Global identification of human transcribed sequences with genome tiling arrays

Elucidating the transcribed regions of the genome constitutes a fundamental aspect of human biology, yet this remains an outstanding problem. To comprehensively identify coding sequences, we constructed a series of high-density oligonucleotide tiling arrays representing sense and antisense strands of the entire nonrepetitive sequence of the human genome. Transcribed sequences were located across the genome via hybridization to complementary DNA samples, reverse-transcribed from polyadenylated RNA obtained from human liver tissue. In addition to identifying many known and predicted genes, we found 10,595 transcribed sequences not detected by other methods. A large fraction of these are located in intergenic regions distal from previously annotated genes and exhibit significant homology to other mammalian proteins.     

The genome of the sea urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.     

Paleogenomics of echinoderms

Paleogenomics propels the meaning of genomic studies back through hundreds of millions of years of deep time. Now that the genome of the echinoid Strongylocentrotus purpuratus is sequenced, the operation of its genes can be interpreted in light of the well-understood echinoderm fossil record. Characters that first appear in Early Cambrian forms are still characteristic of echinoderms today. Key genes for one of these characters, the biomineralized tissue stereom, can be identified in the S. purpuratus genome and are likely to be the same genes that were involved with stereom formation in the earliest echinoderms some 520 million years ago.     

大豆两个MYB 转录因子基因的克隆及表达分析

 【目的】克隆新的植物MYB转录因子基因,进行序列分析,并对其功能进行初步鉴定。【方法】RT-PCR法结合RACE-PCR法分离克隆MYB基因cDNA全长序列;酵母系统检测其转录激活活性;半定量RT-PCR检测目的基因在植物体中的表达情况,及对类黄酮代谢途径中生物合成酶的影响。【结果】根据植物中MYB基因DNA结合域保守区设计简并引物,以大豆品种中豆27的叶片为材料,RT-PCR扩增出两个MYB基因同源片段;据此设计基因特异引物,通过RACE-PCR分离克隆出两个新的MYB基因GmMYBZ1、GmMYBZ2。酵母系统检测表明,GmMYBZ2具有转录激活功能,β－半乳糖苷酶活性为10.35 U;半定量RT-PCR在中豆27的茎和叶中检测到GmMYBZ1的表达,而GmMYBZ2在植物的根、茎、叶及未成熟种子中均有表达;对转基因烟草的RT-PCR检测结果显示,GmMYBZ2的表达可抑制类黄酮代谢途径中PAL、C4H、4CL、CHS、CHI、F4H及FLS等生物合成酶基因的表达。【结论】从大豆栽培品种中豆27中克隆出了两个新的MYB基因GmMYBZ1、GmMYBZ2;功能研究表明,GmMYBZ2可能参与植物类黄酮合成调控。     

稻瘟病菌菌株FJ81278中新发现的一个基因注释及其表达分析

对稻瘟病菌菌株FJ81278进行基因组和转录组分析,发现一个新的特异基因MoHsbA编码的蛋白具有疏水表面结合蛋白A(HsbA)结构域和一个信号肽.进一步的生物信息学分析发现,该基因组中另外7个基因编码的蛋白也具疏水表面结合蛋白结构域.稻瘟病菌中的HsbA蛋白聚集于系统发育树的同一个大的分支上,说明它们具有较近的亲缘关...     

