基于转录组测序数据识别黑猩猩RNA编辑位点

使用转录组测序(RNA-Seq)数据识别黑猩猩RNA编辑位点,探索了RNA编辑的识别机制以及潜在的功能影响．基于黑猩猩RNA-Seq数据与基因组序列的比对信息发现RNA-DNA错配位点,并构建编辑位点候选集．从中滤除基因组或转录组测序质量低的位点,其他的过滤条件包括3′端测不准、覆盖度、SNP位点以及估算的编辑水平．构建二项分布统计模型和Bonferroni多重检验滤除候选集中的随机错误,得到RNA编辑位点．选取落在已知基因上的编辑位点进行功能分析,并用Two Sample Logo软件分析编辑位点上下游序列的特征．识别出黑猩猩12种碱基替换型RNA编辑位点8 334个,其中有41个编辑位点改变原有的氨基酸,另有3个编辑位点落在microRNA(miRNA)潜在靶基因的种子结合区．统计学分析表明,分别有640和872个RNA编辑位点存在组织和性别差异．上下游碱基频率分析表明,多种类型的编辑位点紧邻碱基具有显著偏好．结果显示, RNA编辑在黑猩猩体内大量存在,且潜在具有重要的生物学功能,为进一步深入研究灵长类RNA编辑的机制奠定了基础．     

高通量转录组测序技术在植物雄性不育研究中的应用

植物雄性不育是指植物雄蕊发育受阻不能产生正常有功能花粉的现象。植物雄性不育不仅是生殖生理研究的宝贵材料,也是植物杂种优势利用的重要工具。由于高通量转录组测序技术几乎可以检测细胞内所有mRNA及非编码RNA的信息,已被广泛应用于生命科学研究的各项领域。在植物雄性不育相关研究中,高通量转录组测序技术在不同物种、不同败育类型中的应用已有报道,这为研究者在转录组水平综合了解植物雄性不育的分子机制及代谢网络提供了帮助。本文从测序文库构建策略、差异表达基因、非编码RNA的功能特征等方面综述了高通量转录组测序在植物雄性不育机理方面的研究进展,并探讨了转录组测序技术在花粉败育机制解析及育性相关基因定位中的应用价值,以期为植物雄性不育的相关研究提供参考。     

小花草玉梅高通量转录组测序与花发育基因的挖掘

该研究采用高通量测序技术Illumina HiSeq 2000,对小花草玉梅花器官进行转录组测序,挖掘参与其花发育相关的基因。测序结果得到54 513 822个序列读取片段(reads),包含7 826 726 115bp的碱基序列信息。将测序数据进行序列组装后,获得43 767个单基因簇(unigenes),平均长度为926bp。转录物注释结果显示,28 130条unigenes有同源比对信息。在43 767条unigenes中检测到5 015个SSR位点,且SSR不同重复基序类型中,出现频率最高的是AG/CT,其次是AAG/CTT和ACC/GGT。通过转录因子分析,进一步筛选得到了12个与花发育紧密相关的MADS基因,分别为FUL1,FUL2,AP3-1,AP3-2,AP3-3,PI1,PI2,AG1,AG2,SEP1,SEP3和AGL6。实时荧光定量PCR分析表明,与小花草玉梅正常花相比,全白、绿白相间、五瓣变异、全绿变异花中的FUL1、SEP1,SEP3和AGL6基因均显著上调表达,而12个基因在极端变异花中的表达水平与正常花的差异均不明显。定量结果经主成分分析显示,AGL6、SEP3、FUL1、PI2及SEP1的表达量均为小花草玉梅花形态建成的主要指标。研究结果在一定程度上丰富了小花草玉梅的基因信息,为后续研究其花器官变异的分子机制提供了基础数据。     

