【作者】 程道军；

【导师】 夏庆友； 鲁成；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2003， 硕士

【摘要】 表达序列标签(Expressed sequence tag,EST)是生物基因组中最有价值的基因结构信息。构建生物特定组织、器官或细胞的cDNA文库并进行大规模EST测序分析，能直接获得大量的功能基因结构及表达特征，并以此来构建各种组织器官的基因表达谱。 家蚕是一种重要的经济昆虫，也是鳞翅目昆虫的模式生物。本研究选取了家蚕丝腺、生殖腺、中肠、脂肪体、血液、卵(胚胎期)、卵巢培养细胞等12种不同发育阶段的重要组织器官，分别构建其cDNA文库，再对每个文库进行大规模EST测序分析，以期发现与家蚕性别调控、变态发生、胚胎发育、能量代谢和物质代谢等密切相关的功能基因。家蚕脂肪体担负着体内物质贮存、能量供给和中间代谢等重要生理功能，是典型的变态模式组织。因此，本文主要通过对脂肪体EST数据的分析，研究家蚕变态过程中脂肪体的基因表达情况，获得参与家蚕变态的功能基因群，以期从分子水平阐明家蚕变态发生的作用机制。 1、家蚕cDNA文库的构建 提取高质量的12种家蚕组织器官的总RNA和mRNA，合成带有EcoRⅠ和XhoⅠ粘性突出末端的双链cDNA片段；电泳回收≥500bp的cDNA片段，定向克隆入pBluescript ⅡSK(+)质粒载体。将重组质粒转入受体菌HB101中，得到实验所需的cDNA文库。文库重组率平均为93％，原始库容平均为1.23×10~5克隆／ml，外源插入cDNA片段的大小平均在400-1500bp之间，表明文库质量较高。 2、家蚕大规模EST测序 对家蚕12个组织的cDNA文库进行大规模EST测序。通过去除载体序列、重复序列以及细菌DNA序列等处理，共获得81625条有效的EST序列，序列长度集中在250～550bp之间。 对EST序列进行聚类分析和拼接，共获得24678个UniGene(非重复序列)，其中包括11627个contig(重叠序列)，13051条singletons(独立EST)。contig序列大小集中在200～1200bp之间，由此推测，家蚕中决定蛋白质编码的mRNA长度大部分为1200bp以下。 家蚕EST序列同源性分析的参数标准为：blastx为E≤E-15，Alignment≥30％；blastn为E≤E-10，Alignment≥25％。同源性分析共检索到有7944个UniGene同已知基因数据同源，其中(1)同源且有功能注释的功能已知基因有3899个UniGene、共计32605条EST；(2)有同源但无功能注释的已知序列有4045个UniGene、共计16087个EST；（3）新的序列有16734个UniGene、共计32933个 EST。 家蚕功能已知基因EST数据除了部分与GeneBank中家蚕基因数据同源外，还与果蝇、烟草天蛾、人、小鼠、按故等26个物种的登录基因同源；同时发现了许多参与家蚕性别决定和生长发育调控的重要功能基因，以及与细胞分化、信号传导、组织发生等密切相关的功能基因信息．3、家蚕脂肪体基因表达谱分析3．l脂肪体基因表达概况 家蚕脂肪体cDNA文库测得19030条EST序列，聚类分析获得6337个UniGene，其中包括4262个。。ntig、2075个独立EST．同源性分析发现：（1）功能已知的UniGene有1226个，包括1196个。。ntig、30条勾nde加＊s，共计9264条田T，这些UniGe11e共注释为5肥个基因。（二）已知序列的UniGene有1341个，包括1030个。。ntig、309条singletons，共计2871条EST．m、新的UniGene序列有3770个，共计6895条EST。 502个注释基因中，高丰度基因有117个，包括8179条EST序列：中丰度基因有234个，共包括933条EST序列；低丰度基因有151个。 脂肪体所有己知基因在分子细胞生物学水平上大致可分为细胞骨架蛋白、胞外基质、激素与激素调节、信号转导与细胞调节、膜相关蛋白、转录翻译调节、能量代谢、物质代谢等八类基因。3．2脂肪体己知基因的表达差异 分析表明，幼虫期脂肪体表达较多的是与物质代谢、转录翻译调节、信号转导调节、能量代谢相关的基因；蛹期则是与物质代谢、细胞骨架蛋白、转录翻译调节、膜相关蛋白等相关的基因表达较多。 幼虫期和蛹期脂肪体的基因表达表现出发育阶段上的差异。八类基因中，细胞骨架、胞外基质、信号转导与细胞调节因子、转录翻译调节、能量代谢和物质代谢等六类基因在不同发育时期的表达存在显著差异，差异可信性大于0．999，激素与激素调节以及膜相关蛋白质的表达差异可信性低于0．5。脂肪体已知基因的EST数据分析显示，胞外基质、信号转导与细胞调节、转录翻译调节、能量代谢和物质代谢相关基因在幼虫期表达量较高；而细胞骨架蛋白相关基因在幼虫期表达较低，蛹期较高。幼虫期和蛹期参与激素与激素调节、膜相关蛋白形成的相关基因表达水平相当，差异较小。 幼虫期脂肪体八类已知基因在雌雄个体之间的表达差异不显著，表达水平相当。3．3脂肪体发育特异性EST数据的的生物信息学分析 V 对脂肪体高表达或特异表达可能与家蚕变态相关的EST数据进行了生物信息学分析，包括幼虫期的贮存蛋白（SPI、SPZ、Arylpborin）、30KD protein、collag。n alpbal、yellow－f；蛹期的bllin－binding p。teiu、hemOli。、bombyxin、Innexin、y。flow－Q等更多还原

