意大利蜜蜂工蜂中肠的环状RNA及其调控网络分析

【目的】环状RNA(circRNA)在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在分析意大利蜜蜂Apis mellifera ligustica工蜂中肠circRNA的数量、种类、结构特征和作用,并通过构建和分析circRNA的调控网络探索circRNA的调控功能。【方法】在实验室条件下人工饲养意大利蜜蜂工蜂,利用circRNA-seq技术对意大利蜜蜂7和10日龄成年工蜂中肠样品进行深度测序。利用find_circ软件从质控后的数据中预测circRNA。通过BLAST比对GO和KEGG数据库,对circRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测circRNA靶向结合的miRNA及miRNA靶向结合的mRNA,通过Cytoscape v.3.2.1软件对circRNA-miRNA和circRNA-miRNA-mRNA调控网络进行构建及可视化。通过设计背靠背引物和线性扩增引物RT-PCR对预测出的circRNA进行验证。【结果】意大利蜜蜂工蜂中肠样品的测序平均得到136 463 071条clean reads,去除rRNA后各样品的anchor reads均在136 779 122条及以上。共预测出10 833个circRNA,长度主要介于15~1 000 nt;上述circRNA的类型丰富,其中已注释的外显子circRNA数量最多,分布在西方蜜蜂1号染色体的circRNA数量最多,其次为8号染色体。CircRNA的来源基因可注释到包括结合、细胞进程和细胞在内的45个GO条目,以及包括内吞作用、内质网蛋白加工及核糖体在内的121条KEGG代谢通路,表明circRNA在意大利蜜蜂工蜂中肠的生长、发育、新陈代谢和细胞生命活动等生物学过程中发挥重要作用。进一步构建circRNA-miRNA和circRNA-miRNA-mRNA调控网络,分析结果显示部分circRNA可能作为竞争性内源RNA吸附结合microRNA,从而调控基因的表达水平。最后,对随机选择的3个circRNA的RT-PCR结果验证了其真实存在。【结论】本研究对意大利蜜蜂工蜂中肠中的circRNA进行预测、分析及鉴定。研究结果提供了中肠circRNA的数量、种类、结构特征、作用和调控网络的信息,揭示了circRNA可能通过作用于来源基因和作为竞争性内源RNA在意大利蜜蜂工蜂中肠的生长发育和免疫防御中发挥作用,为深入研究circRNA在意大利蜜蜂中肠发育及胁迫响应过程中的功能奠定了基础。     

东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-10660及其靶基因表达谱

【目的】本研究旨在为探究nce-miR-10660调控东方蜜蜂微孢子虫Nosema ceranae侵染的作用机制提供理论和实验依据。【方法】采用Stem-loop RT-PCR对前期鉴定到的东方蜜蜂微孢子虫nce-miR-10660进行表达验证，再通过Sanger测序验证nce-miR-10660的序列。利用相关生物信息学软件预测和分析nce-miR-10660的靶基因。通过RT-qPCR检测nce-miR-10660及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程中中肠中的表达谱。【结果】Stem-loop RT-PCR和Sanger测序结果分别证实了nce-miR-10660在东方蜜蜂微孢子虫孢子中的表达和真实存在。靶向预测结果显示nce-miR-10660共靶向RRDRP和RCDP 42等9个基因；分别有2和6个靶基因可被分别注释到KEGG数据库中的4条通路和GO数据库中的23个条目。RT-qPCR结果显示，相较于东方蜜蜂微孢子虫侵染后1 d时意大利蜜蜂工蜂中肠中nce-miR-10660的表达量，东方蜜蜂微孢子虫侵染后2 d时意...     

