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王伟科
, 周祖法
WANG Wei-ke, ZHOU Zu-fa
文献标识码: 1671-9964(2016)01-0074-07
文章编号: 1671-9964(2016)01-0074-07
收稿日期: 2015-04-23
网络出版日期: 2016-01-20
版权声明: 2016 上海交通大学期刊中心 版权所有
基金资助:
作者简介:
作者简介: 王伟科(1981-), 男, 硕士, 高级农艺师, 研究方向:食药用菌育种与栽培技术, E-mail:akeok@126.com;
通讯作者: 袁卫东(1969-), 男, 高级农艺师, 研究方向:食药用菌育种与栽培技术, E-mail:ywd0507@126.com
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摘要
为了解灰树花原基形成及子实体分化机制, 采用Illumina测序技术对灰树花菌丝体和原基进行了全转录组测序和数据分析, 对菌丝体和原基中的差异表达基因进行了研究。结果表明, 在菌丝体和原基中分别得到35788532个和32755254个高质量测序标签。差异表达基因分析表明, 两个文库中共有显著性差异表达的基因4094个, 其中在原基中上调、下调的基因数分别为1886和2208个, 只在原基中表达的基因284个。经Blastnr 比对, 在菌丝体与原基阶段差异表达的基因主要与酸性蛋白酶类、凝集素、细胞色素、NADPH-P450还原酶、酯酶、胺氧化酶、克拉维胺合成酶、糖苷水解酶家族相关。在原基中特异表达的基因主要与糖代谢、脂类代谢、核酸代谢及细胞膜、叶绿体膜有关。GO功能富集分析结果表明, 线粒体膜相关基因、谷氨酰胺代谢、脂肪酸生物合成相关基因均上调表达; Pathway 功能富集分析结果表明, 合成核糖体蛋白的基因均上调表达, 表明原基形成时细胞代谢增强, 蛋白质合成量增加。
关键词：
Abstract
The expression profiling of mycelium and primordium of Grifola frondosa was analyzed with high-throughput sequencing technology based on the Illumina Genome Analyzer platform.Totally 35788532 and 32755254 clean reads from mycelium and primordium libraries were obtained.Analysis of differentially expressed genes shows that there are 4094 genes differentially expressed between two libraries, and in the primordium library, 1886 of them are up-regulated while 2208 of them are down-regulated.There are 284 genes that are only expressed in the primordium library.With Blastnr comparison, genes differently expressed between two libraries are found to be related to acid protease and lectin, cytochrome, NADPH-P450 reductase, esterase, amine oxidase, clavaminate synthase, glycoside hydrolase.Genes only expressed in the primordium are found to be related to glucose, lipid, nucleic acids metabolism and cell and chloroplast membrane structure.Gene ontology functional enrichment analysis reveals that most genes related to mitochondrial membrane, glutamine family amino acid metabolic process, fatty acid biosynthetic process are up-regulated.Pathway enrichment analysis shows that genes related to ribosome are all up-regulated, and this indicates that when primordium is developed the cell metabolism speeds up and the protein synthesis increases.
