds

In order to explore the genetic diversity and the classification status of the Chinese domestic pigs at the molecular level, the complete mitochondrial DNA (mtDNA) genomic sequences of 22 Chinese indigenous pig breeds from 6 types were downloaded from GenBank for the analysis using the bioinformatics method. The polymorphism of nucleic acid sequences was analyzed,the molecular phylogenetic tree based on D-loop sequences, Cytb gene, complete coding region mtDNA sequences and the Median-Joining network of the mtDNA D-loop haplotypes were constructed for 22 pig breeds from 6 types. The results showed that a total of 144 mutation sites among 22 pig breeds were detected, 22 haplotypes were discovered, which indicated that Chinese indigenous pig breeds had abundant genetic diversity. According to the complete mtDNA genomic sequences analysis, which suggested that the main mutations was transition, the transition/transversion ratio was greater than 2.0 and all mutations were in line with the neutral mutation. Our findings clearly demonstrated that there was a near genetic distance among 6 types, meanwhile, the shared haplotypes were found. The phylogenetic tree showed that Chinese indigenous pigs from 6 types were diverged from two ancestors.The results indicated that mtDNA D-loop and Cytb gene might serve as molecular markers for phylogenesis, origin and evolution.     

中国地方猪种线粒体基因组遗传多样性及其系统发育分析

为从分子水平上探究中国地方猪种遗传多样性和分类地位,本试验采用生物信息学方法比较了6个类型共22个中国地方猪种的线粒体基因组全序列,分析了其多态性,并构建了6个类型猪种线粒体D-loop区单倍型的网络中介图以及基于线粒体D-loop序列、Cytb基因、完整编码区序列的系统进化树。结果表明,6个类型22个猪种中共检测到了144个多态位点,22种单倍型,说明地方猪种具有丰富的遗传多样性;地方猪线粒体基因组序列中核苷酸变异以转换为主,且Ti/Tv大于转换/颠换比临界值（2.0）,变异位点均符合中性突变。6个类型猪种间遗传距离均较小,且有共享单倍型。系统进化树结果表明,6种类型地方猪种主要聚为两个支系。表明线粒体D-loop序列及Cytb基因均可作为研究种内系统发育、起源进化的分子标记。     

家蚕线粒体基因组1.8kb片段的克隆及序列分析

克隆并测定了家蚕 (Bombyxmori)线粒体基因组 1781bp的EcoRⅠ和HindⅢ双酶切片段序列 ,根据序列同源性比较 ,推测该DNA片段包括 :ND1基因、16SrRNA基因 3′端、tRNALeu(UAG)基因和一个尚待确定的tRNA基因。家蚕与果蝇ND1基因序列同源性约为 81 85 % ,16SrRNA基因 3′端序列同源性约为 74 5 %。在家蚕、果蝇、蜜蜂、蝗虫、卤虫和人 6个物种中 ,线粒体ND1编码的疏水性氨基酸比例较稳定 ,显示了ND1基因的保守性。对上述 6个物种ND1基因序列和 16SrRNA基因 3′端序列进行系统进化分析 ,构建了系统进化树。     

牛性控体外受精冷冻胚胎移植试验

从美国引进的荷斯坦牛性控体外受精冷冻胚胎95枚,以鲁西黄牛、南阳牛、西杂牛、荷斯坦牛为受体进行移植试验.结果显示,移植受体95头,妊娠44头,妊娠率46.3%,流产7头,产母犊37头,效果良好.     