基于高通量测序的麦红吸浆虫转录组分析

麦红吸浆虫Sitodiplosis mosellana(Gehin)是一种世界性的小麦害虫。为获得其转录组信息,本研究采用新一代高通量测序技术Illumina HiSeq TM 2000对麦红吸浆虫成虫转录组进行测序。共获得转录组样本数据量为27.88 G,经分析共获得59257个Unigenes,总长度49861164 bp,最短20 bp,最长29282 bp,平均长度841 bp。将Unigenes序列与NR、NT、Swiss-Prot、KEGG、GO和KOG数据库进行比对(e≤10-10),共获得95029个结果。通过GO功能分类,共有19584个Unigenes在GO数据库中细胞组分、分子功能和生物学过程等3大类50个功能组中找到对应。与KOG数据库进行比对,共有11279个麦红吸浆虫Unigenes被注释,按功能大致可分为26类。通过KEGG pathways分析,共有9110个麦红吸浆虫Unigenes被注释,分别归属于细胞进程、环境信息进程、遗传信息进程、新陈代谢和有机体系统5大类代谢途径,主要包括细胞生长与死亡、细胞运动、信号转导、能量代谢等32类代谢途径。CDS预测发现30088条序列可被编码,占全部基因的50.78%。SSR位点查找发现,在59257个Unigenes中共找到36323个SSR位点,发生率为61.30%。本研究获得的巨大的麦红吸浆虫转录组信息,为麦红吸浆虫的功能基因挖掘提供了重要的信息资源。     

基于高通量测序的玉米象转录组分析

玉米象Sitophilus zeamais是一种世界性的储粮害虫,但其基因信息尚不完善.本研究利用高通量测序平台Illumina HiSeq TM2000对玉米象成虫进行转录组测序,总计获得64358条unigenes,总长度39481752 bp,最短201 bp,最长29046 bp,平均长度613 bp.将unigenes序列在7大数据库Nr、GO、Swissprot、KOG、Pfam、KEGG、TrEMBL中进行注释,分别有25271、20747、16477、12060、9471、3370、25500条unigenes获得注释.通过GO功能分类,共有20747条unigenes在GO数据库中生物学过程、细胞组分、分子功能3大类68个亚类功能组中找到对应.KOG结果显示,12606条unigenes归到25个基因家族.通过KEGG代谢通路分析,共有3370条unigenes被注释,分别归属于细胞进程、环境信息进程、遗传信息进程、新陈代谢和有机体系统5大类代谢途径,共33个亚类274个功能通路.进一步的数据分析,共鉴定出146081个SNP位点和5002个简单序列重复(SSR)位点.本研究获得的玉米象转录组信息,为玉米象的功能基因挖掘提供了重要的信息资源.     

基于转录组测序的扎龙湿地野大麦耐盐性分析

通过转录组测序技术对扎龙湿地野大麦幼苗在200 mmol/L NaCl胁迫2 d后的叶片进行分析。对测序结果进行De novo拼接后,将差异表达基因在GO、KEGG数据库中进行比对注释。结果表明:NaCl胁迫2 d后,对照组与处理组分别获得Unigene序列61038个和46754个,检测到差异表达基因25465个,差异基因GO功能注释到3个大类的55个功能组。差异表达基因被注释到135个Pathway上,直观地显示出NaCl胁迫下野大麦幼苗体内发生调节及改变的代谢过程和信号通路,其中主要涉及光合作用、脯氨酸代谢、叶绿素代谢等途径。通过对野大麦幼苗叶片转录组分析,发掘相关耐盐基因,有助于培育野大麦及其近缘作物新品种,并对揭示植物耐盐性分子机制及相关代谢途径具有重要意义。     

dRNA-seq原理及其在原核生物转录组学研究中的应用

第二代测序技术不仅推进了基因组学的研究, 而且也广泛应用到转录组学的研究中。原核生物转录组学的研究方法主要有Tiling芯片和RNA-seq, 后者因其较高的覆盖率和分辨率以及越来越低廉的成本而逐渐被更多的研究单位采用。根据对mRNA富集方法或rRNA去除方法的不同, 目前RNA-seq方法主要有6种, 其中dRNA-seq由于采用了5′单磷酸依赖的外切酶, 可以特异性地降解加工过的转录本, 从而使初始转录本得到富集, 已被广泛应用到原核生物转录起始位点、小的调控RNA、启动子及操纵子等研究中。文章对dRNA-seq的原理、技术流程及其在原核生物转录组学研究中的应用进行了综述, 并对这一研究方法在现阶段应用的优势和局限性进行了讨论, 也进一步对该方法在未来的发展和应用进行了展望, 期望为国内相关领域研究人员提供参考。     

