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。     

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.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

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。     

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.     

NaCl胁迫下黑麦草种子萌发过程中DNA甲基化与基因表达分析

为了解黑麦草（Lolium perenne）种子在正常条件与NaCl胁迫下萌发过程中DNA甲基化动态变化以及重要盐胁迫相关基因的表达。运用甲基化敏感扩增多态性技术分析对照和150 mmol·L^-1 NaCl处理下黑麦草种子萌发过程（0,1,2,4,7 d）的DNA甲基化水平及动态变化,并通过实时荧光定量 PCR（QRT-PCR）检测8个重要的盐胁迫相关基因的表达情况。结果表明：黑麦草种子萌发过程中甲基化水平呈下降趋势,以双链甲基化方式为主,甲基化变化同时发生在编码序列和非编码序列中。NaCl处理下DNA甲基化程度高于CK,而去甲基化程度低于CK,最终导致NaCl处理下基因组净的甲基化位点数目增加。黑麦草种子耐盐萌发过程中代谢增强,8个盐胁迫相关基因表达量呈上升趋势,而NaCl胁迫延缓了种子萌发进程,在大多数时间点的基因表达低于对照。     

The optimal immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina

The immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. The stability and the cross-species protection of the vaccine were also analyzed. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and the anti-coccidial index (ACI). Chinese Yellow chickens were randomly distributed into corresponding groups (30/group). The challenged, unchallenged and vector control groups were designed. The results illustrated that 25 μg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity. There were no significant differences of ACIs between boosting and non-boosting groups. Storage time and temperature had little effect on the immunizing efficacy of the vaccine. The vaccine could provide partial cross-protection against the challenge with E. necatrix and E. acervulina, but not with E. maxima.     

不同地区及不同季节的柞蚕寄生蝇RAPD分析

利用RAPD技术对不同地区及不同季节发生危害的柞蚕寄生蝇进行了遗传多态性研究。选取11个随机引物对14份供试材料的基因组DNA进行扩增,共扩增出140条稳定条带,其中128(91.43%)条为多态性条带,表明柞蚕饰腹寄蝇(Blepharipa tibialis)、秋柞蚕寄生蝇(未命名)及栗蚕寄生蝇(未命名)间具有丰富的多态性。各材料间的遗传距离在0.0214~0.5143之间,利用UPGMA法进行聚类分析,14份供试材料可聚为两大类:栗蚕(Dic-tyoplocajaponica)寄生蝇与寄生柞蚕的寄生蝇各聚为一类,其中来自河南省的柞蚕饰腹寄蝇自成一类,来自东北地区的柞蚕饰腹寄蝇与秋柞蚕寄生蝇又各自聚为一类。