玉米ZmEXO70s基因家族鉴定及苗期耐热性关联分析

【目的】鉴定玉米EXO70s（ZmEXO70s）基因家族成员,分析其遗传变异与玉米耐热性的关联性,为揭示其在玉米耐热方面的功能和分子机制打下基础。【方法】以拟南芥EXO70s（AtEXO70s）基因家族成员序列为参考,利用MaizeGDB和NCBI数据库从玉米全基因组中鉴定出ZmEXO70s基因家族成员,对其进行基因结构及系统进化分析,根据其在系统发育进化树上的位置对其进行系统命名,并基于转录组测序（RNA-Seq）数据分析ZmEXO70s基因家族成员在不同组织及非生物胁迫下的表达模式。最后,鉴定玉米自交群体AM368中ZmEXO70s基因SNP位点,结合玉米AM368群体苗期耐热存活率进行基因家族关联分析。【结果】从玉米全基因组序列中共鉴定出36个ZmEXO70s基因,分布在10条染色体上,长度为228～3726 bp,编码75～1241个氨基酸残基,蛋白分子量为8.6～136.6 kD,理论等电点（pI）介于4.5～9.9,大部分蛋白pI小于7.0。拟南芥、水稻和玉米的EXO70s蛋白被分为9组（A组～I组）,其中ZmEXO70s蛋白在各组中均有分布。大多数ZmEXO70s基因不含内含子,只有少数基因含有内含子,且内含子数量不同。36个ZmEXO70s蛋白共含8种基序（Motif 1～Motif 8）,主要集中在C端,说明这些蛋白端序列较保守,且Motif 1～Motif 8组成Exo70保守结构域。ZmEXO70s基因在不同组织中的表达水平均存在明显差异,其中ZmEXO70B3基因在雄穗、花药、叶片和花丝中高表达,ZmEXO70C1a基因只在雄穗和花药中高表达,ZmEXO70D2b基因在胚乳和萌发种子中低表达,ZmEXO70G1c基因在花丝和萌发种子中低表达。有23个ZmEXO70s基因至少可响应一种非生物胁迫,说明这些ZmEXO70s基因参与非生物胁迫响应过程。ZmEXO70D2a和ZmEXO70E1基因的遗传变异与玉米苗期耐热性具有显著相关性（P≤0.01）。【结论】ZmEXO70s基因家族成员在系统发育进化上较保守,其基因组复制事件可能发生在禾本科植物分化后,且大多数ZmEXO70s基因被保留,仅部分基因被丢失。ZmEXO70s基因可能在非生物胁迫响应过程中发挥重要作用,ZmEXO70D2a和ZmEXO70E1基因可作为调控玉米苗期耐热性重要候选基因。     

A BAC-based physical map of the major autosomes of Drosophila melanogaster

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.     

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.     

Lack of a role for iron in the Lyme disease pathogen

A fundamental tenet of microbial pathogenesis is that bacterial pathogens must overcome host iron limitation to establish a successful infection. Surprisingly, the Lyme disease pathogen Borrelia burgdorferi has bypassed this host defense by eliminating the need for iron. B. burgdorferi grew normally and did not alter gene expression in the presence of iron chelators. Furthermore, typical bacterial iron-containing proteins were not detected in cell lysates, nor were the genes encoding such proteins identified in the genome sequence. The intracellular concentration of iron in B. burgdorferi was estimated to be less than 10 atoms per cell, well below a physiologically relevant concentration.     

Candidate taste receptors in Drosophila

Little is known about the molecular mechanisms of taste perception in animals, particularly the initial events of taste signaling. A large and diverse family of seven transmembrane domain proteins was identified from the Drosophila genome database with a computer algorithm that identifies proteins on the basis of structure. Eighteen of 19 genes examined were expressed in the Drosophila labellum, a gustatory organ of the proboscis. Expression was not detected in a variety of other tissues. The genes were not expressed in the labellum of a Drosophila mutant, pox-neuro70, in which taste neurons are eliminated. Tissue specificity of expression of these genes, along with their structural similarity, supports the possibility that the family encodes a large and divergent family of taste receptors.     

Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes

Budding yeast grown under continuous, nutrient-limited conditions exhibit robust, highly periodic cycles in the form of respiratory bursts. Microarray studies reveal that over half of the yeast genome is expressed periodically during these metabolic cycles. Genes encoding proteins having a common function exhibit similar temporal expression patterns, and genes specifying functions associated with energy and metabolism tend to be expressed with exceptionally robust periodicity. Essential cellular and metabolic events occur in synchrony with the metabolic cycle, demonstrating that key processes in a simple eukaryotic cell are compartmentalized in time.     

基于高通量转录组测序初步揭示丹参根和叶中丹参酮和丹酚酸含量差异的分子机制

丹参的药用部位为干燥根及茎,叶片不作为药用部位。为了解析丹参不同组织部位药效的差异,本研究使用Illumina高通量测序平台完成丹参药用部位根和非药用部位叶的转录组测序和功能注释,分析比较了根和叶中的差异表达基因,并进行基因的深度挖掘。本研究共获得54.17 Gb Clean Data,各样品Clean Data均达到8.29 Gb以上,Q30碱基百分比在94.37%以上。共检测到表达基因32700个,其中已知基因25607个,新基因7093个;表达转录本共56230个,其中已知转录本24627个,新转录本31603个。根和叶中差异表达基因有6959个,其中3265个基因上调表达,3694个基因下调表达。对丹参酮生物合成通路(二萜代谢通路map00904)和丹酚酸生物合成通路(苯丙烷代谢通路map00940)上差异基因分析,分别获得2个差异表达的PAL基因和2个差异表达的CPS基因,这些差异表达基因可能与丹参酮、丹酚酸主要在丹参根中积累有关。本研究结果为参与丹参酮和丹酚酸生物合成功能基因的挖掘、药效物质次生代谢途径解析提供了丰富的信息。     

