稻瘟病抗性鉴定田土壤宏基因组文库构建及分析

从稻瘟病抗性鉴定田中提取土壤微生物宏基因组DNA,EcoRⅠ和BamHI双酶切后插入到经同样双酶切的pSK 载体中,转化至DH5α感受态细胞,构建了土壤微生物宏基因组文库。随机挑选了9个克隆测序,并通过NCBI的Blastx分析了序列可能所属的物种和基因。结果其中有6个能与库中序列达到较大匹配,3个的分析结果表明可能是新基因,说明所建文库的生物多样性和新基因数较丰富。     

不同日粮类型对绵羊瘤胃微生物木聚糖酶基因多样性的影响

选取安装有永久性瘤胃瘘管的中国美利奴绵羊4只,先后饲喂全粗料日粮和精料型日粮(精粗比3∶2),提取微生物宏基因组DNA。通过PCR产物克隆文库构建、DNA测序和序列分析以研究在不同日粮条件下,绵羊瘤胃微生物糖苷水解酶第10家族(GH10)和第11家族(GH11)木聚糖酶基因的多样性。结果表明,全粗料日粮时分别获得108和137条GH10和GH11家族木聚糖酶基因片段,精料型时分别获得145和235条GH10和GH11家族木聚糖酶基因片段,这些序列分别归属于19、20、53和26个操作分类单元(OUT)。DNA序列分析表明,GH10家族木聚糖酶基因片段与梭菌和拟杆菌来源的木聚糖酶基因有较高的相似性,GH11家族木聚糖酶基因片段与放线菌、麦角真菌来源的木聚糖酶基因有较高的相似性。饲喂全粗料日粮时,有57.9%的GH10家族序列与已知木聚糖酶基因的相似性小于90%;饲喂精料型日粮时,有65.4%的GH11家族序列与已知木聚糖酶基因的相似性小于90%。由此可见,绵羊瘤胃微生物GH10和GH11家族木聚糖酶基因序列具有丰富的多样性,并且大部分为新发现的序列。饲喂全粗料日粮时,更有利于发现瘤胃微生物GH10家族木聚糖酶新序列;饲喂精料型日粮时,更有利于发现GH11家族木聚糖酶新序列。     

Metagenomic profiles of the rumen microbiota during the transition period in low‐yield and high‐yield dairy cows

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low‐yield (LY) or high‐yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA‐based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.     

盐湖土壤微生物总DNA提取方法的比较

为了构建运城盐湖土壤微生物宏基因组文库,文章对盐湖土壤微生物总DNA的提取方法进行了比较和探索,通过改良6种植物DNA的提取方法,分别提取了盐湖土壤微生物总DNA,并采用紫外分光光度法、凝胶电泳、内切酶分析和PCR扩增法检测了各个方法所提取的DNA样品的质量。结果表明：改良后的方法3是较适合提取运城盐湖土壤微生物总DNA的方法。采用该方法所得DNA的OD260/280值为1.883,DNA浓度和得率分别为18.1 ng·μL-1和72.4 ng·g-1;DNA片段长度约为23 kb,琼脂糖凝胶电泳条带清晰,无降解;DNA能被EcoRⅠ完全酶切,并可用于PCR扩增分析。     

草鱼肠道微生物抗生素抗性基因研究

水产养殖过程中抗生素的过度使用导致耐药菌株不断出现,细菌耐药性成了威胁公共安全的一个全球性问题。抗性基因是细菌产生耐药性的根本原因,动物肠道微生物是抗性基因的储存库,然而目前我国对水产养殖动物消化道微生物抗生素抗性基因的研究匮乏。为探究草鱼（Ctenopharyngodon idellus）肠道微生物中抗性基因的类型和丰度,通过提取草鱼肠道内容物的基因组DNA,采用shotgun宏基因组技术进行测序和序列注释,分析草鱼肠道内容物微生物数据,对比抗性基因数据库ARDB筛选出微生物序列中的抗性基因。结果表明,草鱼肠道内容物共有61554个可注释微生物的基因序列,包括409个真核生物序列、88个古细菌序列和61057个真细菌序列;共得到1011个抗性基因,归为123种基因型,包括78种单抗性基因型（679个单抗性基因）,45种多抗性基因型（304个多抗性基因）,多抗性基因数量占抗性基因总数的30.1%,说明草鱼肠道中可能存在大量的多重耐药性微生物;检出草鱼肠道中数量最多的抗性基因是抗大环内酯类抗生素基因MacB,其在国内水环境中研究甚少,可能是因为其单独存在时无法使微生物表现出抗性。本研究统计了草鱼肠道中微生物抗生素抗性基因的种类及数量,为水产养殖中抗生素的使用监管提供基础数据,验证了宏基因组测序技术检测抗性基因的可行性。     