亚致死剂量吡虫啉胁迫对意大利蜜蜂工蜂嗅觉学习行为的影响及其脑部转录组分析

【目的】分析吡虫啉处理对意大利蜜蜂Apis mellifera ligustica嗅觉学习行为及脑部基因转录的影响,为新烟碱类杀虫剂对蜜蜂的负面影响提供依据。【方法】实验室条件下一次性饲喂意大利蜜蜂成年工蜂含有4 ng吡虫啉的50%蔗糖溶液,以饲喂不含吡虫啉的50%蔗糖溶液为对照,通过伸吻反应(proboscis extension response, PER)行为实验测定其对意大利蜜蜂成年工蜂嗅觉学习行为的影响;收集上述测试的意大利蜜蜂工蜂脑部提取RNA,进行RNA-Seq测序和生物信息学分析,利用实时荧光定量PCR挑选6个差异表达基因(DEGs)检测其在脑部的表达量以验证RNA-Seq测序结果。【结果】一次性饲喂意大利蜜蜂成年工蜂含4 ng吡虫啉的50%蔗糖溶液后,意大利蜜蜂的嗅觉学习能力与对照组(饲喂50%蔗糖溶液)相比显著降低。RNA-Seq测序结果显示饲喂意大利蜜蜂含4 ng吡虫啉蔗糖溶液后,处理组与对照组之间共有123个DEGs［校正后的P值(padj)<0.05)］,包括82个下调DEGs和41个上调DEGs。GO聚类分析发现下调DEGs主要富集在S-腺苷甲硫氨酸依赖性甲基转移酶活性、酸性磷酸酶活性、氧化还原酶活性、蛋白质异二聚化活性等,上调DEGs主要富集在跨膜受体活性、分子传感器活性、神经学系统过程等功能条目。KEGG富集分析显示下调DEGs主要富集在核糖体和溶酶体等细胞器、碳代谢和色氨酸代谢等代谢通路及Toll和Imd信号通路,上调DEGs未富集在KEGG代谢通路。荧光定量PCR结果显示测试的6个DEGs相对表达量变化趋势与RNA-Seq测序结果FPKM(fragments per kilobase million)值变化趋势一致,证明RNA-Seq测序结果可靠。【结论】亚致死剂量吡虫啉胁迫意大利蜜蜂成年工蜂显著降低意大利蜜蜂的嗅觉学习能力,影响意大利蜜蜂脑部免疫解毒等相关基因的表达和酶活性及氧化还原等生物代谢过程;亚致死剂量吡虫啉短时胁迫会刺激意大利蜜蜂的嗅觉感觉过程及神经信号传导过程。     

意大利蜜蜂幼虫肠道响应球囊菌早期胁迫的转录组学分析

【目的】球囊菌特异性地侵染蜜蜂幼虫而导致白垩病,该病造成成年蜜蜂数量的大幅下降,从而严重影响蜂蜜等产品的产量。目前,尚无利用二代测序技术研究白垩病的报道。本研究利用RNA-seq技术对正常及球囊菌胁迫后的意大利蜜蜂4日龄幼虫肠道进行深度测序,为解析宿主响应球囊菌胁迫的应答机制提供了重要的参考信息。【方法】利用Illumina Hiseq2500平台对对照组(AmCK)和处理组(AmT)幼虫肠道进行双端(PE125)测序;利用Perl脚本进行下机数据过滤;利用R软件进行测序饱和度分析、计算各样品之间的相关性系数;利用SOAP aligner/soap2软件将未比对上核糖体的读段(Reads)比对到意大利蜜蜂参考基因组;基于GO数据库和KEGG数据库进行GO分类和KEGG代谢通路(Pathway)富集分析。【结果】RNA-seq共得到181 096 194条原始读段(Raw reads),经过滤得到179078764条有效读段(Clean reads)。差异表达基因(DEGs)分析结果显示上调与下调基因的数量分别为4和20个。DEG的基因本体论(Gene ontology,GO)分析结果显示,这些DEGs分布于10个GO分类,除归入结合(Binding)的基因和催化活性(Catalytic activity)的部分基因外,绝大多数GO分类上的基因均表现为下调。DEGs的KEGG代谢通路分析结果显示各有1个DEG分别富集在核糖体和氧化磷酸化且均下调表达。【结论】本研究揭示了意大利蜜蜂幼虫肠道在球囊菌入侵早期的胁迫应答,为解析宿主响应球囊菌胁迫的应答机制奠定了基础。     

