红脚艾蒿的转录组解析

[目的]利用高通量测序技术解析红脚艾(Artemisia rubripes Nakai)的转录组信息特征.[方法]通过高通量测序平台Illumina HiSeq 2500对红脚艾进行转录组测序,通过Trinity软件de novo组装获得Unigene,并基于序列同源性对Unigene进行功能注释,得到红脚艾的转录组信息.[结果]测序数据经过质控后共获得24126043条高质量的reads,通过de novo组装获得173093个转录本,对组装的转录本去冗余后共获得85991个Unigene,平均长度为616.87 bp,N50为925 bp.共有47216个Unigene在NR、KEGG、COG、KOG、GO数据库获得功能注释,40802个Unigene在NR数据库注释,显示红脚艾与向日葵(Helianthus annuus)的单基因匹配率最高,16846个Unigene被KEGG数据库注释到130条代谢途径中,26171个Unigene被注释到25个KOG功能分类中,23203个Unigene被GO注释到生物过程、细胞组成和分子功能三大类51个功能分类,12810个Unigene被注释到25个COG功能分类中.[结论]利用高通量测序技术获得了红脚艾转录组信息特征,这些数据将为后期开展功能基因鉴定、解析化合物次生代谢途径及其调控机制奠定研究基础.     

PCR-genotyping of barley seedlings using DNA samples from tissue prints

Screening of large numbers of plant samples with polymerase chain reaction (PCR)‐based techniques is a central task in marker‐assisted plant breeding and molecular genetics. While PCR and electrophoresis have been streamlined as a result of high‐throughput analysis methods, the collection of plant material and its processing to make it accessible for reliable amplification reactions remain labour‐intensive, costly and not amenable to automation. As an alternative to traditional extraction protocols, DNA can be bound to specially treated paper (FTA‐paper). For PCR analysis, instead of being added as a liquid suspension, DNA is then released from small paper disks that are immersed into the reaction mixture. In the present study, several parameters were investigated for the successful amplification of various single‐copy sequence characterized amplified region markers from barley tissue prints. The results show that the FTA‐paper approach offers a quick, reliable and low‐cost alternative for PCR screening even in a crop plant with a large genome.     

不同地点和复垦方式煤矸石山土壤中丛枝菌根真菌群落变化

以山西省大同市晋华宫人工复垦和自然恢复煤矸石山以及忻州窑自然恢复煤矸石山为研究对象,采用IlluminaMiSeq高通量测序技术对三座煤矸石山土壤AM真菌群落组成进行分析,并测定其土壤理化性质,以探究不同地点煤矸石山不同复垦方式下土壤中丛枝菌根真菌（Arbuscular mycorrhizal fungi, AM真菌）群落组成的差异及其土壤影响因子。结果表明：晋华宫人工复垦煤矸石山和忻州窑自然恢复煤矸石山AM真菌多样性指数（Chao指数和香农-威纳指数）存在显著差异,但晋华宫人工复垦和自然恢复煤矸石山AM真菌群落的多样性指数差异不显著。试验共检测到67个AM真菌OTUs,可鉴定到3个AM真菌属级分类单元,其中球囊霉属（Glomus）在晋华宫人工复垦煤矸石山、晋华宫自然恢复煤矸石山和忻州窑自然恢复煤矸石山中均为最优势菌,多样孢囊霉属（Diversispora）、类球囊霉属（Paraglomus）仅出现在晋华宫自然恢复煤矸石山。多元置换方差分析和多维尺度分析结果表明,晋华宫两种恢复方式的煤矸石山之间AM真菌群落组成差异不显著,而晋华宫与忻州窑两个地点的煤矸石山AM真菌群落组成差异显著。AM...     