海南白唇竹叶青线粒体基因组结构与特征分析

白唇竹叶青蛇(Trimeresurus albolabris)是亚洲竹叶青毒蛇之一,主要分布在东亚和东南亚。为研究海南岛产白唇竹叶青蛇线粒体基因,测定了采自海南文昌的个体线粒体基因组全序列,并与近缘物种进行比较。结果显示,其环状基因组全长17222 bp,包含13个蛋白质编码基因、22个转移RNA基因、2个核糖体RNA基因、2个控制区和1个L-链复制起源。总碱基组成为33.4%A,27.0%T,27.3%C,12.3%G,除8个tRNA基因和ND 6亚基基因在轻链上编码的外,其余基因均分布在重链上。此序列与其他产地的白唇竹叶青蛇相似度为99.61%,与其他竹叶青蛇相似度大于88%,与原矛头蝮蛇相似度大于85%。     

哺乳动物基因组印迹研究进展

基因组印迹即父源或母源等位基因的差异表达,是一种重要的表观遗传学机制。作者就哺乳动物基因组印迹的特征、作用机制及基因组印迹的生物学意义进行综述。     

胚胎植入研究概况

胚胎植入已成为生殖医学研究领域中尚未解决的热点问题。胚胎植入是一个非常复杂的过程,需要胚泡和子宫内膜之间的通讯和相互协同作用,被认为是调控雌性生育和发展避孕方法最理想的靶点和最关键的环节,与人类生殖健康关系极其密切。虽然最近在胚胎植入过程、影响因素、调控因子及细胞与分子机理等研究方面已取得了一些进展,但仍存在不少技术难题有待于进一步深入研究。文章简要综述了胚胎植入的概念、过程以及影响因素与调控因子,在此基础上介绍了胚胎植入的细胞与分子机理方面的研究概况,并对胚胎植入的研究和发展方向进行了探讨。     

基因组印记的研究进展

综述基因组印记的可能发生机制及人和鼠中常见的印记基因，论述了且印记作用的生物学意义。     

用高效液相色谱法检测鸡胚组织基因组DNA甲基化水平变化规律

本试验旨在优化用于基因组DNA甲基化水平检测的高效液相色谱法,并初步探索鸡胚发育过程中不同组织基因组DNA甲基化水平的变化规律。首先,试验从DNA酶解体系和高效液相色谱仪检测条件2个方面改进基因组DNA甲基化水平检测方法;然后,以孵化第8、11、14和17天的科宝500肉鸡胚胎为材料,利用改进的高效液相色谱法测定心脏、肝脏和肌肉的基因组DNA甲基化水平。结果表明:3种组织的基因组DNA甲基化水平均随胚龄增加而升高,且鸡胚发育后期(孵化第17天)肝脏的甲基化水平显著高于心脏和肌肉(P0.05);各胚龄心脏和肌肉的基因组DNA甲基化水平均差异不显著(P0.05)。结果提示,优化后的高效液相色谱法为快速、准确地获取组织基因组DNA甲基化水平奠定了基础;随着鸡胚发育的进行,基因组DNA甲基化水平逐渐升高,且在胚胎发育后期肝脏甲基化水平较高。     

一种获得重组传染性法氏囊病病毒的新方法

采用 RT- PCR技术 ,从传染性法氏囊病病毒 (IBDV)细胞适应株 GZ911中扩增了 A片段的 2个片段 GA5和 GA3,从中国分离的 IBDV超强毒株 L X中扩增了 B片段的 2个片段 L B5和 L B3。将 GA5和 GA3克隆到质粒p Bss K的 Eco R / Kpn 位点 ,将 L B5和 L B3克隆到 p Bss K的 Eco R / Xba 位点 ,获得携带 GZ911完整 A片段基因的质粒 GA- p Bss K和携带 L X完整 B片段基因的质粒 L B- p Bss K。然后将 GZ911的 A片段 c DNA和 L X的 B片段c DNA分别插入带有巨细胞病毒立即早期加强子 /启动子的质粒 p AL TER- MAX中 ,获得重组质粒 GA- p AL TER和L B- p AL TER。用 GA- p AL TER和 L B- p AL TER共转染鸡胚成纤维细胞 (CEF) ,获得具有感染活性的重组 IBDV,命名为 r IBDV- GZ L X。该方法的建立为在体外对 IBDV的基因组进行遗传操作 ,进而彻底了解 IBDV基因的结构与功能的关系打下了基础。