采用高通量测序技术研究抗生素对小鼠肠道菌群的影响

目的 探讨不同浓度抗生素对小鼠肠道菌群多样性和结构的影响,并预测相关功能变化。方法 15只SPF级ICR小鼠随机分为正常组、低浓度抗生素组和高浓度抗生素组,连续灌胃5 d后,采集小鼠新鲜粪便样本。利用Illumina MiSeq测序平台,对细菌的16S rRNA V3‒V4区进行高通量测序,并对测序结果进行生物信息学分析。结果 高、低浓度抗生素组小鼠肠道菌群组成与正常组存在明显差异。与正常组相比,高剂量组小鼠肠道肠球菌属、志贺埃希菌属相对丰度显著升高（t=‒2.71,P=0.026;t=‒2.30,P<0.05）;分节丝状菌属、拟普雷沃菌属相对丰度显著降低（t=2.88,P=0.020;t=2.49,P=0.037）,理研菌属极显著降低（t=3.79,P=0.005）。低剂量组小鼠肠道菌群变形菌纲成为优势菌,芽胞杆菌属、粪球菌_2、苏黎世杆菌属、普雷沃菌属_2、普雷沃菌属_7、志贺埃希菌属、沙雷菌属和放线菌属等相对丰度显著升高（均P<0.05）;梭杆菌属、泛菌属极显著升高（t=‒3.19,P=0.013;t=‒3.50,P=0.008）;分节丝状菌属、理研菌属相对丰度显著降低（t=2.69,P=0.028;t=2.33,P=0.048）。PICRUSt功能预测分析显示,抗生素组显著增加人类疾病、细胞过程和环境信息处理功能层的基因拷贝数,显著降低有机系统、遗传信息处理和代谢功能层的基因拷贝数。结论 广谱抗生素能破坏小鼠肠道的微生态平衡,有必要深入研究抗生素对心血管、免疫性、感染性及神经退行性疾病发展的潜在作用。     

金缕梅亚科ITS序列分析及其系统学意义

根据金缕梅亚科22属（活塞花属Embolanthera除外）代表种的nrDNA ITS序列数据构建了分子系统树。结合形态解剖证据,金缕梅亚科可分为3个族,即①Ji木族LoropetaleaeZhangtrib.nov.,包括蜡瓣花属Corylopsis、Maingaya、Matudaea、活塞花属、四药门花属Tetrathyrium和Ji木属L;②DicorypheaeZhangtrib.nov.,包括毛枝花属Trichocladus、Dicoryphe、Neostrearia、Ostrearia、Noahdendron、秀柱花属Eustigma、牛鼻栓属Fortunearia、山白树属Sinowilsonia、Molinadendron;③金缕梅族Hamamelideae,包括Fothergilla、金缕梅属Hamamelis、Parrotiopsis、水丝利属Sycopsis、Parrotia、银缕梅属Shaniodendron、蚊母树属Distylium和拟母树属Distyliopsis。     

不同分娩方式对晚期早产儿肠道菌群的影响

目的 探讨不同分娩方式对晚期早产儿肠道菌群定植的影响。方法 以胎龄（周）为340/7～366/7的15例晚期早产儿为研究对象,根据分娩方式分为自然分娩组（n=8）和剖宫产组（n=7）。收集早产儿出生后3 d、7 d、14 d的粪便标本,应用高通量测序技术对细菌16S rRNA可变区中的V4区进行测序,分析肠道菌群多样性及组成结构。结果 （1）自然分娩组晚期早产儿粪便标本菌群多样性指数逐渐上升,剖宫产组的多样性指数较平稳,两组相比差异无统计学意义;（2）45份粪便标本中共检测出10个菌门,均以变形菌门、厚壁菌门、放线菌门和拟杆菌门为优势菌门,两组晚期早产儿生后变形菌门、拟杆菌门所占比例逐渐降低,厚壁菌门、放线菌门呈增多趋势。两组相比,剖宫产组7 d、14 d时拟杆菌门的相对丰度显著低于自然分娩组（Z=‒2.896,P=0.004;Z=‒2.120,P=0.040）,变形菌门相对丰度仅在7 d时显著高于自然分娩组（Z=‒2.190,P=0.030）;（3）两组研究对象中,除自然分娩组14 d时以双歧杆菌属为优势菌属外,余下均以肠杆菌属为优势菌属。相比于自然分娩组,在7 d时剖宫产组拟杆菌属所占比例显著降低（Z=‒2.806,P=0.005）,肠杆菌属所占比例显著升高（Z=‒2.199,P=0.030）。结论 剖宫产能显著影响婴儿早期肠道菌群的定植,降低肠道中早期拟杆菌的水平。     