意大利蜜蜂工蜂中肠响应Nosema ceranae胁迫的高表达基因分析

东方蜜蜂微孢子虫Nosema ceranae是专性寄生蜜蜂中肠上皮细胞的单细胞真菌,对意大利蜜蜂(意蜂)具有较强的侵染性。本研究利用RNA-seq技术对正常10 d (Apis mellifera ligustica control group,Am CK)和N. ceranae胁迫10 d的意蜂工蜂中肠(Apis mellifera ligustica treatment group,Am T)进行测序,共得到160 847 237 100条原始读段,过滤后得到1 066 955 298条有效读段。主成分分析结果显示Am CK与Am T测序样品的组内重复性较好,组间的基因表达模式差异明显。GO分类结果显示,Am CK与Am T的前100位高表达基因(HEGs)分别分布于32和33个GO terms,基因富集数最多的均为代谢进程。KEGG代谢通路(pathway)富集分析结果显示,Am CK的前100位HEGs富集在21个pathways,基因富集数最多的是内吞作用、信号通路和嘌呤代谢; Am T的前100位HEGs富集在26个pathways,基因富集数最多的是内吞作用、RNA转运和蛋白质的内质网加工。前100位HEGs的Venn分析结果显示Am CK与Am T的共有HEGs为87个,特有HEGs分别均为13个。研究结果揭示了N. ceranae胁迫意蜂工蜂中肠过程中的基因表达谱信息,也为解析N. ceranae致病的分子机理提供了有益信息和线索。     

意大利蜜蜂铁蛋白基因AmFer3HCH的克隆及其在温度胁迫下的应答

【目的】本研究旨在克隆鉴定意大利蜜蜂Apis mellifera ligustica铁蛋白基因Am Fer3HCH,分析其在意大利蜜蜂应对温度胁迫时的组织表达变化,探讨铁蛋白Am Fer3HCH在蜜蜂应对环境温度胁迫中的作用。【方法】PCR扩增意大利蜜蜂铁蛋白基因Am Fer3HCH的cDNA序列,并对其编码氨基酸序列进行生物信息学鉴定;实时定量PCR分析该基因在不同温度(40℃和5℃)胁迫下的意大利蜜蜂工蜂成虫胸、中肠和脂肪体中的表达变化。【结果】PCR扩增获得意大利蜜蜂铁蛋白基因Am Fer3HCH,cDNA片段大小为507 bp,其核苷酸序列与GenBank意大利蜜蜂基因组数据库中的铁蛋白基因Fer3HCH(GenBank登录号:GB55416)的完全一致,确认为Am Fer3HCH基因序列。Am Fer3HCH鉴定为重链型铁蛋白,在系统进化上聚类于重链型铁蛋白分支,其氨基酸序列具有重链型铁蛋白特有的Fe2+氧化酶活性中心,含有铁蛋白家族成员所共有的水合氧化铁成核中心和铁离子通道。实时定量PCR分析显示,与对照组相比在短时高温(40℃,0~48 h)胁迫过程中,Am Fer3HCH在意大利蜜蜂工蜂成虫胸、中肠和脂肪体中的表达水平整体呈先上升、后下降再上升的趋势;在低温(5℃,0~6 h)胁迫处理过程中,中肠和脂肪体中Am Fer3HCH基因的表达量呈先上升后下降趋势,而在胸中表达量逐渐下降。【结论】意大利蜜蜂铁蛋白Am Fer3HCH为重链型铁蛋白,在意大利蜜蜂对极端温度胁迫的应答中扮演了重要的角色。     

基于中肠代谢组学意大利蜜蜂工蜂枣花病的发病机理研究

【目的】利用代谢组学分析患枣花病意大利蜜蜂Apis mellifera ligustica工蜂中肠内代谢物变化,挖掘出与蜜蜂枣花病相关的重要代谢通路,旨在揭示蜜蜂枣花病发病机制,为枣花病靶向药物的研发提供理论依据。【方法】分别利用液相色谱-质谱联用(liquid chromatography-mass spectrometry, LC-MS)和气相色谱-质谱联用(gas chromatography-mass spectrometry, GC-MS)技术对患枣花病和健康意大利蜜蜂工蜂进行中肠非靶向代谢组检测;通过主成分分析(principal component analysis, PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA),以变量投影重要性(variable projection importance, VIP)>1.0,差异倍数(fold change, FC)>2.0和P<0.05为标准筛选差异代谢物,并对其进行KEGG注释和通路富集;利用生...     

