不同试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较

筛选能高效提取烟丝DNA,并能获取其微生物群落多样性较高的试剂盒。选择3种不同的试剂盒提取烟丝DNA,比较DNA的纯度和浓度,并将提取的DNA进行原核生物16S rRNA基因V3~V4区和真菌ITS2区的PCR扩增及扩增子高通量测序,进而比较烟丝样品微生物群落多样性和结构差异。结果表明：（1）DNeasy? PowerPlant? Pro Kit植物基因组提取试剂盒(P)提取的烟丝DNA浓度为(103.67±1.7) ng/μL,显著高于其他试剂盒(P<0.01),且3种试剂盒提取的DNA纯度均达到合格水平。（2）3种试剂盒提取的DNA样本均能反映样本中原核生物群落多样性与结构,从门分类水平看,试剂盒P检测到11个门类,E.Z.N.A.TM HP Plant DNA Kit植物基因组提取试剂盒(M)检测到8个门类,DNeasy? PowerSoil? Kit土壤基因组提取试剂盒(S)检测到5个门类;从属分类水平看,试剂盒P检测到83个属,试剂盒S检测到71个属,试剂盒M检测到69个属。（3）3种试剂盒均能检测到Unclassified类群、子囊菌门Ascomycota、接合菌门Zygomycota和Others等4个真菌门类,但试剂盒P检测到的类群比例最高;在属分类水平上,试剂盒P检测出30个属,试剂盒S检测出20个属,试剂盒M检测出11个属。DNeasy? PowerPlant? Pro Kit植物基因组提取试剂盒(P)能有效提取烟丝及其所含微生物的总DNA,并能检测出更高的烟丝微生物群落多样性,推荐该试剂盒用于烟丝样品中微生物群落多样性与结构的分析。     

4个不同苯甲醇含量茶树品种的转录组分析

香气是茶叶品质决定因子之一,苯甲醇作为一种芳香族醇,参与茶叶香气物质形成。本研究选用4个苯甲醇含量不同的茶树品种,分别是‘薮北’(苯甲醇含量低)、‘湘妃翠’、‘牛皮茶’(苯甲醇含量中等)和‘铁香茗’(苯甲醇含量高),通过Illumina Hiseq^TM2000高通量测序技术对上述4种茶树一芽二叶的转录组进行测序,通过Blast搜索比对,共有151 198条Unigene获得了基因注释。4种茶树在新陈代谢路径中注释的基因数最多,其中次生代谢物生物合成和萜类和聚酮化合物代谢途径可能与茶树苯甲醇合成有关,通过对4种茶树样品的Unigene与NR数据库比对,发现5条可能编码茶树苯甲醇合成途径的关键酶β-葡萄糖苷酶基因,这些相关研究为分析茶树香气功能基因提供理论指导,为香气相关候选基因的发掘提供重要依据。     

健康和患病卵形鲳鲹肠道菌群结构的差异

为研究病害发生后卵形鲳鲹肠道菌群结构的差异及其与养殖环境的关系,分析不同样品中的菌群组成和细菌多样性。实验采用Illumina HiSeq高通量测序技术和生物信息学分析手段,分别构建了健康鱼水样、患病鱼水样、健康鱼肠道、患病鱼肠道和饵料颗粒5个样品中菌群的16S rRNA基因测序文库。研究表明,与健康鱼肠道细菌组成相比,患病鱼肠道中螺旋体门相对丰度显著升高,厚壁菌门和拟杆菌门相对丰度显著降低;患病后肠道中的细菌种类仅为健康鱼肠道细菌种类总数的54.94%;健康卵形鲳鲹肠道菌群中有73.46%的可操作分类单元(OTUs)与水体样品中OTUs一致,有70.58%与饲料样品一致,而患病后分别只有17.98%和38.95%的OTUs与水体和饲料样品一致。健康卵形鲳鲹肠道中黑海弧菌的相对丰度为17.19%,患病后该菌的相对丰度大幅提高,达到78.90%;健康鱼肠道中鳆发光杆菌占比54.53%,患病鱼肠道中不含鳆发光杆菌。健康和患病鱼肠道菌群种类组成类似,但存在一定差异,病害的发生会导致肠道细菌多样性显著降低。健康鱼肠道菌群与养殖环境和颗粒饲料中菌群组成关系密切,而患病后鱼肠道细菌多样性受环境中菌群的影响较小。黑海弧菌在患病鱼肠道中的大量增殖可能是引起其患病的重要原因。     

抗A型塞内卡病毒天然产物筛选及颉颃作用机制研究

【目的】从天然产物库中筛选具有颉颃A型塞内卡病毒(Senecavirus A,SVA)活性的天然产物并研究其颉颃作用机制。【方法】利用荧光素酶重组塞内卡病毒(rSVA-NLuc)与BHK-21细胞,结合荧光素酶高通量筛选技术,建立抗SVA药物体外筛选平台。从天然产物库中筛选浓度为10μmol/L时具有抑制荧光素酶活性效应的天然产物,并进一步利用实时荧光定量RT-PCR验证其抑制活性,通过细胞毒性试验确定其最大无毒浓度。选择病毒感染周期的吸附、入胞、复制、组装释放4个主要过程,利用实时荧光定量RT-PCR、50%组织细胞感染量(TCID₅₀)测定等进行天然产物分子颉颃机制研究。【结果】从包含560种天然产物的分子库中筛选出16种候选抗SVA活性分子,通过实时荧光定量RT-PCR及细胞毒性检测,鉴定出4种安全、有效的天然产物,分别为20S-原人参三醇((20S)-protopanaxatriol)、蕈青霉素(paxilline)、方胆碱(fangchinoline)、竹红菌乙素(hypocrellin B)。作用机制研究显示,20S-原人参三醇能抑制SVA感染过程中的...     

