微卫星DNA在动物亲子鉴定中的应用及研究进展

本文概述了微卫星DNA进行亲子鉴定的基本原理与方法,将其用于亲子鉴定的优点以及研究进展,最后对未来的应用进行了展望。     

基于COI基因构建西北太平洋常见鱼类DNA条形码参考数据库

西北太平洋以其独特的地理位置和复杂的海洋环境,孕育了极为丰富的渔业资源,成为全球海洋生物多样性保护和渔业资源管理的热点区域。为了更有效地进行鱼类种类识别和多样性的调查,本研究建立了西北太平洋常见鱼类DNA条形码本地数据库。基于线粒体COI基因,对2023年6-8月在西北太平洋海域所采集的307份样品进行扩增和测序,共获得7目13科20属25种鱼类的COI基因序列。77.96%的COI序列在公共数据库中都能比对到高相似度序列。种间平均遗传距离为0.233,种内平均遗传距离为0.003,种间遗传距离是种内遗传距离的77.67倍,且能够形成明显的条形码间隙,不存在物种区分困难的现象。基于COI基因序列构建的系统发育树显示,同一属的鱼类首先聚为一支,随后与同一科的鱼类聚为一支,最后,同目不同科的鱼类聚为一支。综上所述,COI基因具有物种特异性,能够有效的区分西北太平洋常见鱼类物种,本数据库的初步建立,有利于后期利用环境DNA技术进行西北太平洋鱼类多样性的监测和调查,为西北太平洋海域生物多样性保护、资源管理和种群动态监测提供了技术支持。     

虹鳟倍性鉴定SSR标记筛选与应用研究

虹鳟(Oncorhynchus mykiss)是我国冷水性鱼类的主要养殖品种。与二倍体相比,虹鳟三倍体具有生长速度快、肉质好和不育等优点。目前,鉴定虹鳟倍性主要是通过流式细胞术进行DNA含量分析,但这种方法需要采集鱼类血液或组织样本,会对其造成伤害。为了实现利用微创方法收集样本鉴定虹鳟倍性,本研究利用PCR扩增以及电泳分离技术对153个微卫星标记(SSR标记)进行分析,其中139个SSR标记能够在二倍体和三倍体中成功扩增,筛选出132个SSR标记具有多态性,最终鉴定出7个标记在三倍体中表现出较高的变异性。通过在52个已知倍性水平的参考样本和48个未知倍性的样本上进行验证,进一步从中筛选出3个SSR标记(SSR1054、SSR1056和SSR1468)足以区分倍性水平,并且根据3个标记序列重新设计了3对具有良好稳定性的引物序列进行倍性分析应用。本研究所筛选的SSR标记解决了现有虹鳟倍性鉴定中存在的技术流程复杂、耗材昂贵的问题,并有助于不同倍性虹鳟的遗传多样性研究。     

Inheritance and reliability of random amplified polymorphic DNA-markers in two consecutive generations of common carp (Cyprinus carpio L.)

Random amplified polymorphic DNA (RAPD) markers have been used in a variety of genetic studies in fisheries and aquaculture. Most population studies are performed without preliminary data demonstrating the Mendelian inheritance and reproducibility of RAPD markers. In this study, the inheritance and reproducibility of RAPD markers was examined in two consecutive generations of common carp, Cyprinus carpio L. Variability and segregation of RAPD markers were investigated in one F₁ progeny and three F₂ progenies. Seventy-four RAPD markers were generated by five primers using DNA extracted from the initial ornamental (koi) common carp female and wild-type colour common carp male. Fifty-five of these RAPD markers were transmitted to the F₁ progeny and the inheritance patterns were analysed. Twenty RAPD markers were fully reproducible and demonstrated dominant simple Mendelian inheritance patterns in two consecutive generations. Twenty-four RAPD markers were not reproducible in all progenies. Thirteen markers displayed inheritance ratios in the progenies that did not fit simple Mendelian inheritance patterns. Non-reproducibility of RAPD markers and distorted ratios may be caused by the absence of amplification, poor amplification or by the appearance of artefact bands. Random amplified polymorphic DNA markers with poor reproducibility and non-Mendelian inheritance can lead to misinterpretations of data in population studies, resulting in errors in the estimation of genetic diversity within and between individual populations. Therefore, it is recommended to first identify the set of reproducible RAPD markers that demonstrate Mendelian inheritance before application of the RAPD technique in population studies.     