高通量转录组测序技术及其在鳞翅目昆虫上的应用

转录组是指组织或者细胞在某一特定状态下转录出来的所有RNA的集合。高通量第2代测序技术使转录组学的研究模式发生了巨大的改变,所衍生出的转录组测序迅速成为研究非模式生物的先进技术。转录组测序能够在整体水平上探究细胞内基因表达的种类和数量,揭示在特定条件下机体生理生化发生过程以及其中的分子机理。本文简要阐述了转录组测序技术的基本概念、技术流程与原理,详细介绍了转录组测序在解决鳞翅目昆虫的分类、毒理、发育、与寄主互作以及非编码RNA调控等问题上做出的贡献,并对该技术现存的困难进行了系统的阐述并对其未来的发展趋势作出了简要的预测与剖析。     

Complete genome sequence of Vibrio vulnificus MO6-24/O

Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.     

IVET-based identification of virulence factors in Vibrio vulnificus MO6-24/O

Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V. vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of approximately 18,000 resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of 1.6 x 10(6). Two hours after infection, kanamycin was administered at 200 microg per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.     

Identification of the cadBA operon from Vibrio vulnificus and its influence on survival to acid stress

By a transposon-tagging method, cadBA genes encoding a lysine/cadaverin antiporter and a lysine decarboxylase were identified and cloned from Vibrio vulnificus. The deduced amino acid sequences of cadBA were 64-97% similar to those reported from other Enterobacteriaceae. Functions of cadBA genes on acid tolerance were assessed by comparing acid tolerances of V. vulnificus and its isogenic mutants, whose cadBA genes were separately inactivated by allelic exchanges. The results demonstrated that gene products of cadBA contribute to acid tolerance of V. vulnificus, and that their contribution is dependent on prior exposure of cells to moderately acidic pH.     

Isolation and characterization of hemolysin mutants of Vibrio vulnificus

Abstract The importance of the cytolysin/hemolysin in the virulence of Vibrio vulnificus was investigated using both the naturally occuring virulent and avirulent colony variants and ethylmethane-sulfonate generated mutants. Both virulent and avirulent isogenic morphotypes produced similar amounts of hemolysin. Two mutants deficient in the production of hemolysin and negative for CHO cell activity were characterized and their virulence for mice was examined. Non-hemolytic mutants were found to be as virulent as their parent strain. It is concluded that the hemolysin produced by V. vulnificus is not required for the full virulence of this pathogen.     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Nonribosomal peptide synthase is responsible for the biosynthesis of siderophore in Vibrio vulnificus MO6-24/O

Vibrio vulnificus produces siderophores, lowmolecular- weight iron-chelating compounds, to obtain iron under conditions of iron deprivation. To identify genes associated with the biosynthesis of siderophore in V. vulnificus MO6-24/ O, we screened clones with mini-Tn5 random insertions for those showing decreased production of siderophore. Among 6,000 clones screened, nine such clones were selected. These clones contain the transposon inserted in VV2_0830 (GenBank accession number) that is a homolog of a nonribosomal peptide synthetase (NRPS). There is an another NRPS module, VV2_0831, 49-bp upstream to VV2_0830. We named these two genes vvs (Vibrio vulnificus siderophore synthetase) A and B, respectively. Mutation of either vvsA or vvsB showed a decreased production of siderophore. The expression of an NRPS-lux fusion was negatively modulated by the presence of iron, and the regulation was dependent on Fur (ferric uptake regulator). However, the expression of the NRPS genes was still not fully derepressed in the iron-rich condition, even in fur-null mutant cells, suggesting that some other unknown factors are involved in the regulation of the genes. We also demonstrated that the NRPS genes are important for virulence of the pathogen in a mice model.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N -butanoyl-homoserine-lactone (C₄-HL) by both reporter strains and by HPLC-high-resolution MS. C₄-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain–heart infusion) and its production was enhanced when blood factors were added to MSWYE. C₄-HL was detected in vivo , in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.