家蚕未受精卵表达序列标签（EST）分析

【作者】 邱咏梅；

【导师】 夏庆友；

【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】 家蚕是丝绸业的生物基础，通过EST测序从分子水平上获得大量数据，可为家蚕基因组结构和绝大部分基因提供信息，为进一步进行家蚕功能基因的研究奠定基础。 家蚕的EST数据的收集在新基因的发现、基因敲除的研究和基因芯片的制备以及发育分析等方面具有重大的价值。本实验以家蚕肾脏形卵和正常形卵的未受精卵为材料建立了cDNA文库，这些未受精卵的细胞中含有大量的早期胚胎发育所必需的信息。 母性基因是指这些未受精卵中所含有的大量影响后代发育的一类基因，这些基因在卵子发生过程中表达，在卵子发生过程或胚胎发育过程中翻译。母性基因在胚胎的早期发育阶段起着至关重要的作用，主要功能是决定轴的分化、卵裂方式的形成和形态的发生。在昆虫的早期发育中，胚胎的前后轴和背腹轴分别独立地由母性基因产物决定。这些母性基因主要编码转录因子或调控蛋白，它们的产物通常形成一定的浓度梯度并产生特异的位置信息，以进一步激活一系列合子基因的表达。随着这些基因的表达，胚胎被分成不同的区域。每个区域表达特异性基因的组合，沿前后轴或背腹轴形成一定的体节模式。未受精卵中的mRNA及蛋白质均属于母性基因产物。 本实验首先将获得的原始数据屏蔽载体序列，然后再去除低质量和小于100bp的小片段。这些EST序列再采用clustalW软件进行拼接。将获得的EST序列的六个阅读框用BLASTX软件与GeneBank中1，230，998条非冗余的蛋白质序列数据库进行比对，用BLASTN软件与GeneBank中1，453，916条核苷酸序列数据库进行比对，寻找具有相似性的蛋白质和核苷酸序列。对只知氨基酸序列但功能未知的基因，运用SMART软件分析其结构域，从而推测所获得的表达序列标签可能的性质和功能。另外这些数据还与家蚕EST数据库SILKBASE http://www.ab.a.u-tokyo.ac.jp/silkbase/中的28个cDNA文库的EST序列进行比对。对于已知基因则进入http://www/geneontology.org网址进行基因功能分类。 本实验最终成功获得391条EST序列，平均读长659hp。用。lusterb比较分析后拼接得到374个非重复序列，其中最大的由4条EST组成，最长的为1958hp。374条非重复序列中 15条是 contig，118条Singletons是正常型未受精卵中的 EST序列，其余 24条是肾脏形未受精卵文库中的。通过大规模的 EST测序和分析，获得了大量功能基因的信息。经生物信息学分析发现，171个与己知基因具有较高的相似性（己知基因），25个与己知序列具有较高的相似性（新基因），其余178个为低度相似或没有相似性的序列（未知基因）。 未受精卵文库提供了大量的母性基因的信息，本实验共获得3个与己知的母性基因高度相似的 EST序列，分别是 BmVLG、。apu和 annexin hi3。其余则主要是一些与DNA复制，能量代谢，信号传导以及胚胎发育中个体形成等相关的基因相似的序列。这些有用的信息为母性基因功能的研究提供了序列信息，为进一步研究家蚕胚胎早期发育的调控提供基础数据，同时还对家蚕功能基因组的研究具有重要推动作用。更多还原

【Abstract】 Silkworm (Bombyx mori) is a basic biologic unit of the sericulture, EST(expressed sequence tag) sequencing can obtain detailed information concerned on molecular level and supply the silkworm’s genomic structure and the information of most genes. It is a significant case study valuable for the further achievement.The collections of silkworm’s EST database have great contribution in discovery of new genes and study of genes kick off and preparation of gene chip. This study constructed cDNA libraries from the silkworm’s kidney and normal unfertilized eggs ,these cells of unfertilized eggs express plenty of information regarding with early embryo development.Maternal genes are the decisive genes which are stored in unfertilized eggs and affect offspring’s development. They transcript during oogenesis and translate during oogenesis or embryogenesis. Maternal genes are responsible for controlling the axis specification, cleavage pattern, morphogenetic movements in the early embryo development. In the development of insects, the anterior-posterior and dorsal-ventral of embryo are determined by maternal genes’ products independently. These maternal genes mainly encode transcript fators or regulative proteins, their products usually form specific concentrative gradient and produce locational information. The information activates the expression of a series of zygotic genes, then the embryo is devided to different regions. The combination of specific genes expressed at different parts form definite segment polarity through anterior-posterior or dorsal-ventral. The mRNAs and proteins in the unfertilized eggs are maternal genes’ products.The primary data were masked out the vector sequence, then wiped off low quality and smaller than 1OObp sequences. These EST sequences were assembled by clusterW. The six frame translations of the EST sequences were compared to the GenBank non-redundant collection of 1,230,998 protein sequences using the function blastx. In addition, the EST sequenceswere compared to the 1,453,916 non-redundant nucleotide sequences in GeneBank using the function blastn. Through blastx and blastn to find similarity between ESTs with protein or nucleotide sequences, We also used SMART to analyse the domains of some genes, in which amino acid sequence have known, with unknown functions, then deduced their characters and functions. In addition, the EST sequences were compared with the 28 cDNA databases’s EST sequences in silkbase. Functional catalogue of known genes were classified by Gene Ontology.At last, we have successfully sequeced 391 ESTs , with an average length of 659bp . After assembling by clusterW , we obtained 374 unigenes, the largest unigene was composed of four ESTs, the longest unigene was 1958bp. There are 15 contigs in the unigenes, 118 singlets are ESTs from normal unfertilized eggs, the other 241 are ESTs from kidney unfertilized eggs.Through large scale sequencing and analysing, we obtained lots of information about functional genes. The bioinformatic analyse found that 171 unigenes have significant similarities with known genes, 25 have significant similarities with known EST sequences, the other 178 have low or no similarities with known genes or EST sequences.The unfertilized eggs cDNA libraries provide a lot of maternal information, most of them except three known maternal genes are relate to replication, metabolism, conduction and embryo development, the three maternal genes are BmVLG,capu and annexin bl3. These useful information provide sequence information for the study of functional genes and maternal genes’ function and is helpful for basic data of regulation during the early embryo development of silkworm and accelerate the study of silkworm’s functional genome.更多还原