Keywords：
灰树花(Grifola frondosa)又名栗子蘑、莲花菌、贝叶多孔菌, 在分类系统中属于担子菌亚门、层菌纲、非褶菌目、多孔菌科[1]。其子实体肉质柔嫩、口味鲜美, 富含氨基酸、多种维生素、微量元素、多糖与生物活性物质, 是近年来开发的珍奇药食两用菌之一[2]。研究表明, 从灰树花中提取的多糖具有抗肿瘤、抗氧化、抗HIV 病毒、增强免疫功能等多种生物活性[3]。
灰树花原基的形成是其生活史中重要的生理过程, 对灰树花的品质及出菇有重要影响[4]。原基的形成不仅需要一定的环境条件和营养条件, 更是由一系列基因协调作用的结果[5]。研究灰树花原基形成时的差异表达基因, 对阐明灰树花子实体形成的分子机理具有重要的意义。
通过对灰树花菌丝体、原基2个不同生长发育期转录组的比较研究, 筛选出2个时期差异表达的基因, 并对这些差异表达的基因进行生物信息学分析, 旨在发现控制灰树花原基形成和子实体生长的功能基因, 为通过基因工程培育优质、高产的灰树花新品种提供理论依据。
供试菌株为灰树花小黑汀, 引自山东泰安。菌丝长满菌包后移入出菇大棚培养7~10 d, 待菌丝扭结形成原基后, 收集菌丝体与原基样品。
用Trizol法分别提取菌丝体与原基总RNA, 并用RNAeasy plant mini kit对提取的总RNA进行纯化, 70 ℃变性2 min后, NanoDrop ND-2000检测其浓度、琼脂糖凝胶电泳分析RNA的完整性[6]。检测合格的RNA用于mRNA的富集及cDNA的合成。
用Ultra RNAlibrary prep kit for illumina进行文库构建, 纯化后Agilent high sensitivity DNA kit检测文库插入片段大小, 定量后Illumina HiSeq TM 2000对建好的测序文库进行测序[7]。
使用Trinity(版本r20131110, 默认参数)对RNA-seq的原始reads数据进行拼接, 并使用测序序列(reads)比对软件Tophat v2.0.5将clean reads分别比对到NR数据库[8]。后续分析都基于上述比对结果, 然后统计clean reads比对到NR数据库的分布情况。
基因表达量的计算使用FPKM数值[9], FPKM代表每百万个比对上的reads中每kb外显子的片段数, 用于衡量基因的表达水平, 判断差异表达显著的方式为满足条件:表达量变化倍数>2且FPKM差值>20, 或表达量变化倍数>4且FPKM差值>2。
对不同样本中差异表达的基因进行基因本体(gene ontology, GO)分析能确定差异表达基因的功能[10]。卡方检验P值≤0.05 条件的GO 条目被认为是显著富集的GO条目。
使用http://www.genome.jp/tools/kaas/对灰树花原基和菌丝体差异表达基因进行Pathway显著性富集分析。卡方检验P值≤0.05的Pathway确定为不同样本间差异表达的基因所参与的最主要代谢途径和信号转导途径。
灰树花菌丝体和原基RNA经过NanoDrop定量后, 样本浓度(表1)完整性及28S∶18S(图1)均符合转录组测序质量要求, 可用于开展下一步试验。
通过高通量测序, 在菌丝体和原基样本库中分别得到了35913116个和32873980个原始测序标签(raw reads)。原始数据去除低质量的标签后, 在菌丝体和原基这2个文库得到高质量测序标签(clean reads)总数分别为35788532和32755254。将所有高质量测序标签与参考基因序列进行比对, 在菌丝体和原基文库中, 在参考序列上有唯一比对位置的测序序列分别为22085081和21547780, 占高质量标签种数的61.7%和65.78%。整段比对到外显子的测序序列总数为22061957和21537230(表2)。
表1 灰树花菌丝体、原基RNA浓度
Tab.1 Concentration of RNA from mycelium and primordium of Grifola frondosa
| 样品 Sampels | 浓度 Concentration | 单位 Unit | OD260/280 | OD260/230 |
|---|---|---|---|---|
| 1-JST | 266.4 | ng·μL-1 | 2.14 | 1.92 |
| 2-YJ | 697.8 | ng·μL-1 | 2.16 | 2.21 |
图1 灰树花菌丝体、原基RNA电泳检测1-JST表示菌丝体, 2-YJ表示原基
Fig.1 Electropherogram of RNAs from mycelium and primordium of Grifola frondosa1-JST mean mycelium, 2-YJ mean primordium
菌丝体共有41858个表达基因, 原基中有34255个表达基因, 最终在菌丝体和原基数据库中, 共发现显著差异表达的基因4094个, 其中在原基中上调1886个, 下调2208个, 只在菌丝阶段表达的基因915个, 只在原基中表达的基因284个。