土壤微生物宏基因组研究现状——基于Citespace文献计量分析

以2005—2020年中国知网(CNKI)和Web of Science数据库为文献来源,采用文献计量方法对土壤微生物宏基因组学研究现状进行统计分析。结果表明,中外学者采用高通量技术进行土壤微生物宏基因组研究的报道持续增加,研究对象以细菌为主,其次是真菌;研究的生境涉及根际、沉积物等。英文文献的研究机构和作者之间合作紧密,而中文文献的研究机构与作者之间的合作以中科院体系为主。在文献质量方面,以美国学者所发表文献的引用率最高,而中国学者所发文献中引用率排名靠前的文献较少。未来基于高通量测序的土壤微生物宏基因组研究应进一步加强机构与人员合作,注重学科交叉,以创新的思维在基因、功能等不同水平上全面分析了解土壤微生物,以深入挖掘其生态学功能,为生态系统的可持续发展提供服务。     

Characterization of antimicrobial resistance genes in Enterobacteriaceae carried by suburban mesocarnivores and locally owned and stray dogs

The role of wildlife in the dissemination of antimicrobial‐resistant bacteria and antimicrobial resistance genes (ARGs) in the environment is of increasing concern. We investigated the occurrence, richness and transmissibility potential of ARGs detected in the faeces of three mesocarnivore species: the coyote (Canis latrans), raccoon (Procyon lotor) and Virginia opossum (Didelphis virginiana), and of stray and owned dogs in suburban Chicago, IL, USA. Rectal swabs were collected from live‐captured coyotes (n = 32), raccoons (n = 31) and Virginia opossums (n = 22). Fresh faecal samples were collected from locally owned (n = 13) and stray dogs (n = 18) and from the live‐captured mesocarnivores, when available. Faecal samples and rectal swabs were enriched to select for Enterobacteriaceae and pooled by mesocarnivore species and dog type (owned or stray). Pooled enriched samples were then analysed for the presence of ARGs using shotgun sequencing. The three mesocarnivore and stray dog samples had twice as many unique ARGs compared to the owned dog sample, which was partly driven by a greater richness of beta‐lactamase genes (genes conferring resistance to penicillins and cephalosporins). Raccoon and stray dog samples had the most ARGs in common, suggesting possible exposure to similar environmental sources of ARGs. In addition to identifying clinically relevant ARGs (e.g. bla_CMY and qnrB), some ARGs were linked to the class 1 integrase gene, intI1, which may indicate anthropogenic origin. Findings from this pilot investigation suggest that the microbial communities of suburban mesocarnivores and stray dogs can host ARGs that can confer resistance to several antimicrobials used in human and veterinary medicine.     