犬牙周病的临床研究进展

牙周病(periodontal disease, PD)是由细菌在牙齿表面聚集形成的牙菌斑和牙结石逐渐侵蚀牙周组织,引发周围组织炎症的一类疾病。根据疾病的发展进程,可分为齿龈炎与牙周炎2个阶段。在小动物临床疾病中牙周病尤为常见,世界范围内约有80%的成年犬患有该病。由于饲主对于犬的口腔保健意识薄弱且该疾病隐秘性较高,患犬就诊时往往已出现牙周支持组织不可逆的损伤,细菌及其毒力因子通过血液循环进入全身从而引发系统性疾病的风险大大提高。但目前犬牙周病的核心致病菌属、菌群失调的原因与发病机制尚不明确。因此,笔者基于高通量测序方法的病原学研究结果,分析与疾病发展相关的微生物群。同时,整合目前世界通用的最新犬牙周病临床诊断分级标准及犬牙周病的中西医防治方案,分析犬牙周病与全身系统性疾病之间的相关性,系统综述犬牙周病在国内外小动物临床领域的研究进展,以期为该类疾病的临床诊疗提供理论参考,推进兽医师在临床实践中更高效地开展该类疾病的诊疗工作,并为犬牙周病相关的临床研究提供思路。     

High-Throughput Screening of a Marine Compound Library Identifies Anti-Cryptosporidium Activity of Leiodolide A

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.     

高通量测序分析凡纳滨对虾育苗期水体菌群结构特征

为了解凡纳滨对虾育苗期正常苗池水体菌群结构和多样性变化,实验采用Illumina MiSeq高通量测序水体细菌16S rDNA V4区,比较幼体在无节V期(N5)、溞状Ⅱ期(Z2)、糠虾Ⅰ期(M1)与仔虾Ⅰ期(P1)时的水体菌群特征。结果显示,水体菌群Shannon多样性指数随幼体发育先降后升,而Pielou均匀度指数除在N5时稍高外,在其余阶段都很接近。育苗期间,变形菌门为水体绝对优势菌群(50.0%~88.0%),尤其红杆菌科从N5到P1阶段分别占24.2%、61.6%、43.3%和51.8%;拟杆菌门在各期丰度均9.7%,其中腐螺旋菌科以5.3%~16.9%优势分布于后3个阶段;放线菌门在后3个期都为优势类群,并以微杆菌科为主,丰度分别达5.6%、37.6%和10.8%;而疣微菌门只在P1时表现为优势(5.3%)。苗池水体核心菌属主要由洛克氏菌、海命菌、栖东海菌、亚硫酸杆菌和2个分类未定属构成。可见水体菌群呈高度多样而动态变化,且N5菌群明显区别于后3个阶段。育苗期间水体主要环境因子总体波动较小,对优势操作分类单元(OTU)丰度分布无显著影响。研究表明,凡纳滨对虾健康苗池水体菌群随幼体发育和饵料投喂而明显更替,其中核心菌群的潜在功能对维持水体生态系统稳定起重要作用,可为对虾育苗益生菌与健康苗池指示菌的筛选提供依据。     

Integrated analysis reveals an association between the rhizosphere microbiome and root rot of arecanut palm

The rhizosphere microbial community is crucial to plant health. Many studies have explored the association between the rhizosphere microbiome and plant disease. However, few studies have focused on root rot in arecanut palm, a disease causing devastating effects and thus resulting in economic losses that considerably affect the development of the arecanut industry. Here, rhizosphere samples were collected from both healthy arecanut palm plants and root-rotted arecanut palm plants, and the microbial communities were analyzed using high-throughput sequencing. The root-rotted samples exhibited distinct microbial community richness, diversity, and composition compared with the healthy samples, which was associated with pH according to the Mantel test. Identified potential plant pathogens, including Proteobacteria, Bacteroidetes, Chytridiomycota, and Mortierellomycota, were significantly enriched in the root-rotted samples. In contrast, potentially beneficial plant microbes, such as Acidobacteria and Gemmatimonadetes, were significantly depleted in the root-rotted samples. Co-occurrence networks were constructed to further identify microbial relationships in the root-rotted samples. These findings revealed ecological imbalance among beneficial bacteria in the root-rotted samples. The present study therefore provides an integrated view of the association between the microbial community and root rot in arecanut palm.     

生草栽培对南丰蜜橘园土壤微生物群落结构与功能特征的影响

为探究橘园生草对土壤微生物群落结构与功能特征的影响,本研究以南丰蜜橘(Citrus reticulita Blanco cv.Kinokuni)为研究对象,设置行间播种白三叶草(Trifolium repens)、黑麦草(Lolium multiflorum)、自然生草和清耕4个处理,采用荧光定量PCR技术和Illum...     

蛋白质定向进化的高通量筛选方法

定向进化是改造蛋白质分子的有效新策略。创建突变库的方法已经有很多而且比较通用,而建立有效的高通量的筛选方法是蛋白质定向进化成功的关键。本文综述了近几年发展起来的用于定向进化的高通量的文库筛选方法,介绍了各种展示技术及流式细胞分选技术的原理及其在文库筛选中的应用,分析了存在的问题及发展趋势。