为进一步了解菌丝体阶段与原基阶段差异表达基因的情况, 对两者具有明显差异表达的4094个基因进行分析, 对同源比对(blastnr)得到的有确切功能的基因列于表3(明显差异表达前20个基因)。从表中可知, 在菌丝体及原基阶段差异表达的基因主要与酸性蛋白酶类、凝集素、细胞色素、NADPH-P450还原酶、酯酶、胺氧化酶、克拉维胺合成酶、糖苷水解酶家族相关。其中凝集素能凝集细胞并参与糖结合作用; 酯酶协助参与降解纤维素、半纤维素和木质素; 胺氧化酶参与分解氨基酸; 糖苷水解酶能作用各种糖苷或寡糖使糖苷键水解, 参与糖的代谢。上述基因表达量的变化可能在原基的形成中起到一定的作用。
表2 两个样本中标签分布一览表
Tab.2 Distributions of tags from two libraries
| 数据产出及比对 Data output and comparison | 菌丝体 Mycelium | 原基 Primordium |
|---|---|---|
| 原始序列标签 Rawreads | 35913116 | 32873980 |
| 高质量测序标签 Cleanreads | 35788532 | 32755254 |
| 未比对上的标签 Unmapped reads | 9028139 | 7574132 |
| 非唯一比对位置标签 Mapq<mapq_cut(non-unique) | 4665894 | 3623751 |
| 唯一比对位置标签 Mapq>=apq_cut(unique) | 22085081 | 21547780 |
| 第一端测序标签 Read-1 | 11010040 | 10756337 |
| 第二端测序标签 Read-2 | 11075041 | 10791443 |
| 比对到基因组上正列 Reads map to‘+’ | 10981873 | 10704107 |
| 比对到基因组上负列 Reads map to‘-’ | 11103208 | 10843673 |
| 整段比对到外显子的序列 Non-splice reads | 22061957 | 21537230 |
| 分段比对到两个外显子上的序列 Splice reads | 23124 | 10550 |
| 双端测序序列定位到基因组上相对 的距离符合测序片段的长度分布 Eads mapped in proper pairs | 19597840 | 19326088 |
为了进一步研究原基阶段特异基因的表达情况, 对原基中特异表达的284个基因进行了同源比对(blastnr)。284个基因中在NCBI 中比对到的同源基因绝大多数是功能未知的假定蛋白(hypothetical protein)或预测蛋白(predicted protein)。比对得到有确切功能的基因与糖代谢、脂类代谢、核酸代谢及细胞膜、叶绿体膜有关(表4)。其中, E1脱氢酶及转酮醇酶在戊糖磷酸循环中起着重要作用, HAD水解酶在核酸代谢过程中起重要作用。研究该类基因可能有助于揭示原基的形成。
在富集到的前20个具有明显差异的Go Term中, 有13个显著富集的Go Term富集在参与分子功能(molecular function)本体上, 有5个显著富集的Go Term富集在参与生物过程(biological process)本体上, 有2个显著富集的Go Term富集在参与细胞组成(cellular component)本体上。其中, 在细胞组成本体中, 与线粒体膜成分相关的基因均上调表达; 在生物过程本体中, 与谷氨酰胺代谢、脂肪酸生物合成相关的基因也均上调表达(表5)。
表3 菌丝体、原基中差异表达基因的同源比对
Tab.3 Blastnr of differently expressed genes between mycelium and primordium libraries
| 基因ID Gene ID | 菌丝中表达值 Expression value of mycelium | 原基中表达值 Expression value of primordium | Q值 Q value | 同源基因及其功能 Homologous gene and its function |
|---|---|---|---|---|
| unigene23873 | 483.4 | 6.0 | 0.023 | 酸性蛋白酶(gi|392558359|)(gb|EIW51548.1|) Acid protease |
| unigene13929 | 11.6 | 5860.5 | 0.037 | 凝集素(gi|18478668|)(gb|AAL73235.1|) |
| unigene30943 | 186.4 | 1.4 | 0.037 | DUF1479结构域蛋白(gi|392564554|)(gb|EIW57732.1|) DUF1479 domain containing protein |
| unigene12210 | 8.2 | 2412.5 | 0.042 | 凝集素(gi|50980806|)(gb|AAT91249.1|) lectin |