蛋白质序列中的关联规则发现及其应用

随着蛋白质序列-结构分析中使用的机器学习算法越来越复杂,其结果的解释和发现过程也随之复杂化,因此有必要寻找简单且理论上可靠的方法。通过引入原理简单、理论可靠、结果具有很强实际意义的关联规则发现算法,找到了蛋白质序列中数以万计的模式。结合实例演示了如何将这些模式应用于蛋白质序列分析中,如保守区域发现、二级结构预测等。同时根据这些结果构建了一个二级结构规则库和一种简单的二级结构预测算法,实验结果表明,约81%的二级结构可以由至少一条关联规则预测得到。     

The impact of next-generation sequencing on genomics

This article reviews basic concepts,general applications,and the potential impact of next-generation sequencing(NGS)technologies on genomics,with particular reference to currently available and possible future platforms and bioinformatics.NGS technologies have demonstrated the capacity to sequence DNA at unprecedented speed,thereby enabling previously unimaginable scientific achievements and novel biological applications.But,the massive data produced by NGS also presents a significant challenge for data storage,analyses,and management solutions.Advanced bioinformatic tools are essential for the successful application of NGS technology.As evidenced throughout this review,NGS technologies will have a striking impact on genomic research and the entire biological field.With its ability to tackle the unsolved challenges unconquered by previous genomic technologies,NGS is likely to unravel the complexity of the human genome in terms of genetic variations,some of which may be confined to susceptible loci for some common human conditions.The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come.     

儿茶素与金属离子相互作用及其生物学意义

儿茶素的医学保健功效已经得到广泛证实,其与金属离子的相互作用也随之成为无机生物化学和医学等交叉研究领域的热点。综述了儿茶素与金属离子的相互作用及其生物学意义,并对其研究前景进行了展望。     

Studies on rDNA ITS1 Regions of Soybean and Its Wild Relatives

The rDNA internal transcribed spacer 1 (ITS1) regions of wild soybean (Glycine soja), semiwild soybean (G. gracillis), perennial wild soybean (G. tomentella, G. tabacina) and two accessions of cultivated soybean (G. max) were amplified by PCR and cloned. The copy number in soybean genome was about 2 × 103. Sequence analysis showed that the G/C content of G. soja (61.40%), G. gracillis (61.40%), G. max (61.40%), G. tabacina (58.11%) and G. tomentella (59.01%) were very similar to that of Phaseolus radiatus (59.81%), and the G/C content of G. tabacina was the lowest one in all known ITS1 re- gions of plants. Maxium-homology analysis proved that the ITS1 sequence of soybean was the most homologous to its counterpart of P. radiatus. It was implied that the homology of ITS1 regions of relative species were related with the genetic relationships among these species. Sequence analysis disclosed that there were two conserved sequences (GACCCGC- GAA) and (GCGCCAAGGAA) in all sequenced ITS1 regions of plants.     

Synthesis,Characterization, and Biological Study of 3-Trifluoromethylpyrazole Tethered Chalcone-Pyrrole and Pyrazoline-Pyrrole Derivatives

The present study illustrates the design and synthesis of new series of 3-trifluoromethylpyrazole tethered chalcone-pyrrole and pyrazoline-pyrrole derivatives. All compounds were further screened for in vitro cytostatic activities on full NCI 60 cancer cell lines at National Cancer Institute, USA. Compounds (2E)-3-(1H-pyrrol-2-yl)-1-{4-[3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}prop-2-en-1-one ( 5a ) and (2E)-1-{3-methyl-4-[3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-3-(1H-pyrrol-2-yl)prop-2-en-1-one ( 5c ) displayed significant antiproliferative activity (Growth Percentage: −77.10 and −92.13, respectively at 10 μM concentration) against the UO-31 cell lines from renal cancer and were further selected for assay at 10-fold dilutions of five different concentrations (10⁻⁴ to 10⁻⁸ M). Both compounds 5a and 5c exhibited promising antiproliferative activity (GI₅₀: 1.36 to 0.27 μM) against leukemia cancer cell lines HL-60 and RPMI-8226, colon cancer cell lines KM-12; breast cancer cell lines BT-549. Moreover, both compounds 5a and 5c were found to be non-cytotoxic (LC₅₀>100) against HL-60, RPMI-8226, and KM-12 cell lines. Remarkably, GI₅₀ values of compounds 5a and 5c were identified as more promising than sunitinib against most cancer cell lines. In silico study of compounds 5a and 5c exemplified the desired ADME properties for drug-likeness as well as tighter interactions with VEGFR-2. Hence, compounds 5a and 5c would be good cytotoxic agents after further clinical study.     