结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性

【目的】蜜蜂球囊菌(Ascosphaera apis)是一种专性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在利用PacBio单分子实时(singlemoleculereal-time,SMRT)测序技术对蜜蜂球囊菌孢子(AaS)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)以及长链非编码RNA (long non-coding RNA,lncRNA)进行鉴定和分析,进而揭示蜜蜂球囊菌孢子中转录组的复杂性。【方法】采用Suppa软件对蜜蜂球囊菌孢子中基因的AS事件进行鉴定。通过RT-PCR对不同类型的AS事件进行验证。采用TAPIS pipeline对蜜蜂球囊菌孢子基因的APA位点进行鉴定。利用MEME软件分析孢子全长转录本的poly(A)剪接位点上游50bp的序列特征并鉴定motif。联用CPC和CNCI软件和比对Swiss-prot数据库的方法预测lncRNA,取三者的交集作为高可信度的lncRNA集合。进一步比较lncRNA和mRNA的转录本长度,外显子数量与长度,内含子长度,GC含...     

西方蜜蜂工蜂蛹中期响应低温胁迫的差异表达基因分析

【目的】本研究旨在分析西方蜜蜂Apis mellifera工蜂蛹中期可能存在的低温应对机制以及低温对中期蛹发育的不利影响。 【方法】对西方蜜蜂工蜂8 d封盖子进行低温(20℃)处理96 h(T),以未经低温胁迫的8 d封盖子为对照(CK),通过转录组测序(RNA-seq)并筛选差异基因(differentiallyexpressed genes, DEGs);对DEGs进行GO分类和KEGG通路分析。利用RT-qPCR对随机选取的5个DEGs的表达量进行验证。【结果】西方蜜蜂8 d封盖子低温(20℃)胁迫96 h的DEGs有1 101个;GO分类发现DEGs富集数最多的条目为代谢过程(142个DEGs)和细胞过程(142个DEGs),其次是结合(131个DEGs),催化活性(120个DEGs)和单有机体过程(118个DEGs);KEGG通路分析结果显示,DEGs显著富集于与氧化损伤密切相关的过氧化物酶体通路、D-谷氨酰胺代谢通路、D-谷氨酸代谢通路和谷胱甘肽代谢通路;此外,与激素调控相关的昆虫激素代谢通路、mTOR信号通路和FOXO信号通路上也有DEGs显著富集。随机挑选的5个DEGs的RNA-Seq与RT-qPCR结果一致。【结论】本研究发现低温状态下西方蜜蜂中期蛹主要通过体内激素调控发育进程,使其减速或停止发育以应对低温;低温通过影响其抗氧化酶表达量进而可能积累氧化损伤,从而对羽化后蜜蜂产生影响。本研究结果有助于理解发育阶段温度生态幅狭窄的昆虫对于低温的应对机制及低温对其的不利影响。     

ame-miR-14在意大利蜜蜂工蜂幼虫肠道发育过程的调控作用

【目的】本研究旨在揭示ame-miR-14在意大利蜜蜂Apis mellifera ligustica工蜂幼虫肠道发育过程的调控作用。【方法】通过Stem-loop RT-PCR和Sanger测序分别验证ame-miR-14在意大利蜜蜂工蜂6日龄幼虫肠道中的表达和序列真实性。饲喂ame-miR-14的模拟物(mimic-ame-miR-14)和抑制物(inhibitor-ame-miR-14)及其相应的阴性对照mimic-NC和inhibitor-NC对ame-miR-14分别进行过表达和敲降,利用RT-qPCR检测意大利蜜蜂工蜂4-6日龄幼虫肠道中ame-miR-14的表达量。利用生物信息学软件预测ame-miR-14的靶基因并进行相关分析。利用RT-qPCR检测ame-miR-14的过表达和敲降后其靶基因FoxO和Hedgehog在4-6日龄幼虫肠道中的相对表达量。【结果】ame-miR-14在意大利蜜蜂工蜂6日龄幼虫肠道中真实存在和表达。相较于饲喂mimic-NC,饲喂mimic-ame-miR-14后ame-miR-14表达量在意大利蜜蜂工蜂4-6日龄幼虫肠道中均为显著上调;相...     

Uniform hypothesis of cell behavior--movement, contact inhibition of movement, adhesion, chemotaxis, phagocytosis, pinocytosis, division, contact inhibition of division, fusion.