| unigene41873 | 2.3 | 81.1 | 0.042 | 细胞色素P450(gi|395327404|)(gb|EJF59804.1|) Cytochrome P450 |
| unigene45937 | 1.7 | 67.0 | 0.042 | NADPH-P450还原酶(gi|392559250|)(gb|EIW52435.1|) NADPH-P450 reductase |
| unigene27346 | 223.5 | 5.9 | 0.044 | IlvN结构域蛋白(gi|395334382|)(gb|EJF66758.1|) IlvN domain containing protein |
| unigene28323 | 3.5 | 162.9 | 0.044 | 酯酶(gi|395328576|)(gb|EJF60967.1|) esterase |
| unigene41874 | 2.1 | 68.9 | 0.044 | 细胞色素P450(gi|395327404|)(gb|EJF59804.1|) Cytochrome P450 |
| unigene45936 | 2.2 | 73.6 | 0.044 | NADPH-P450还原酶(gi|392559250|)(gb|EIW52435.1|) NADPH-P450 reductase |
| unigene41875 | 3.0 | 170.3 | 0.048 | 细胞色素P450(gi|395327404|)(gb|EJF59804.1|) Cytochrome P450 |
| unigene14635 | 3.5 | 118.4 | 0.052 | 真菌疏水蛋白结构(gi|390605204|)(gb|EIN14595.1|) Fungal hydrophobin |
| unigene47574 | 248.0 | 1.3 | 0.056 | 胺氧化酶(gi|392563541|)(gb|EIW56720.1|) Amine oxidase |
| unigene19870 | 22.8 | 653.1 | 0.058 | 真菌疏水蛋白结构(gi|395323171|)(gb|EJF55662.1|) Fungal hydrophobin |
| unigene36089 | 1.8 | 34.7 | 0.058 | 细胞色素P450(gi|390599619|)(gb|EIN09015.1|) Cytochrome P450 |
| unigene30525 | 0.8 | 13.6 | 0.06 | 克拉维胺合成酶(gi|392569544|)(gb|EIW62717.1|) Clavaminate synthase-like protein |
| unigene36088 | 1.5 | 27.1 | 0.06 | 细胞色素P450(gi|390599619|)(gb|EIN09015.1|) Cytochrome P450 |
| unigene39627 | 14.6 | 1.0 | 0.061 | 类CDF转运蛋白(gi|395334914|)(gb|EJF67290.1|) CDF-like metal transporter |
| unigene30523 | 0.5 | 14.7 | 0.063 | 克拉维胺合成酶(gi|392569544|)(gb|EIW62717.1|) Clavaminate synthase-like protein |
| unigene2984 | 69.6 | 0.3 | 0.065 | 糖苷水解酶家族(gi|392564918|)(gb|EIW58095.1|) Glycoside hydrolase family protein |
菌丝体和原基中Pathway 显著性富集分析见表6, 在富集到的前11个具有明显差异的pathway中, 核糖体蛋白代谢途径中的全部基因均上调表达, 即核糖体数量在增多, 核糖体是蛋白质的合成场所, 核糖体数量在原基阶段增加, 表明原基形成时蛋白质合成增加。另外, 萜类生物合成途径、苯丙氨酸、酪氨酸、色氨酸生物合成途径均上调表达, 氧化磷酸化途径、花生四烯酸代谢途径、脂肪酸代谢相关途径基因基本上调表达, 表明在原基形成时期细胞代谢旺盛。
表4 原基中特异表达基因的同源比对
Tab.4 Blastnr comparison of genes only expressed in the primordium
| 同源基因功能 Functions of homologous genes | 同源基因Homologous gene |
|---|---|
| 糖代谢相关 Glucose metabolism | E1脱氢酶及转酮醇酶结构域蛋白(gi|395325950|)(gb|EJF58365.1|), dehydrogenase E1 and transketolase domain-containing protein 醛脱氢酶(gi|392561496|)(gb|EIW54677.1|), Aldehyde dehydrogenase MFS通用底物转运蛋白(gi|392561630|)(gb|EIW54811.1|) MFS general substrate transporter |