利用AFLP分析西南特色猕猴桃的遗传多样性

为分析西南地区特色猕猴桃(Actinidia Lindl.)的遗传多样性,建立并优化了猕猴桃DNA 多态性分析的AFLP 体系,AFLP 标记体系为300 ng 基因组DNA 用EcoR Ⅰ/Mse Ⅰ (15 U/5 U)于37℃下酶切2 h,加接头后的混合物稀释5 倍用于预扩增,预扩增产物再稀释10 倍后进行选择性扩增.结果表明,筛选的22 对引物进行扩增反应,共得到979 条条带,其中多态性条带为649 条.采用优化的AFLP 体系,对西南地区的10 种猕猴桃50 个样本进行了遗传多样性分析,包括普通品种中华猕猴桃、美味猕猴桃,及特色品种硬毛猕猴桃、毛被猕猴桃、革叶猕猴桃、四萼猕猴桃、狗枣猕猴桃、葛枣猕猴桃、京梨猕猴桃和紫果猕猴,结果表明种内和种间的聚类关系明显;斑果组(sect. Maculatae)和净果组(sect. Leiocarpae)的种间有聚类交叉,并呈现地理区域性分布.     

环形RNA全长组装与定量工具的研究进展

环形RNA是一种广泛存在于真核细胞的内源性RNA,由前体RNA反向剪接而成,不具有5’末端帽子和3’末端poly(A)尾巴,呈封闭环状结构。环形RNA通过miRNA海绵结合等方式参与基因表达调控等许多重要的生物学过程。环形RNA可以通过可变剪接产生不同的环形RNA转录本,因此获取环形RNA转录本内部全长序列信息以及对环形RNA内部可变剪接产物进行精确定量是揭示环形RNA调控功能的前提。生物信息学工具能够高效便捷的处理高通量测序数据,被普遍用来鉴别和分析环形RNA。本文介绍了环形RNA的产生机制以及功能特性,对环形RNA检测、全长序列组装以及定量相关计算工具进行综述。     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

枯草杆菌启动子－信号肽序列的克隆及序列分析

利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序,结果表明,克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。     

罗伊乳杆菌对小鼠血清细胞因子含量和肠道菌群的影响

目的 探讨罗伊乳杆菌对小鼠免疫力及肠道菌群的影响。方法 将50只昆明系小鼠按体重随机分成5组：空白对照组、低剂量组、中剂量组、高剂量组和发酵上清液组。空白对照组灌胃等量生理盐水,其余各组分别灌胃罗伊乳杆菌菌液及发酵液上清21 d后,眼球取血收集血清,采用ELISA法检测IL-2、IFN-γ、IgA和IgG含量;采用16S rRNA基因高通量测序分析小鼠肠道菌群变化。结果 与对照组相比,其余各组小鼠血清IL-2、IFN-γ、IgA和IgG的水平均明显升高,粪便菌群丰度有所增高,多样性降低。与对照组相比,低剂量组和中剂量组小鼠粪便菌群结构无显著差异,而高剂量组和发酵上清液组小鼠粪便菌群结构差异显著。结论 罗伊乳杆菌可以增强小鼠免疫力,提高菌群丰度,改变小鼠肠道菌群结构。     

Impacts of low coverage depths and post-mortem DNA damage on variant calling: a simulation study

Background

Massively parallel sequencing platforms, featuring high throughput and relatively short read lengths, are well suited to ancient DNA (aDNA) studies. Variant identification from short-read alignment could be hindered, however, by low DNA concentrations common to historic samples, which constrain sequencing depths, and post-mortem DNA damage patterns.

Results

We simulated pairs of sequences to act as reference and sample genomes at varied GC contents and divergence levels. Short-read sequence pools were generated from sample sequences, and subjected to varying levels of “post-mortem” damage by adjusting levels of fragmentation and fragmentation biases, transition rates at sequence ends, and sequencing depths. Mapping of sample read pools to reference sequences revealed several trends, including decreased alignment success with increased read length and decreased variant recovery with increased divergence. Variants were generally called with high accuracy, however identification of SNPs (single-nucleotide polymorphisms) was less accurate for high damage/low divergence samples. Modest increases in sequencing depth resulted in rapid gains in total variant recovery, and limited improvements to recovery of heterozygous variants.

Conclusions

This in silico study suggests aDNA-associated damage patterns minimally impact variant call accuracy and recovery from short-read alignment, while modest increases in sequencing depth can greatly improve variant recovery.

