以次世代定序資料重組南美白蝦的轉錄基因體並探討其基因表現

Abstract

本研究以現今台灣蝦類養殖的主要蝦種:南美白蝦(學名: Litopenaeus vannamei，又名白蝦、白對蝦)為對象，利用 RNA-Seq技術，定序白蝦幼蝦與造血組織、血細胞等組織的轉錄體，並整合目前發布在 NCBI上的白蝦高通量定序資料，進行組裝(de novo Assembly)，希望能夠最完整的表現出白蝦的轉錄體資訊。經過初步的鹼基讀序品質檢查後，由平均長度為90 bps，共168 million條短讀序開始，利用trinity 進行組裝，共組裝出19萬餘條，濾除掉序列高度相似的組裝序列後，得到約 165,000條的重組序列。經轉譯產物預測分析取得每一組裝序列的最長開放讀架(open reading frame) ，共可轉譯出 62,000 餘條的蛋白質序列，其中有將近 40% 的序列在 nr 資料庫中找到對應 (blastp, e value cutoff: 1E-5)。分析這些對應序列的物種組成，發現 80% 以上為節肢動物門昆蟲綱與甲殼類的相近物種。與KEGG資料庫的比對結果顯示，約有 40% (23,700 /62,026) 以上的組裝序列能在 KEGG中找到對應資料。另以SignalP 與 tmHMM分析蛋白質序列，其中有1,198 條被預測為為膜蛋白，3,730條可能為具訊息胜肽 (signal peptide)。 透過定序資料映射(Mapping) 回組裝所得的轉錄體模板後，便能獲得各組資料中每條重組序列的表現量,進而計算不同狀況下的基因表現差異。本研究分析HPT，HC，PL三個組織之間的差異，並以LPS( Lipopolysaccharide，脂多醣)來模擬細菌感染的情境，透過對不同時間點的血細胞與造血組織檢體的定序資料進行基因表現叢集分析 (clustering Analysis)。在HPT，HC，PL的叢集分析結果，可以看到HC與HPT兩組有較高的內皮組織穿膜遷移(Transendothelial Migration)的活性；另外，觀察Astatine、Antimicrobial peptide、Prophenoloxidase三個標的基因在各組間的表現，發現三者皆在血細胞組有明顯表現，並觀察到Astakine在血細胞實驗組中於注射後六小時，有相對的高量表現，而Antimicrobial peptide與Prophenoloxidase卻恰好相反，在在注射後六小時為最低表現。經由比較叢集分析所得到的表現模式，與血細胞Astakine的表現模式一致的基因還有fushi tarazu-factor 1及zinc metalloproteinase，推論可能是Astakine表現時，還有其他的協同因子共同參與。 最後，我們將組裝序列及註解結果、後續的分析資料整合，建構為線上資料庫，提供即時的轉錄體序列與功能分析查詢，預計將能對甲殼類相關研究社群帶來助益，並對白蝦轉錄體有更深的認識及瞭解，進而有助於整體水產研究深度與廣度的提昇。

Whiteleg shrimp (Litopenaeus vannamei), also known as Pacific white shrimp, is one of the major aquaculture species in Taiwan and around world, while the information of molecular regulation and basic gene knowledge on whiteleg shrimp are poor. In this study, we aim to apply Next-Generation Sequencing (NGS) technology to get the most updated and well annotated transcriptome of the Litopenaeus vannamei. Starting from 168 million illumina read pairs with average length around 90 bps from RNA-Seq libraries of post-larvae whole body (PL), hematopoietic tissue (HPT), hemocyte (HC), and larvae (composed by several development stages), we perform Trinity (de novo assembler) to assemble 190 thousand contigs in average length equal to 893 bps. For decreasing of redundant contigs, the assembly were further merged by CD-HIT. Finally, 165,000 contigs around were representative as whole transcriptome of whiteleg shrimp in this study. Sixty-two thousand putative protein products were predicted with at least length >30 residues. Overall 38.5% of the protein products were annotated by nr (E value cutoff: 1E-5). Among these annotated contigs, eighty percent of the best matched sequences are from Phylum Arthropoda. Meanwhile, forty percent of putative proteins can map on KEGG database. A single dose of lipopolysaccharide (LPS) administration was applied for mimicking the immune response upon bacteria challenge in shrimps. RNA-Seq data from hematopoietic tissue and hemocytes were mapped to the shrimp transcriptome assembly to calculate the expression profiling. The differentially expressed genes (DEGs) were identified and further clustered to reveal the specific expression patterns. In this approach, three important immune genes, Astakine, Antimicrobial peptide, and Prophenoloxidase, are found with high expressions in hemocyte. The expression of Astakine was peaked at six hours after LPS administration, but the expression of Antimicrobial peptide and Prophenoloxidase were in opposite way. Genes like fushi tarazu-factor 1 and zinc metalloproteinase were also found in the same expression patterns by the clustering analysis, suggesting the functional cooperation with Astakine in LPS induced response. Next-Generation Sequencing (NGS) undergoes vigorous development in recent years. Compare to the traditional sequencing, NGS has lower cost and can fetch a large amounts of data with the high efficiency and benefit. In this study, we successfully applied this approach and build a pipeline to recover almost all of the known white shrimp transcripts in NCBI Unigene set and discovered more non-protein coding genes. By integration of all the assembly with annotations, the web database for transcriptome of Litopenaeus vannamei is built and it will be shared to research community worldwide with free access.