4种周年轮作模式对耕作层土壤微生物的影响

为明确不同轮作模式下耕作层土壤微生物群落的变化,揭示水旱轮作有益于蔬菜可持续生产的潜在机理。本文设计4种常见的轮作模式,在同一地理区域固定试验田块进行长期轮作试验,并利用高通量测序技术测定土壤中真菌的ITS1区域序列和细菌的16S rDNA的V3-V4可变区序列,进行土壤微生物多样性分析。结果表明：Alpha多样性分析表明,在4种周年轮作模式下,耕作层土壤真菌群落多样性发生了显著变化,水旱轮作模式(SH)的Shannon指数显著高于嫁接模式(JJ)和蔬菜轮作LZ模式(LZ)的,而与蔬菜轮作LD模式(LD)的无显著差异。耕作层土壤细菌群落多样性和丰富度均发生显著变化,SH模式的Shannon指数和ACE指数均显著高于LD模式的,且SH模式的Shannon指数显著高于其他模式的。主坐标分析表明,4种周年轮作模式间,耕作层土壤微生物群落发生显著分化,SH模式和LZ模式均各自成组,JJ模式与LD模式聚为一组。组间物种差异显著性分析表明,SH模式有7个属真菌和26个属细菌的丰富度显著高于其他模式的,LD和LZ模式分别有4个属真菌和7个属细菌的丰富度显著高于其他模式的,JJ模式只有1个属细菌的丰富度显著高于其他模式的。不同轮作模式长期周年轮作下耕作层土壤微生物群落发生了显著变化。长期水旱轮作提高了土壤微生物群落多样性,增加了有益菌种类和相对丰度,更有利于蔬菜可持续生产。     

基于整合高通量绝对定量法的土壤微生物多样性分析

目前,常用的高通量测序技术只能得到微生物群落结构的物种种类和相对丰度,而整合高通量绝对定量法（iHAAQ）结合了高通量测序技术和qPCR技术,可以进一步计算得到微生物群落结构的绝对含量。相对丰度和绝对含量均是描述微生物群落结构的必备指标,其中相对丰度适合描述和评价微生物群落结构在单个样本内微生物之间的关系,而绝对含量更适于描述和评价微生物群落结构真实的数量变化以及在样本间微生物之间的关系。本研究基于发表的香蕉土传病害、再造沙地农业生态系统和微生物抑制剂作用3篇文献的高通量测序和qPCR数据,通过整合高通量绝对定量法获得3篇文献中古菌域、细菌域和真核生物域的真菌三类微生物群落结构的绝对含量数据,并进一步计算出这三类微生物整体（简称三域微生物）的绝对含量和相对丰度,从而对土壤微生物群落结构不同物种的相对丰度和绝对含量进行分析,旨在更加深入、准确地揭示土壤微生物群落及其生态功能。结果表明：（1）土壤中细菌的物种丰度和绝对含量高于古菌和真菌,在三域微生物中占据主导地位,缺乏绝对含量指标可能造成对微生物群落结构变化理解的偏差;（2）基于相对丰度和绝对含量数据分析,微生物群落结构α多样性指数结果相同,但β多样性指数结果不同;（3）在香蕉土传病害和再造沙地农业生态系统研究中,三域微生物的PCoA结果与细菌的结果较为相似,表明这两项研究中三域微生物群落结构主要受细菌的影响,但在微生物抑制剂的研究中未发现类似结果。整合高通量绝对定量法可以应用于所有同时进行高通量测序和qPCR测序的研究,在未来的土壤微生物群落结构研究中,采用高通量绝对定量法开展三域微生物群落的物种种类、相对丰度和绝对含量的整体评价,具有重要的生态学意义。     