小麦PIN基因家族的鉴定及表达分析

【目的】PIN（puroindoline）是植物所特有的一类蛋白家族,对控制小麦籽粒硬度有重要功能。分析小麦PIN家族成员在全基因组的分布、结构及进化,研究其在不同组织的表达特异性以及在不同硬度种子的表达模式,为阐明小麦PIN基因家族的生物学功能奠定基础。【方法】根据已报道的小麦PIN基因和大麦HIN基因,利用BLASTP和HMM方法,在最新发布的小麦中国春参考序列中鉴定小麦PIN基因家族成员。利用UniProt、URGI、PFAM、CDD、expVIP等数据库,采用Clustal X、MEGA 7.0、ExPASy、MEME、GSDS、TB tools、GraphPad Prism5等软件进行生物信息学分析。采用qRT-PCR方法检测TaPIN基因家族在不同籽粒硬度小麦样品中的表达情况。【结果】共鉴定出19个小麦PIN基因,集中成簇分布于第1、5和7染色体同源群,编码148—327个氨基酸,编码蛋白相对分子量为16.39—37.19 kD,等电点为6.35—9.34。通过结构域和系统发育分析,可将19个TaPIN基因分为A和B两大类。大部分TaPINs基因仅有1个外显子,没有内含子,顺式作用元件分析发现其上游序列包含大量抗逆和种子发育相关的调控元件。转录组分析表明该基因家族在小麦籽粒中相对表达量很高,而在根茎叶等其他组织几乎不表达。实时荧光定量PCR表明,各基因间相对表达量差异显著,TaPIN9和TaPIN10表达量较高。随着小麦籽粒硬度降低,TaPIN9和TaPIN10表达上调,且表达比例增加,而TaPIN16和TaPIN6则呈现相反的趋势。【结论】小麦籽粒硬度的调节以Pina和Pinb为主,该基因家族其他成员也具有相同结构域,推测也具有相似功能,但受表达量低的限制,对籽粒硬度影响较小。从该基因的进化关系看,粗山羊草与小麦亲缘关系最近,其次是燕麦、黑麦和大麦。     

鲤CYR61基因的克隆、表达及系统进化树的构建

通过鲤(Cyprinus carpio)基因组序列与斑马鱼(Danio rerio)CYR61基因编码区全序列的比对得到了CYR61 A、CYR61 B和CYR61 C共3条序列。分别克隆、测序得到其开放阅读框(Open Reading Frame,ORF)分别是1 158 bp、1 053 bp、1 107 bp,CYR61 A和CYR61 C得到完整编码区,都是由5个外显子和4个内含子构成,分别编码385、368个氨基酸,其理论等电点值分别是7.83、8.54,分子量(Mw值)分别为42.55 ku、40.83 ku。鲤3个CYR61基因具有高度同源性,并且均含IB、vWC、TSP1、CT 4个模块。分子系统学分析表明鲤CYR61基因与其他物种CYR61基因具有高度同源性,且CYR61 C与其他高等物种的CYR61同源性更高,利用MEGA 5.0计算出CYR61 A、CYR61 B和CYR61 C的分化时间早于鲤和斑马鱼的物种分化。CYR61 C在13个组织中均有表达,在卵巢中表达最高,肠、脑、鳃次之,在其他组织中表达较低且在心脏中表达最低。CYR61 A在精巢、肠、鳃中表达较高。CYR61 B在精巢和脑中表达最高,肠、肌肉、卵巢次之。实时荧光定量PCR分析CYR61 3个复制在鲤胚胎发育时期的表达,都是在0 h相对表达量最高,显著降低至18 h或24 h,随后逐渐升高并在6 d时下降。     

