基于宏基因组学的转基因棉田土壤微生物功能多样性分析

[目的]研究转基因棉田土壤微生物的功能多样性。[方法]采用直接法提取转基因棉根际土壤微生物总DNA,用限制性内切酶BamHI进行部分酶切,回收2.0～4.0 kb大小的片段插入到pUC19/BamHI脱磷载体上,转化到DH5α高效感受态细胞,构建宏基因组文库。随机挑取10个克隆序列测序,并通过NCBI预测可能存在的开放式阅读框和进行序列比对分析。[结果]该文库的外源插入片段平均长度为2.4 kb,90%以上克隆都含有插入片段,10个序列中存在着多种功能的开放式阅读框,包括一些水解酶、脱氢酶、脂解酶、修饰酶、抗生素及与电子传递链相关的酶类。[结论]该研究可为研究转基因棉大面积野外种植以后的生态安全性提供参考。     

叶围微生物宏基因组文库的构建和分析

宏基因组文库技术是开发利用未培养微生物基因资源的有效途径。采用CTAB-SDS法直接从植物叶面沉积样品中提取基因组DNA,构建了以puc19为载体的基因组文库,共获得8126个阳性克隆,文库DNA总容量约30.1MB,平均插入片段长度为3.8kb。叶围微生物基因组文库的成功构建,对于以后研究植物和叶围微生物的关系有重要意义。     

橡胶林土壤宏基因组文库的构建及分析

构建包含了7 680个克隆子的橡胶林土壤宏基因组文库,76.2%的质粒含有外源DNA,插入片段的平均大小约为4.3 kb,DNA文库的容量约为32.25 MB.随机挑取20个克隆子进行测序,NCBI的Blastx分析结果发现,3号与不能培养的微牛物(Candidatus Kuenenia)有一定的同源性.5号与Rickettsiella grylli的同源性达83%,可以初步推断其功能为吡哆醛生物合成裂解酶Pdxs.11号和16号与分类地位尚未确定的细菌(Solibacter)有同源性,随机测序的这20个序列中,部分序列除与裂解酶、蛋白酶、激酶等蛋白有一定的同源性外,有5个与功能尚不清楚的蛋白有一定的同源性,其中13号与假定蛋白pEA 28_O1的同源性达100%.     

Two New Cytotoxic Indole Alkaloids from a Deep-Sea Sediment Derived Metagenomic Clone

Two new indole alkaloids, metagenetriindole A (1) and metagenebiindole A (2), were identified from deep-sea sediment metagenomic clone derived Escherichia coli fermentation broth. The structures of new compounds were elucidated by spectroscopic methods. The two new indole alkaloids demonstrated moderately cytotoxic activity against CNE2, Bel7402 and HT1080 cancer cell lines in vitro.     

山羊瘤胃微生物宏基因组文库中一个新型纤维素酶 基因的克隆与表达

通过功能筛选方法,从安徽白山羊瘤胃微生物宏基因组文库中筛选得到了6个纤维素酶的阳性克隆。对其中的1个阳性克隆进一步进行亚克隆和序列分析,获得一个新型纤维素酶基因开放阅读框,通过BLAST对基因全序列进行分析比较,发现其与来自Bacteroides cellulosilyticus DSM 14838的hypothetical protein具有51%的序列相似性和65%的同源性。并以p ET28a(+)为载体、Escherichia coli BL21(DE3)为宿主菌,对新型酶基因进行高效重组表达,并对该酶表达条件进行优化。     

南极中山站附近海水微生物多样性和宏基因组分析

以中国第34次南极科考采集的中山站附近海域5个站点海水样品为研究对象,通过Illumina高通量测序,鉴定微生物组成和微生物群落结构,发现中山站取样的海水中变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)最为丰富,其次为蓝细菌(Cyanobacteria),属水平上黄杆菌属(Flavobacterium)和嗜冷杆菌属(Psychrobacter)含量最多,均超过20%。首次在南极海水中发现UBA1315属微生物,功能注释分析显示含有丰富的DNA修复基因,可能有助于适应南极恶劣环境。对中山站和罗斯海上层海水以及南北极不同地理位置海水微生物比较发现,变形菌门、拟杆菌门和蓝细菌在所有海水中普遍存在,变形菌门为优势菌,蓝细菌在南极海水中含量丰富,罗斯海上层海水比中山站海水微生物多样性丰富,且主要菌群丰度不同。初步揭示了中山站附近海域微生物群落组成,为后续进一步研究南极中山站海水微生物资源调查和功能开发研究提供一定的基础数据。     

Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.     