| 脂类代谢相关 Lipid metabolism | 酮脂酰合成酶结构域蛋白(gi|395326786|)(gb|EJF59192.1|) ketoacyl-synt-domain-containing protein |
| 核酸代谢相关 Nucleotide etabolism | HAD水解酶(gi|395328072|)(gb|EJF60467.1|) HAD hydrolase |
| 细胞膜蛋白 Cell membrane protein | DUF6结构域蛋白(gi|392559753|)(gb|EIW52937.1|), DUF6-domain-containing protein NAD(P)结合蛋白(gi|392588861|)(gb|EIW78192.1|) NAD(P)-binding protein |
| 叶绿体膜蛋白 Chloroplast membrane protein | 磷酸丙糖转运蛋白家族(emb|AJ437264.1) phosphate translocator-like protein |
| 其他 Others | 细胞色素(gi|395325039|)(gb|EJF57468.1|), cytochrome 叶绿素A-B 结合蛋白PF00504(gb|EF576336.1|), Chlorophyll A-B binding protein PF00504 类克拉维胺合成酶(gi|395330466|)(gb|EJF62849.1|), Clavaminate synthase-like protein 乌头酸水合酶(gi|4029334|)(emb|CAA76360.1|) Aconitate hydratase |
食用菌子实体的形成和发育, 是食用菌育种研究的主要领域[1]。子实体由原基诱导分化而来, 对菌丝体与原基形成过程中基因表达差异的研究有助于进一步了解原基形成、分化机制[12]。灰树花转录组序列不含内含子及其他非编码序列, 可识别表达丰度较低的RNA, 对灰树花菌丝体、原基进行全转录组测序, 组建并比较两者完整的基因表达谱, 有助于我们更深入研究灰树花原基形成机制, 为食用菌灰树花的分子育种奠定基础。
采用新一代Illumina高通量测序技术, 对灰树花菌丝体和原基进行了全转录组测序, 共发现两者具有显著差异表达的基因4094 个, 其中有1886个基因在原基中上调表达, 有2208个基因在原基中下调表达, 这些差异表达的基因主要涉及酸性蛋白酶类、凝集素、细胞色素、NADPH-P450还原酶、酯酶、胺氧化酶、克拉维胺合成酶、糖苷水解酶家族等。同时分析得到284个只在原基中表达的基因, 这些基因分别与糖代谢、脂类代谢、核酸代谢及细胞膜、叶绿体膜结构相关。
通过GO功能富集分析和Pathyway显著性分析, 发现灰树花原基形成时细胞代谢、蛋白质合成均升高, 这表明灰树花原基形成是一个复杂的生理过程, 在多种能量代谢的调控下合成了新蛋白质。本研究得到的灰树花菌丝体及原基形成时差异表达基因, 将为今后进一步探索其原基形成的分子机制奠定基础。
表5 菌丝体与原基GO功能富集分析
Tab.5 Significantly enriched GO terms of mycelium and primordium
| 基因本体条目 Gene ontology entries | 分类 Classification | P值 P value | 上调基因数 Up-regulated genes | 下调基因数 Down-regulated genes |
|---|---|---|---|---|
| 结合ATP ATP binding | 分子功能 Molecular function | 9.9E-06 | 74 | 30 |
| 酸酐水解酶活性 Hydrolase activity | 分子功能 Molecular function | 2.1E-07 | 46 | 5 |
| 胞内成分 Intracellular part | 细胞组成 Cellular component | 3.1E-07 | 91 | 24 |
| 结合过渡金属离子 Transition metal ion binding | 分子功能 Molecular function | 9.8E-09 | 102 | 52 |
| 线粒体膜成分 Mitochondrial membrane part | 细胞组成 Cellular component | 9.9E-08 | 6 | 0 |
| 结合金属离子 Metal ion binding | 分子功能 Molecular function | 1.6E-11 | 117 | 60 |
| 结合核酸 Nucleic acid binding | 分子功能 Molecular function | 8.0E-13 | 115 | 24 |