油棕SAUR基因家族的全基因组鉴定及生物信息学分析

【目的】挖掘并鉴定油棕生长素上调小RNA(SAUR)基因家族成员,并对其进行生物信息学分析,为油棕生长素信号转导机制的研究提供参考。【方法】根据拟南芥SAUR基因序列,在油棕全基因组数据库中同源序列搜索,并利用CDD和Pfam数据库进行SAUR蛋白结构域分析,剔除无保守结构域的序列,最后利用生物信息学软件对油棕SAUR基因家族成员进行可视化分析。【结果】从油棕全基因组中共鉴定出40个SAUR基因家族成员(EgSAUR1~EgSAUR40),有31个基因在油棕14条染色体上呈不均匀分布,其中4号染色体上分布的基因最多为7个;除EgSAUR14和EgSAUR24基因含有2个外显子外,其余38个基因均仅含1个外显子,无内含子。40个EgSAURs蛋白亚细胞定位于细胞核,其二级结构均由α-螺旋、β-转角、无规卷曲和延伸链组成,以无规卷曲和α-螺旋所占比例较高,其中有11个EgSAURs蛋白呈酸性,29个EgSAURs蛋白呈碱性。40个油棕SAUR蛋白被划分为四大类,其中第Ⅰ(除EgSAUR27蛋白外)、Ⅱ(除EgSAUR3和EgSAUR22外)、Ⅲ、Ⅳ类蛋白均有Auxin_inducible保守结构域。40个EgSAURs蛋白共含10种保守基序(Motif),其中Motif 2存在于所有基因成员中。在花中特异表达的EgSAURs基因(EgSAUR4、EgSAUR9、EgSAUR10、EgSAUR24、EgSAUR29、EgSAUR30、EgSAUR32和EgSAUR33)数量最多,其次为根、茎和叶,在中果皮中特异表达的基因数最少。【结论】基因重复和组织表达特异性是油棕SAUR家族基因的两大特点,推测该家族基因对油棕花的生长发育及授粉受精过程发挥调控作用。     

Cloning and Molecular Identification of A Fatty Acid Desaturase 2 Gene in a and C Genome of Brassica Species

The fatty acid desaturase 2 (fad2) gene was proven to be a major locus for high oleic acid (C18:1). Brassica napus is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa and Brassica oleracea. B. napus contains multiple copies in genome for most of the genes, including fad2 genes. The research cloned nine fad2 genes from 3 varieties of B. rapa and 3 varieties of B. oleracea, respectively. Alignment of the nine fad2 sequences from B. rapa and B. oleracea detect-ed 6 single nucleotide polymorphic sites, which resulted in 6 amino-acid substitutions. The nucleotide substitutions at position 743 bp in the fad2-A gene and position 947 bp in the fad2-C gene were used as 3’ end of al ele-specific primers. In use of the al-lele-specific primers to amplify fad2 gene, we could identify if the fad2 gene originated from A genome or C genome. Besides, the research found that fad2 genes in C genome are more conserved in evolutionary process than those in A genome. The fad2 expression data reported in this study revealed that fad2-A from B. rapa was not only expressed in siliques same as fad2-C from B. oleracea, but also expressed in a high level in stems. Not even the less, fad2 gene from B. napus was expressed higher in roots and flowers. Al these results provided evidences that fad2, though it was expressed differently in B. rapa and B. oleracea, but it was regulated by the same approach in B. napus.     

苹果MdMYB9、MdMYB11表达及其蛋白互作分析

【目的】克隆苹果(Malus domestica)中的TT2同源基因MdMYB9和MdMYB11,分析它们的序列特征,并对这两个基因在不同组织器官及光诱导条件下的表达特性及蛋白互作进行分析,为进一步解析这两个基因的功能奠定基础。【方法】采用同源克隆的方法分离得到MdMYB9和MdMYB11;利用DNAMAN软件对这两个蛋白的分子量、等电点等进行预测及对氨基酸序列进行分析,同时利用MEGA4.0软件构建这两个蛋白与拟南芥R2R3家族MYB蛋白的系统进化树;利用定量PCR检测这两个基因在不同组织器官、不同发育时期苹果果皮及种子和光照处理条件下的表达特性;利用酵母双杂交检测这两个MYB蛋白与花青苷合成相关蛋白MdbHLH33之间的互作关系。【结果】序列分析显示,MdMYB9开放阅读框长度为873 bp,编码290个氨基酸残基,与拟南芥TT2序列同源性为38.13%;MdMYB11开放阅读框为861 bp,编码286个氨基酸残基,与TT2同源性为32.44%;蛋白结构分析显示,MdMYB9和MdMYB11蛋白的N端都含有保守的R2R3功能域;进化树分析显示,这两个MYB蛋白都与拟南芥原花青苷合成相关蛋白TT2聚在同一个分支;荧光定量结果表明,MdMYB9和MdMYB11在苹果根、茎、叶和花中均有表达,但各器官中表达水平存在差异;同时,这两个基因在不同发育时期的种子及果皮中都有表达,其中均在发育中期表达水平最高;此外,光照诱导条件下MdMYB9和MdMYB11的表达变化无明显差异。酵母双杂交结果显示,MdMYB9和MdMYB11均能够与花青苷合成相关蛋白MdbHLH33相互作用。【结论】苹果MdMYB9和MdMYB11属于R2R3类MYB蛋白,在不同组织器官及不同发育时期的果皮和种子中都有表达,并与MdbHLH33蛋白相互作用。