水稻-油菜周年耕作方式对土壤微生态的影响

为探索土壤微生态对稻-油系统周年耕作方式的响应机制,利用湘北洞庭湖区长期免耕直播土壤,设置稻-油轮作试验,通过比较双免耕双直播和旋耕抛栽土壤中酶活性、微生物数量、群落多样性和菌群功能等指标的差异,研究不同耕作方式对土壤微生态的影响。宏基因组测序表明,细菌群落在2种耕作方式下组成相似,双免耕双直播下细菌多样性更高;真菌群落在不同耕作方式下有不同的真菌类型,但旋耕抛栽下真菌多样性更高。菌群功能分析显示,需氧和兼性厌氧原核微生物相对丰度在双免耕双直播中较高;与氮代谢相关的原核微生物相对丰度在双免耕双直播中较高,而与碳代谢相关的原核微生物的相对丰度在旋耕抛栽中较高。可培养微生物数量测定表明,免耕土壤中可培养细菌、真菌、放线菌和硝化细菌的数量都较旋耕极显著降低,而氨化细菌数量极显著增加。酶活性分析表明,双免耕双直播土壤中的脲酶活性比旋耕抛栽提高了58.12%,而转化酶、磷酸酶和纤维素酶的活性分别降低了28.57%、11.00%和57.73%。本研究表明稻-油轮作双免耕双直播的耕作方式有利于提高土壤中细菌的多样性、需氧和兼性厌氧原核微生物的相对丰度,有利于土壤中氮的转化和利用,为洞庭湖区稻-油系统合理耕作方式的选择提供依据。     

瘤胃元基因组BAC文库研究

目前，传统的分离培养技术在瘤胃微生物的研究中仍存在一些无法逾越的障碍。元基因组文库（metagenomiclibraries）及相关技术的发展，为探讨环境中未培养微生物以及复杂的微生物间关系创立了一个新的平台。将该技术应用于瘤胃微生物研究有着广阔的前景。本文综述了元基因组文库技术在瘤胃微生物研究中的应用潜力，同时介绍了初步应用BAC文库研究瘤胃微生物工作的一些进展。     

Diversity in metagenomic sequences reveals new pathogenic fungus associated with smut in Job's tears

Smut is a serious disease in Job's tears, also known as adlay, and contributes to the reduction of crop yield in agricultural fields. In this study, the key pathogenic fungi in adlay smut disease were first identified by internal transcribed spacer region(ITS) high-throughput sequencing and then used to elucidate the composition and diversity of the fungal community in adlay smut. Results indicate that an abundance of operational taxonomic units(OTUs) were detected in the infected involucres of flowering plants and the OTUs were classified to nine phyla, 20 classes, 45 orders, 90 families and 119 genera. A total of 4 986 OTUs clustered together, sharing six core OTUs in all samples, while 145 OTUs were shared among four geographical groups. The Shannon and Simpson indices ranged from 0.137–1.629 and 0.357–0.970, respectively. Community diversity ranked as Anshun(AS)Qinglong(QL)Xingren(XR)Xingyi(XY) among the four geographical groups by Shannon and Simpson indices, exhibiting complex community diversities among accessions and geographical groups. The richness and diversity data imply a weak relationship between the accession community richness and geographical origins of samples. Two closely related fungal genera, Sporisorium and Ustilago, were implicated as causes of smut disease. The genus Sporisorium appears to be more commonly found among accessions and thus is more likely to be the fungal pathogen causing smut in adlay. This work can facilitate strategies to control and prevent smut infection to improve adlay yield.