The cell has been represented as a charged liquid drop. Contrary to the DLVO-theory, the effect of the surface potential upon the value of the interfacial tension of the cell membrane has also been taken into consideration. The cell membrane has visco-elastic properties and its constituents may move against each other. Cell movement is caused by the appearance of a small number of the electrically charged constituents of the cell membrane on the leading edge of the cell. This produces a local decrease in the surface tension and the cell membrane expansion. At the moment of contact between two cells proton transfers occur between the strongly negatively charged microvilli of one cell and the body of the other, analogous to a condenser breakdown. This, through the effect on the surface tension, causes contact inhibition of movement. The distribution of the proton dissociable groups modifies the interaction between the cells (differentiation) and between the cell and the substratum (adhesion). Adsorption of the charged compounds at the surface of the cell membrane, decreasing the surface potential and increasing the surface tension, causes the phenomena of chemotaxis, phagocytosis and pinocytosis. Cell division, considered in the terms of the surface energy, requires an adequate supply of considerable quantities of energy inversely proportional to the surface potential value. In case of a reduction of the distance between the cells, their surface potential and the energetic barrier of the cell division processes increases, and causes contact inhibition of cell division. Due to their high charge, division of neoplastic cells is inhibited much later than division of normal cells, or is completely ininhibited due to geometric conditions. Fusion of the cell membrane in the intra-cellular and intercellular processes is a reverse process in relation to the cell division.     

Reliability of Analyses of Hg,Fe, Ca,K, P,pH, Alkalinity,Conductivity, Hardness and Colour from Lakes

The aim of this report has been to present results concerning analytical quality controls of Hg analysis of fish and sediment, analyses of Fe, Ca, total-P, K, pH, alkalinity, conductivity, colour and hardness (Ca + Mg) of lake water samples. Despite the fact that these are standard parameters in many regular water control programs, there are major differences in the reliability with which these parameters can be determined. The focus here is on an overall inter-laboratory comparison between the parameters. Six laboratories have been involved in the analysis. Selected results: pH gives the lowest (average) relative standard deviation (error), about 2 %; conductivity gives an error of about 5–7 %; alkalinity yields an average error of as much as 13–25 %, which is the largest among the parameters studied here; colour also gives a high error, 9–15 %; hardness gives a relative standard deviation of about 6–7 %. Of the other parameters (i. e., Hg, Fe, Ca and P), Hg gives the best reliability and Fe and P the lowest. To have knowledge of the reliability of the analytical data is of paramount importance in most control programs and research projects.     

The Effect of Age and Gender on Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Sr, V, and Zn Contents in Rib Bone of Healthy Humans

The effect of age and gender on major, minor, and trace element contents in the intact rib bone of 80 relatively healthy 15–55-year-old women and men was investigated. Contents or upper limit of contents of 16 chemical elements in the rib bone were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Mean values (M?±?SΕΜ) for the mass fraction of Ba, Ca, Cu, Fe, K, Li, Mg, Na, P, S, Sr, and Zn (milligram per kilogram of dry bone) were as follows: 2.54?±?0.16, 171,400?±?4,050, 1.35?±?0.22, 140?±?11, 1,874?±?71, 0.049?±?0.011, 2,139?±?38, 5,378?±?88, 75,140?±?1,660, 1,881?±?51, 291?±?20, and 92.8?±?1.5, respectively. The upper limits of contents of Al, B, Mn, and V were <7.20, <0.65, <0.36, and <0.03, respectively. Statistically significant tendency for the Ca, Mg, and P content to decrease with age was found in the human rib bone, regardless of gender. The mass fraction of Fe in the male rib bone increases with age. It was shown that higher Ca, Mg, Na, P, and Sr mass fractions as well as lower Fe content were typical of female ribs as compared to those in male ribs.     

Distribution of N,P, K,Ca, Mg,S, Zn,Fe, Mn and Cu in groundnut

Summary Concentration of N, P, K, Ca, Mg and S in summer groundnut crop was higher than in kharif while Zn, Fe, Mn and Cu contents were higher in summer crop. Kernel's N, P and Zn; Leaflet's Ca and Mn; Stem's K and Fe; Root's S and Cu and Petiole's Mg contents were highest. Shell's N, P, K, Mg, S, Zn and Cu; Kernel's Ca, Fe and Mn contents were the least. N, P, K, S, Zn and Cu concentrations decreased linearly as the crop grew. Ca, Mg, Fe and Mn concentrations did not display any distinct pattern. Ca concentration was positively correlated with pod yield in both the seasons.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.