豬胚早期發育階段大規模表現序列標籤之功能性基因體分析及其註解

Abstract

哺乳動物埋殖前胚發育經歷許多關鍵及獨特的轉變，諸如胚源性基因體轉錄作用發軔，特有的胚葉細胞緊密化作用及細胞分化之初始等，而這些生理作用受到埋殖前胚特定發育階段次序性基因表現所影響，以維持其正常生長功能。本研究之主旨為利用功能性基因體分析工具進行豬埋殖前胚發育階段表現序列標籤（expressed sequence tags, ESTs）之身份註解。 本研究利用豬不同發育階段：四細胞、八細胞、桑椹胚及早期囊胚基因庫之cDNA序列進行分析。經過多重序列品質確認步驟可得24,605條高品質EST序列。之後，根據序列相似性進行群集分析，產生1,327個群集(clusters)及1,736條單一序列(singletons)。於不同cluster組合而得之序列組合片段(contigs)與singletons一併利用blastn演算法比對搜尋National Center for Biotechnology Information (NCBI)之 non-redundant nucleotide sequence (nt) database以進行序列身分確認。此外，比對搜尋UniGene database（物種：豬、小鼠）以確認其特定表現組織。而經上述流程未有身份註解之contigs及singletons進一步利用Open Reading Frame Finder (ORF Finder)取得其轉譯序列，以期能於蛋白質層次得到相關資訊。另具身份註解之ESTs則利用基因功能分類體系(Gene Ontology , GO)資料庫進行基因功能分類。 結果顯示核醣體蛋白質(ribosomal protein, RP)基因於豬桑椹胚階段活化，並於早期囊胚時期大量表現，而分析結果指出其活化目的主為負責細胞代謝及生合成之基礎功能，據此可推測RP基因為支持埋殖前胚正常發育之基石。於四細胞期活化之非RP基因其功能註解主要為負責核苷酸鍵結(nucleic acid binding)，據此推測基因於此時期活化係為了協助豬發生於四細胞期之胚源性轉錄體活化。而八細胞期非RP基因之主要功能分類為蛋白質鍵結(protein binding)及核醣體之構築(structure constituent of ribosome)，此結果顯示其可能於此時期活化以協助母源性蛋白質降解，胚源性蛋白質轉錄活化。桑椹胚時期活化之非RP基因功能註解則集中於細胞溝通(cell communication)及細胞增生(cell proliferation)二功能分項，而此二者正為推動胚葉細胞緊密化作用(compaction)之重要功能，由此可知，活化基因於桑椹胚階段之緊密化作用乃至後續囊胚形成扮演重要角色。另分析結果指出，412個contigs及singletons之轉譯序列長度＞50 amino acid residues，此對於後續蛋白質身分之搜尋比對甚有助益。 利用生物資訊工具進行cDNA基因庫之身分及功能註解對於了解埋殖前胚發育階段表現基因有其必要性，而此研究策略亦有助於實驗室篩選於埋殖前胚發育階段特異表現基因進行後續分析。

Preimplantation development of mammalian embryo is marked by many critical and unique events, such as starting of zygotic transcription, the first blastomere compaction and initiation of cell differentiation. Sequential gene expression influences the physiological processes through specific stages of embryogenesis for normal functionality of mammalian early development. The objective of this study was to annotate the gene identity of large-scale expressed sequence tags (ESTs) expressed in the preimplantation period of pig by using bioinformatics tools of functional genomic analysis. This study has adopted the cDNA sequences of the EST libraries cloned from porcine embryos during various stages of early development including 4-cell, 8-cell, morula and early blastocyst. The cDNA sequences were filtered using several quality control criteria and resulted in 24,605 high quality EST sequences. Thereafter, these sequences were clustered into groups according to sequence similarity to produce 1,327 clusters and 1,736 singletons. Tentative consensus sequences (contigs) assembled in different clusters along with singletons were searched by blastn against the non-redundant nucleotide sequence (nt) database of National Center for Biotechnology Information (NCBI) for gene identification; then, these sequences were searched against UniGene database (species: mouse and pig) to confirm the specific tissues in which they were expressed. Those contigs and singletons without known identity were translated to amino acid sequences using Open Reading Frame Finder (ORF Finder) to provide more information on protein level. In order to classify the functionality of sequences with known identity, the list of gene identity was analyzed for functional annotation using Gene Ontology (GO) database. Results showed that expression of ribosomal protein (RP) genes were increased at morula stage, and abundantly activated at early blastocyst stage with the functional annotations related to metabolic and biosynthetic processes. It suggests that RP genes might play a fundamental role for supporting the development of porcine early stage embryo. The non-RP genes expressed at the 4-cell stage were mainly annotated to nucleic acid binding which might be responsible for activation of zygotic genome transcription during this stage. The major functions of those non-RP genes activated at the 8-cell stage were protein binding and structure constituent of ribosome demonstrating that they might work for degradation of maternal protein and initiation of embryonic-derived protein translation. Those genes expressed at the morula stage were numerously mapped to cell communication and cell proliferation indicating that cell-cell interaction was activated at this stage for compaction to prepare the blastocyst formation. There were 412 contigs and singletons without known identity could be translated to amino acid sequence with greater than 50 amino acid residues, which might be helpful in further annotation of such type of EST sequences. Annotation of these cDNA libraries using bioinformatics tools is essential for identifying the expressed genes of early embryogenesis with their functionality. Therefore, such strategy of study would facilitate laboratory to select interesting genes which involve in early embryogenesis for further studies.