【Abstract】 Expressed Sequence Tag (EST) is important structural information of gene in the genome of organisms. Large-scale EST sequencing of cDNA libraries from specific Tissues, organ or cell can be directly used to analyze the expressed gene structure and charateristics, discover new gene,and construct the gene expressed profile of many tissue by ESTs.Silkworm is an important economic insect and a model organism of Lepidoptera insect In this study, silkland, middle gut, fat body, blood, egg(embryonic period) and cultured cell were selected to construct cDNA libraries. It is expected to discover a lot of genes associated with sex regulation, metamorphosis, embryonic development and metabolism by Large-scale EST sequencing and analyzing of these cDNA libraries.In addition, the function of fat body is protein storage, providing energy, Intermediary metabolism, it also was model Tissue during silkworm metamorphosis period. The function genes associated with it will be acquired and molecular mechanism of metamorphosis will be analyzed.1. Construction of silkworm cDNA libraryTotal RNA and mRNA of high quality were isolateted ,Double-stranded cDNA with EcoRI And XhoI overhang extension were synthesized and recovered. They were recombinanted with Plasmid. The cDNA library were obtained by transforming the recombinant into the host cell HB101.The recombination rate of these library was an average 93%.Primary sink size obtained 1.23X105 and its insert size ranged from 0.4-1.5kb.2. Large-scale EST sequencing and data analysiscDNA clone from the silkworm cDNA library were partial sequenced (EST sequencing),81625 ESTs sequence were obtained after removing the vector sequence,repeat sequence and germ DNA.The length of EST sequences ranged front 0.25-0.55kb.24678 unigene contained 11627 contig and 13951 singletons were gained by assembling, contig length was 0.2-1.2kb.We predicted that most of coding genes of silkworm were less than 1.2kb.The parameter of bioinformatics analysis of EST were: blastx, E<E-15,Alignment>30%;The parameter of bioinformatics analysis of EST were: blastx, E<E-15,Alignment>30%; blastn,E<E-10,Alignment>25%. There were 7944 unigene(32605 ESTs) matched with known genes, 4045 unigene (16087 ESTs) matched known sequences and 16734 UniGene(32933 ESTs) matched no sequences.The ESTs of known gene of silkworm have significant similarities with gene of silkworm and Drosophila melanogaster, Manduca Sexta, Anopheles gambiae, Homo sapiens and Mus Musculus in GeneBank. A lots of Functional gene associated with sex regulation, metamorphosis, embryonic develop and metabolism etc. were discovered.3. Study on gene expressed profile of fat body3.1 Characterization of gene expressed profile of fat body6337 unigene contained 4262 contig and 2705 singletons were gained. There were 1226 unigene (9264 ESTs) matched known genes,1341 unigene(2871EST) matched with known sequences and 3770 UniGene (6895 EST) matched no any sequences.Among the 502 known genes, there were 117 high abundant genes,234 middle abundant genes and 151 low abundant gene.All known genes of fat body grouped into cytoskeletal, extracellular matrix, Energy metabolism , hormones and hormonal regulation, signal transduction and cell regulation, transcription and translation, membrane-associated and substantial metabolism.3.2. Gene Expressed difference of fat bodyThe categories of gene most highly expressed in fat body during larval period were those participating in substance metabolism, and the next were those related with transcription and translation, signal transduction and cell regulation, energy metabolism; During pupal state, genes highly expressed were related with substance metabolism, cytoskeletal, transcription and translation, membrance-associated.Gene expression in fat body showed difference during metamorphosis development states. Among the eight kind of genes, expression of gene linked to cytoskeletal, extracellular matrix, energy metabolism ,signal transduction and cell regulation, transcription and tra更多还原