腹泻仔猪肠道病毒组学分析

为了解上海周边地区腹泻仔猪肠道病毒组学特征,特别是冠状病毒流行情况,采用病毒宏基因组学方法对6个猪场的90份疑似病毒感染导致腹泻粪样进行检测,并通过Geneious和MEGA7.0等生物信息学软件对获得的完整的α冠状病毒属中的PEDV ORF3基因序列进行遗传进化分析。数据显示,腹泻仔猪肠道病毒群落组成主要由小RNA病毒科(Picornaviridae)(63.67%)、星状病毒科(Astroviridae)(12.68%)、杯状病毒科(Caliciviridae)(4.07%)、细小病毒科(Parvoviridae)(2.51%)等组成。在唯一一个检测出PEDV阳性的猪场中,腹泻仔猪PEDV阳性率高达43.33%(13/30)。基因序列遗传进化分析显示,在本研究获得的3个PEDV ORF3基因序列中,有1个属于G2基因型,与其他参考PEDV病毒株相似性较高(96.44%～99.70%);另外2个ORF3基因序列属于G1基因型,相似性相对较低(95.11%～99.41%)。与PEDV传统株CV777蛋白序列比较分析发现,除piglet91株产生F2S突变以外,三株PEDV存在一致性突变,分别如下:V21A、I70M、V79I、F80V、L85I、L92F,这一特性,有可能是PEDV传统株与流行株基因型的鉴别依据。上海周边地区腹泻仔猪肠道病毒组成丰富,在不同猪群中,不同病原感染的情况有所差异,除了PEDV外,星状病毒和杯状病毒也是仔猪腹泻的主要病原。PEDV的ORF3基因与传统分离株相比具有独特的分子特征,新检测到的毒株突变位点与病毒毒力的关系有待于进一步研究。研究结果有助于了解腹泻仔猪肠道的病毒谱,并为仔猪病毒病防控提供一定的基础数据。     

小鼠不同肠道段内容物和粪便中微生物的宏基因组测序和比较分析

随着二代测序技术的迅速发展,从宏基因组角度了解环境微生物成为可能。目前,动物肠道微生物研究大多以粪便作为实验样品,对肠道内部微生物情况了解甚少。研究以小鼠为模型,分别对十二指肠、空回肠、盲肠、结肠4个部分内容物进行了宏基因组测序和分析。结果显示,(1)在门水平上,十二指肠比其他肠段厚壁菌门减少、变形菌门增多;(2)在属水平上,大肠的优势菌属为螺杆菌、真细菌等,小肠优势菌属为链球菌、大肠杆菌、梭菌、肠球菌等,而且粪便与大肠优势菌分布趋势类似;(3)大肠和小肠肠道微生物相似度为0.62~0.77,粪便与各肠段微生物相似度为0.7~0.9;(4)粪便和肠道内容物细菌功能差异同其生理功能有一定关联。研究表明,小肠内容物与大肠内容物和粪便中微生物有明显差异,尽营粪便微生物与大肠内容物微生物类似。这一结果暗示着粪便微生物不能完全代表动物宿主肠道微生物的真实情况。     

Characterization of the bacterial community in different parts of the gut of sea cucumber (Apostichopus Japonicus) and its variation during gut regeneration

Bacterial community plays an important role in keeping the health status of the host. A study on the characteristics of gut bacterial community of sea cucumber (Apostichopus japonicus) not only helps improve the current aquaculture model, but promotes healthy and rapid development of the aquaculture industry as well. Therefore, taking pond‐cultured sea cucumber (A. japonicus) as the studying object, the metagenomic and 16S rRNA sequencing technology were used in this study to explore the characteristics of bacterial community in different parts of the gut of sea cucumber, as well as during gut regeneration after evisceration. The results showed that the compositions of bacterial community are different in varying parts of the gut of sea cucumber (A. japonicus). Specifically, bacterial community in the midgut and hindgut are highly similar, showing significantly diversified bacterial species compared to the foregut. GO annotation indicated that the foregut is associated with richer catalytic activity and binding than the midgut and the hindgut. According to the KEGG annotation, metabolism‐related genes are mainly concentrated in the foregut, while genes related to signal transduction and the immune system are mostly annotated in the midgut and hindgut. During the gut regeneration stage, the structure of bacterial community varied greatly in different stage of the regeneration stage, with significant differences between the earlier and later stage. The dominant bacteria in the earlier stage is Rubritalea, and that in the later stage is Arcobacter. Besides, there were Loktanella, Thalassobacter and Phaeobacter in the gut throughout the entire regeneration stage. Cupriavidus, Hellea, HTCC2207, Methylophaga, Methylotenera, Stenotrophomonas and Tenacibaculum were only present in the earlier stage, and gradually disappeared in the later stage due to improving gut functions. The abundance and diversity of bacteria in the gut were higher in the earlier regeneration stage than that in the later stage, with a peak between the 15th and 25th day of the regeneration stage. At 45th day, the abundance and diversity became stable.