| 谷氨酰胺家族氨基酸代谢过程 Glutamine family amino acid metabolic process | 生物过程 Biological process | 1.4E-05 | 8 | 0 |
| 肽酶活性 Peptidase activity | 分子功能 Molecular function | 6.6E-11 | 27 | 25 |
| 结合阳离子 Cation binding | 分子功能 Molecular function | 2.9E-11 | 117 | 60 |
| 脂肪酸生物合成过程 Fatty acid biosynthetic process | 生物过程 Biological process | 4.8E-06 | 4 | 0 |
| 单糖分解代谢过程 Monosaccharide catabolic process | 生物过程 Biological process | 7.7E-06 | 2 | 5 |
| 胞内蛋白代谢过程 Cellular protein metabolic process | 生物过程 Biological process | 2.2E-06 | 41 | 2 |
| 小分子代谢过程 Small molecule metabolic process | 生物过程 Biological process | 6.7E-06 | 21 | 10 |
| 金属内肽酶活性 Metalloendopeptidase activity | 分子功能 Molecular function | 1.1E-06 | 9 | 1 |
| 水解酶活性 Hydrolase activity | 分子功能 Molecular function | 1.5E-09 | 120 | 62 |
| 结合嘌呤核苷三磷酸 Purine ribonucleoside triphosphate binding | 分子功能 Molecular function | 3.2E-07 | 95 | 32 |
| 氧化还原酶活性 Oxidoreductase activity | 分子功能 Molecular function | 1.1E-16 | 141 | 59 |
| 依赖NTP嘌呤解旋酶活性 Purine NTP-dependent helicase activity | 分子功能 Molecular function | 2.6E-07 | 17 | 3 |
| 单价无机阳离子跨膜运输活性 Monovalent inorganic cation transmembrane transporter activity | 分子功能 Molecular function | 1.1E-05 | 6 | 5 |
表6 菌丝体与原基pathway 显着富集分析列表
Tab.6 List of pathway enrichment analysis of mycelium and primordium
| 代谢途径 Metabolic pathway | P值 P value | 上调基因数 Up-regulated genes | 下调基因数 Downk-regulated genes |
|---|---|---|---|
| 氧化磷酸化途径 Oxidative phosphorylation | 7.2E-05 | 12 | 1 |
| 萜类生物合成途径 Terpenoid backbone biosynthesis | 0.000118 | 6 | 0 |
| 核糖体 Ribosome | 1.8E-06 | 21 | 0 |
| 花生四烯酸代谢途径 Arachidonic acid metabolism | 6.0E-05 | 4 | 1 |
| 莨菪、哌啶、吡啶类生物碱合成 Tropane, piperidine and pyridine alkaloid biosynthesis | 5.7E-05 | 5 | 2 |
| mRNA检测途径 mRNA surveillance pathway | 0.000155 | 14 | 0 |
| 次级代谢产物生物合成途径 Biosynthesis of secondary metabolites | 2.4E-07 | 50 | 15 |
| 色氨酸代谢 Tryptophan metabolism | 0.000225 | 14 | 5 |
| 丙氨酸、天冬酰胺、谷氨酸代谢相关 Alanine, aspartate and glutamate metabolism | 0.000194 | 6 | 1 |
| 脂肪酸代谢相关 Fatty acid metabolism | 3.4E-05 | 13 | 1 |
| 苯丙氨酸、酪氨酸、色氨酸生物合成途径 Phenylalanine, tyrosine and tryptophan biosynthesis | 0.000238 | 8 | 0 |
The authors have declared that no competing interests exist.
