建構轉錄體分析平台來探討海水溫度變化對珊瑚共生體之影響

造礁珊瑚(reef-building coral)跟共生藻(Symbiodinium spp.)特殊的細胞內共生(endosymbiosis)關係使得他們得以分佈在熱帶海域的透光區，形成了特殊的珊瑚生態系。但由於過度地海岸開發，環境污染及全球氣候變遷造成的海水環境的變化，影響此種共生關係並威脅著珊瑚生態系。因此，本研究利用次世代定序(Next Generation Sequencing, NGS)的技術，以總體轉錄體學(Meta-transcriptomics)的分析方法，建立完整的分析平台，研究造礁珊瑚與共生藻在海水環境變化時，共生關係的調整與改變，並提供線上資料庫，讓相關研究學者快速掌握後續研究的線索。本論文在第一章，回顧珊瑚及其共生藻類的相關文獻，並論述以次世代定序資料作為基礎，如何以總體基因體學的方法切入分析混雜多種生物的序列資料。第二章，則是說明依據次世代特性及珊瑚共生體分析需求，如何設計出完整的分析平台。在接下來兩章，則是分別基於溫度的變化，利用本平台研究外在環境對造礁珊瑚與共生藻的共生關係所造成的影響。本論文第二章，詳述本分析平台所使用的轉錄體定序(RNA Sequencing, RNA-Seq)的方式及如何處理片段雜訊，得到高品質的序列資料，並說明所選擇的主要序列組裝工具 - Trinity 其演算法原理及序列處理原則。在序列組裝完成後，根據實驗需求，選擇了 NCBI 的 nr 資料庫、KEGG 訊息傳導資料庫及 Pfam 蛋白質功能模組資料庫，可以提供完整的功能性註解幫助分析平台解析。最後，依據資料及實驗目的，設計具搜尋，資料圖形化，功能分析(functional enrichment)的分析平台，才能從這大資料裡面獲取有價值的資訊。實驗方面共分為兩部份，分別探討『高溫』及『溫度變動』對珊瑚造成的影響（第三、四章）。其中第三章探討『高溫』對珊瑚所造成的影響，使用由台灣屏東後壁湖所採集的細枝鹿角珊瑚(Pocillopora damicornis)樣本，在實驗室分別以 27°C 及 30°C 的溫度控制，各分為三個實驗缸做重複實驗，在2週及36週的溫控實驗後，從每一個實驗缸取出樣本抽取 RNA 以 RNA-Seq 的方式產生次世代定序資料，經序列組裝註解後，將結果整合線上分析平台於 http://symbiont.iis.sinica.edu.tw/coral_pdltte/ 以提供後續分析比較。結果顯示，在顯著表現差異基因 (differentially expressed genes, DEG)上，在高溫組中，珊瑚calmodulin及CAMK受到抑制，可能造成細胞內鈣離子濃度上升，促使熱休克蛋白(heat shock protein)生成，高溫壓力下的共生藻，geranylgeranyl reductase表現上升而促使葉綠素合成。推論珊瑚及其共生藻分別以不同生理調控的方式，抵銷高溫所形成活性氧化物質(Reactive oxygen species, ROS)的破壞。本論文第四章則探討『溫度變動』對珊瑚所造成造成的影響，使用對溫度變化敏感的尖枝列孔珊瑚(Seriatopora hystrix)為實驗物種，分別從溫度變化較大的後壁湖及溫度較穩定的後灣採集樣本，兩個地區的樣本均分成六個培養缸，並各自以三缸為一組進行定溫(26ºC)及變溫(23-29ºC以六小時為周期)的實驗，為期一週，也是從每缸取出樣本抽取 RNA 以 RNA-Seq 的方式產生序列資料，經序列組裝註解後，將結果整合線上分析平台於 http://symbiont.iis.sinica.edu.tw/s_hystrix/ 以提供後續分析比較。結果顯示，相對於溫度變動的影響而言，由不同地區採樣的珊瑚，其顯著表現差異基因較多，且集中於共生藻的基因差異，顯示短期且在一定範圍內的溫度變化，對珊瑚基因表現影響並不大，推論這兩個地區的尖刺列孔珊瑚其內共生的共生藻種類不同，需要進一步研究。在處理次世代定序的經驗中，發現龐大而詳細的資料增加了分析的難度，僅僅從生物資訊工具所產生的結果也無法滿足研究人員對序列資料需要了解的深度，於是想要建立分析平台，讓次世代定序資料可以充分反映實驗設計的成果。次世代定序資料經過合適的組裝，再輔以本平台所設計的分析及圖形化呈現，的確讓次世代定序的分析流程變得更系統化，更方便使用。未來可以基於新的分析需求在平台上建立相關工具，增加序列資料的價值，更可以把開發平台的經驗，使用在不同的研究物種與題材，來加速研究的進展。