     

高通量扩增子测序分析生防细菌对丹参根际和根表土壤真菌群落多样性的影响

利用高量测序方法探究生防细菌对丹参植株根际和根表土壤真菌群落多样性的影响。

向丹参植株根部施入生防细菌DS-R5,培养45 d后采集根际和根表土壤样品提取总DNA,扩增样品基因组DNA的V4―V5区后进行双端测序,利用生物信息学解析生防细菌对丹参植株根际和根表土壤真菌群落多样性的影响。

菌株DS-R5处理后增加了根际和根表土壤真菌群落的多样性和丰度;根际土壤共有物种种类大于根表土壤,说明菌株DS-R5处理后根际土壤处理与对照物种种类更接近,而对根表土壤中的微生物物种影响较大。真菌群落结构组成分析结果表明,不同土壤样品在门水平上共有优势真菌主要有子囊菌门、接合菌门、担子菌门和未分类;相比根表土壤对照样品,根表土壤处理样品中子囊菌门丰度下降了13.0%,接合菌门丰度升高了69.2%;根际土壤处理样品相比根际土壤对照样品,子囊菌门和接合菌门丰度分别升高了5.9%和8.9%,但二者差异无统计学意义。在属水平上,根表土壤样品经菌株DS-R5处理后提高了有益菌属的丰度,同时降低了有害菌属的丰度。

丹参植株施入生防细菌后,改变了根际土壤和根表土壤中微生物群落结构和多样性,本研究结果可以为利用生防细菌防控丹参根腐病提供理论参考。

     

The Impact of Non-cultivated Biodiversity on Enzyme Discovery and Evolution

The search for novel enzymes with biotechnological potential in the fine chemical, food and feed, detergent and cosmetics industries is driven by the need to improve existing processes and applications, to design novel processes for innovative products or intermediates or to avoid intellectual property related operative restrictions. Strategies for obtaining novel biocatalysts will be based on screening natural biodiversity or a combination of nature derived scaffolds and optimization by directed evolution technology. Considering the enormous potential of in vitro mutational and recombinatorial strategies to alter genes and improve enzyme properties, we propose that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance.     

测序深度对RNA编辑识别算法的影响

RNA编辑是重要的转录后修饰过程,目前已有多种算法用于识别RNA编辑,本文主要研究小鼠中测序深度对RNA编辑识别算法的影响,从而为RNA编辑的研究给出建议的方法. 本文使用STAR比对软件将小鼠的RNA-seq数据进行序列比对,然后使用GATK识别SNV,并用Separate Method、GIREMI、RNAEditor 3种方法识别出RNA编辑位点. 最后对3种方法识别RNA编辑位点的共同部分、识别效率、识别稳定性、识别与测序深度的关系进行分析. 结果发现3种方法识别的编辑位点数目差异大,共有位点较少,随着测序深度的增加,识别的RNA编辑位点数也在增加. 结果表明RNA编辑识别算法在小鼠中的识别性能与测序深度呈正相关.     

The Impact of Non-cultivated Biodiversity on Enzyme Discovery and Evolution

Abstract

The search for novel enzymes with biotechnological potential in the fine chemical, food and feed, detergent and cosmetics industries is driven by the need to improve existing processes and applications, to design novel processes for innovative products or intermediates or to avoid intellectual property related operative restrictions. Strategies for obtaining novel biocatalysts will be based on screening natural biodiversity or a combination of nature derived scaffolds and optimization by directed evolution technology. Considering the enormous potential of in vitro mutational and recombinatorial strategies to alter genes and improve enzyme properties, we propose that it might be advantageous to select improved molecular starting points before embarking on the arduous walk through sequence space towards optimized performance     

Diversity of epiphytic fungi on the surface of Kyoho grape berries during ripening process in summer and winter at Nanning region,Guangxi, China

The two-harvest-per-year farming system allow table grape to be harvested a year both in summer and winter in southern China. Herein, we used high-throughput sequencing to investigate the diversity of fungi on grape fruits surface during the ripening process in summer and winter at subtropical Nanning region, Guangxi, China. The results showed that 23 fungal species existed in all samples. Among them, the five most dominant species were Cladosporium ramotenellum, Pseudozyma aphidis, Gyrothrix spp., Gibberella intricans and Acremonium alternatum, with abundance from 61.62 % to 91.26 %. Analysis using the student's t-test for Shannon index indicated that components of fungal community varied significantly between the two ripening seasons. The dominant genera of core fungal community were Cladosporium, Gyrothrix, Paramycosphaerella, Acremonium, Penicillium and Tilletiopsis in the summer and Cladosporium, Pseudozyma, Gibberella, Colletotrichum, Sporobolomyces, Rhodosporidium, Alternaria and Aspergillus in the winter. Overall, fungi diversity on grape fruits surface at Nanning showed significantly differences between different ripening seasons. Our results ennrich the understanding of epiphytic communities of grape fruits in subtropics.     

“反向免疫学”与免疫功能基因的发现

人类基因组测序的结束为生命科学领域展开了全新的一章.尽管已经得到了人类基因组的序列,但是隐藏在DNA序列中的功能基因,它们之间的相互作用以及对整个机体的意义大部分有待发掘.近些年来形成的“反向生物学”为免疫功能基因的发现、功能的研究及其应用价值提供了一套全新的技术与思路.