Effect of Aspergillus niger NBC001 on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most important pests causing considerable damage to soybean (Glycine max (L.) Merr.) around the world. Biocontrol provides a strategy for sustainable nematode control. Previously, Aspergillus niger NBC001 was isolated from the cysts of Heterodera spp. and able to control H. glycines and promote the growth of soybean in a pot experiment. In this study, the effects of NBC001 on H. glycines density and on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field were studied. The results showed that NBC001 could suppress H. glycines by 31.7% in the field. High-throughput sequencing analysis showed that NBC001 had no significant influence on soybean rhizosphere microbial community structure, indicating that seed coat-dressing with the concentrated culture filtrate of NBC001 was safe for the soil ecological environment. In addition, high-throughput sequencing results demonstrated that at 10 days post transplantation, NBC001 increased the abundances of Actinobacteria and Acidobacteria, but decreased the abundances of Bacteroidetes and Gemmatimonadetes at the phylum level. Meanwhile, the abundances of Phyllobacterium, Ralstonia and H16 were increased, while the abundances of Adhaeribacter, Gemmatimonas, Sphingomonas, Flavisolibacter were suppressed by application of NBC001. However, at 90 days post transplantation, NBC001 only increased the abundances of Aeromicrobium and RB41 whereas it decreased the abundance of H16. The results indicated that application of NBC001 increased the relative abundances of the beneficial microorganisms such as Actinobacteria, Acidobacteria, Aeromicrobium and Phyllobacterium in the soil. In summary, NBC001 is an eco-friendly biocontrol agent for H. glycines control.     

广西天桃木土壤细菌群落多样性特征

2020年6月,以广西南宁市、崇左市扶绥县、百色市田阳区的原生天桃木Mangifera persiciformis为研究对象,采用PacBio第三代高通量测序技术,对天桃木土壤细菌群落多样性进行了研究,并通过测定土壤化学性质,分析天桃木土壤优势细菌群落与土壤化学因子的关系.结果表明:在门水平上,共存在23门细菌,其中变...     

猪组成型雄烷受体激动剂高通量药物筛选模型的建立

构建猪组成型雄烷受体(pgCAR)激动剂体外高通量筛选模型。利用RT-PCR技术从猪肝脏总RNA中扩增出pgCAR基因序列,将测序后正确的pgCAR片段连接到pcDNA 3.1 vector上构建表达载体pcDNA 3.1-CAR;将合成的5个拷贝NR1(Nuclear receptor sites)插入PGL3-TK-promoter构成报告质粒(NR1)5-TK-luc,与PRL-TK报告质粒构成双荧光报告系统。用PEI转染技术将表达载体与报告质粒共转染HepG 2细胞株中,通过检测荧光素酶基因表达状况评价具有生物活性配体化合物对pgCAR激动活性。阳性药苯妥英钠明显提高荧光素酶的表达,最大上调倍增数可达约2.53倍,并且在一定浓度下阳性药与相对荧光酶的活性表达有较好量效关系。     

Screening Mangrove Endophytic Fungi for Antimalarial Natural Products

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14–16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.     

茶园土壤真菌群落组成及影响因素研究

茶园土壤真菌群落多样性是评价茶园土壤环境质量与健康的重要依据。以代表性古茶山森林、现代茶园和古茶园土壤为研究对象,采用Illumina平台Hiseq 2500高通量测序技术,对真菌18S rDNA区段测序,结合土壤养分水平分析,探究不同类型土壤真菌群落组成及其与养分状况的关联性。结果表明：子囊菌、担子菌、接合菌、壶菌和球囊菌5个门的相对丰度占真菌群落的97.17%~99.67%,Cryptococcus、Archaeorhizomyces和Mortierella 3个属在森林和茶园土壤中分布广泛;土壤真菌群落多样性和丰度与土壤pH、C:N和Olsen-P的相关性明显高于有机质、碱解氮和有效钾;茶园土壤真菌数量和多样性优于森林土壤;属最佳分类水平上,森林土壤优势群落Hannaella、Trichoderma、Alternaria、Herpotrichiellaceae、Russula、Membranomyces、Gliocladiopsis、Penicillium等丰度与土壤C:N含量呈显著正相关关系,茶园土壤优势群落：Cryptococcus、Archaeorhizomyces、Fusarium、Exophiala、Mortierella和Metarhizium等丰度与土壤pH、有机质、碱解氮、有效钾和Olsen-P呈显著正相关关系。茶树种植及年限和不同茶山土壤pH及碳氮磷钾养分状况与茶园土壤真菌群落结构及多样性关系密切。