關鍵字

次世代定序；總體轉錄體學；造礁珊瑚；共生藻；序列組裝；細枝鹿角珊瑚；尖枝列孔珊瑚

並列摘要

Reef-building corals for a mutualistic relationship with photosynthetically active dinoflagellates of the genus Symbiodinium that allows them to dominate high light regions of Earth’s tropical seas. Coral reef ecosystems are threatened by excessive coastal development, environmental pollution, and global climate change, and changes in water quality can lead to the disintegration of the coral-dinoflagellate symbiosis. To gain a better understanding of how changes in temperature, in particular, affect such mutualistic associations, next generation sequencing (NGS) was used to profile the meta-transcriptomes (population of all expressed mRNAs) of all “compartments” of the coral “holobiont” (i.e., coral host+Symbiodinium+bacteria) exposed to 1) elevated temperatures over a long-term timescale and 2) variable temperatures over a short-term one. To uncover the gene mRNAs responsive to temperature change, comprehensive analytical platforms were designed for each experiment; both such platforms were made available to the public as interactive websites. In the first chapter, we review the literature on coral and their symbiotic algae, and discuss how to analyze NGS datasets of multi-compartment organisms (i.e., meta-transcriptomes). In the second chapter, we discuss the various components of the NGS analytical pipeline/platform, as well as the experimental methods. In the next two chapters, we present the data from the two aforementioned experiments with a specific focus on uncovering which cellular pathways of the coral-dinoflagellate endosymbiosis are most affected by elevated and variable temperature exposure. In the second chapter, we introduce NGS and discuss how to deal with sequencing errors in order to produce the highest quality sequence datasets. We also discuss various algorithms and facets of Trinity, a popular NGS sequence assembly tool and one that was used to assemble the transcriptomes of this dissertation. The assembled contigs were functionally annotated by via alignment to the following three databases: NCBI’s “nr” , 2) KEGG , and 3) Pfam. Finally, we implemented a variety of ways to query the datasets: 1) via keyword search, 2) via BLAST search, 3) via functional enrichment, and 4) via statistical parameter filtering. In first experiment, the influence of high temperature on the model reef coral Pocillopora damicornis were explored. P. damicornis specimens originally collected from Houbihu (HBH), Nanwan Bay, Taiwan were exposed to either a control (27°C, n=3) or elevated (30°C, n=3) temperature. After 2 and 36 weeks of temperature exposure, samples were sacrificed for RNA extraction followed by NGS-based RNA-Seq. The Symbiodinium populations were relatively more affected than the host corals in which they resided at the mRNA level, and the differentially expressed genes (DEGs) tended to differ between compartments. An interactive website (http://symbiont.iis.sinica.edu.tw/coral_pdltte/) has been designed to serve as a depository for the sequence data set. In second experiment, the influence of variable temperatures on a coral symbiosis was addressed. Seriatopora hystrix, a temperature-sensitive species, was collected from an upwelling reef in Nanwan Bay, Houbihu, as well as from a non-upwelling site, Houwan. The samples from each region were divided into six tanks and exposed to either a stable temperature (26ºC, n=3) or a variable temperature profile (23-29ºC over a 6-hr cycle, n=3) for one week. After one week of treatment exposure, RNAs were extracted from 12 samples (3/treatment x site of origin group) and sequenced with an Illumina platform. The results revealed that transcriptome varied more across sampling sites than temperature regimes. Also, an interactive website (http://symbiont.iis.sinica.edu.tw/s_hystrix/) has been designed to serve as a depository for the sequence data set. NGS datasets are immense in size and notoriously difficult to analyze; labs using different pipelines may very well come to entirely different conclusions from another lab handed the same sequence dataset. Such analytical difficulties are compounded in meta-transcriptomic analyses, where mRNA populations are of mixed origin. We therefore believe the analytical platforms and websites developed as part of this dissertation have dramatically advanced the meta-transcriptomics field and may be useful to not only those studying coral-dinoflagellate endosymbioses, but also others interested in profiling the transcriptomes of environmental samples in which RNAs may have emerged from a plethora of different species. Ideally, others will adopt this same pipeline such that meta-transcriptome analyses become more streamlined in the near future, allowing for easier comparison across species, sequencing platforms, and laboratories.

並列關鍵字

meta-transcriptome next-generation sequencing ； Pocillopora damicornis ； reef-building coral ； sequence assembly ； Seriatopora hystrix ； Symbiodinium ； temperature

參考文獻

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國際替代計量

建構轉錄體分析平台來探討海水溫度變化對珊瑚共生體之影響

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