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Abstract Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.
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We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays鈩 formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix 庐; Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.
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We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 4109恪52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 309銇05 untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 01— 105 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.
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Abstract Unified, structured vocabularies and classifications freely provided by the Gene Ontology (GO) Consortium are widely accepted in most of the large scale gene annotation projects. Consequently, many tools have been created for use with the GO ontologies. WEGO (Web Gene Ontology Annotation Plot) is a simple but useful tool for visualizing, comparing and plotting GO annotation results. Different from other commercial software for creating chart, WEGO is designed to deal with the directed acyclic graph structure of GO to facilitate histogram creation of GO annotation results. WEGO has been used widely in many important biological research projects, such as the rice genome project and the silkworm genome project. It has become one of the daily tools for downstream gene annotation analysis, especially when performing comparative genomics tasks. WEGO, along with the two other tools, namely External to GO Query and GO Archive Query, are freely available for all users at http://wego.genomics.org.cn. There are two available mirror sites at http://wego2.genomics.org.cn and http://wego.genomics.com.cn. Any suggestions are welcome at wego@genomics.org.cn.
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草菇菌丝体与原基差异表达基因分析 [J].DOI:10.5376/gb.cn.2012.01.0003 URL 摘要
采用Solexa测序技术,对草菇菌丝体和原基进行了数字基因表达谱（DGE）测序,在菌丝 体和原基文库中分别得到5701781个和5659262个高质量测序标签（cleantags）,对应的标签种数（distinct clean tags）分别为85626和95363。将所有高质量测序标签与参考基因库进行比对,在菌丝体和原基文库中,占标签种数的43.32%和52.57%的 标签可以唯一定位（map）到参考序列上,占标签种数的21.65%和21.47%的标签可以被定位到基因组序列上。最终,被菌丝体和原基标签唯一定位的 基因数（unambiguous tag-mapped genes）分别为14794和15534。差异基因分析显示,两个文库中共有显著性差异表达的基因4163个,其中在原基中上调、下调的基因数分别为 2486和1677,只在原基中表达的基因321个。经过Blastnr比对,在原基中特异表达的基因,涉及蛋白质（氨基酸）合成与代谢、糖代谢、脂类代 谢和抗逆反应等多个代谢途径。GO功能富集分析结果表明,葡萄糖、己糖和乙醇等代谢途径大部分基因下调表达。Pathway功能富集分析结果表明,合成核 糖体蛋白的基因均下调表达,表明原基形成时细胞代谢减弱,蛋白质合成量减小。
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以金针菇(Flammulina velutipes)1123菌株的单孢 W23基因组为参考完成其菌丝和原基转录组测序与数据分析，研究两样本间差异基因，并对差异基因进行 GO功能和 KEGG Pathway 显著性富集分析。结果表明：两个样本中共有显著性差异表达的基因3310个，其中在原基中上调、下调的基因数分别为1686、1624个，只在原基中表达 的基因有26个。GO功能分析结果表明，在原基中膜封闭腔、内膜系统、细胞器腔、核糖核蛋白复合体、翻译调节器、发育过程、免疫系统过程、多细胞生物过程 这8个GO基本单元中的差异基因全部呈现上调表达。Pathway 功能富集分析结果表明，核糖体与DNA复制中的差异基因全部呈现上调表达，糖酵解途径中从D-葡萄糖转化成丙酮酸过程相关的11个差异基因全部呈下调表 达，磷酸戊糖途径中相关的13个差异基因中有11个基因呈下调表达。
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