Tth DNA聚合酶基因的分段PCR克隆及其表达载体构建

利用计算机软件对取自ＧｅｎＢａｎｋ的嗜热栖热菌ＴｈｅｒｍｕｓｔｈｅｒｍｏｐｈｉｌｕｓＨＢ－８的ＤＮＡ聚合酶基因（Ｔｔｈ）进行分析,并设计了两组引物进行ＰＣＲ,分段克隆该基因。将两个ＰＣＲ产物同时连接到另一个克隆载体上,使之成为完整基因,最后将完整基因克隆到表达载体上,构建了Ｔｔｈ基因的表达质粒,使之适合在以大肠杆菌为寄主的细胞中进行表达。     

湖南省山羊脑多头蚴的线粒体nad1和nad4基因的序列测定及种系发育分析

本研究旨在阐明脑多头蚴湖南分离株线粒体烟酰胺腺嘌呤二核苷酸( NADH)脱氢酶亚单位1基因(nad1)部分序列(pnad1)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位4基因(nad4)部分序列(pnad4)的遗传变异情况,并用pnad1和pnad4序列重构脑多头蚴与其它带科绦虫的种群遗传关系.利用聚合酶链反应(PCR)扩增脑多头蚴的pnad1和pnad4,应用ClustalX 1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAstar5.0中的Megalign程序进行同源性分析.结果显示所获得的pnad1和pnad4序列长度分别均为666和887 bp,湖南分离株与已知多头带绦虫位于同一分枝.由于脑多头蚴pnad1和pnad4序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为脑多头蚴的分子流行病学和其相关疾病的诊断奠定基础.     

Genetic analysis of clinical and vaccine strains of Bordetella pertussis by Pulsed-Field Gel Electrophoresis (PFGE), Multi Locus Sequence Typing (MLST) and serotyping

In spite of high vaccination coverage in the Expanded Program of Immunization (EPI), pertussis has not been eradicated yet and the re-emergence of the disease is still reported worldwide. The genetic divergence study of circulating clinical strains of Bordetella pertussis among the population with high vaccination coverage is a useful tool to have an insight in the understanding of genetic patterns of this bacterium and deviation of them from vaccine strains. Different methods are accessible for studying of Bordetella pertussis that can perform appropriate assessment between populations. Strains used in this study were a collection of two pertussis vaccine strains used to create killed pertussis vaccine over years at Razi Vaccine and Serum Research Institute, 10 clinical and 2 reference strains (ATCC9797 and Tohama I) in Multilocus Sequence Typing (MLST), Pulsed-Field Gel Electrophoresis (PFGE), and serotyping. The genetic profiles of vaccine working and master seeds showed no important change(s) in frequencies of fingerprint types investigated in the vaccine strains and had homogeneity in PFGE method where the clinical isolates showed diversity in genetic profile. Serotyping method showed that all of 10 clinical strains expressing Fim 3. In MLST study, seven housekeeping genes including adk, pgm, fum C, tyr B, gly A, pep A and icd were analyzed which showed no changes in the sequence of clinical and vaccine strains with 100% homology. The genes that cause pathogenicity like ptxC, tcfA and fhaB were also evaluated and the results illustrated heterogeneity in the vaccine and circulating strains.     

锥—46抑制[^3H]次黄嘌呤掺入伊氏锥虫核酸的研究

用液体闪烁计数法对锥—46的抗锥虫作用的机理做了初步研究。发现T-46与Berenil很相似,均可显著抑制[~3H]次黄嘌呤掺入伊氏锥虫,抑制作用与浓度及时间呈正相关。T-46和Berenil对DNA合成的IC_(60)分别为1.33和1.73μg·ml~(-1)。本实验还提示T-46抗锥虫作用可能与损伤DNA模板有关。     

Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

Interleukin (IL)-10 exerts potent anti-inflammatory effects by suppression of both T-help (Th) 1 and Th2 cells. Previous studies have reported that IL-10 can ameliorate various inflammatory disorders. The present study was performed to examine whether IL-10 plasmid DNA could suppress development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice, as an initial step towards the development of an appliance for use in dogs with AD. Intradermal injection of IL-10 plasmid DNA markedly inhibited the development of AD-like skin lesions, as evidenced by a marked decrease in skin symptoms and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Moreover, relative mRNA expression levels of IL-4 and interferon-γ in the skin lesions of mice injected with IL-10 plasmid DNA were also decreased compared with those of control mice. Of note, higher serum IL-10 levels in mice injected with IL-10 plasmid DNA were maintained compared with those in control mice. Taken together, the results indicate that IL-10 plasmid DNA can suppress the development of AD-like skin lesions by suppressing both Th1 and Th2 cell responses. Beneficial effects of IL-10 plasmid DNA may be expected in dogs with AD.