Real-Time PCR-Based Gene Expression Analysis in the Model Reef-Building Coral ＂Pocillopora Damicornis＂: Insight from a Salinity Stress Study

即時聚合酶鏈鎖反應分析細枝鹿角珊瑚之基因表現：鹽度壓力研究

梅菲爾得．安德森(Anderson B. Mayfield) ； 樊同雲(Tung-Yung Fan) ； 陳啟祥(Chii-Shiarng Chen)

《Platax》 2013卷 (2013/12) Pp. 1-29

https://doi.org/10.29926/PLATAX.201312_2013.0001

摘要

細枝鹿角珊瑚的廣泛分布全球，使其逐漸成為研究全球氣候變遷時所選擇的珊瑚代表物種。為了更進一步發展可有效確實瞭解珊瑚細胞內對環境改變之反應的分子生物技術，本研究從三個角度深度探討珊瑚對壓力的細胞分子反應。一，鹽度壓力實驗。樣本暴露在五種不同的濃度下（24，28，32（控制組），36或40 psu）經過不同時間長度（1，2，或3小時）後測量下述基因的基因表現：三個mitogen-activated protein kinase (MAPK) genes；stress-activated protein kinase 2 (P38)，extracellular signal-regulated kinase 2 (erk2)，and big MAPKl (bmkl)。本研究發現在1小時28 psu的處理下p38和erk2大量的增加其基因表現。此結果可能代表這兩個蛋白質和珊瑚細胞初期如何應對低鹽度壓力環境有關並可能為未來研究珊瑚如何對環境變動反應之重要分子目標。二，本研究建立臺灣南部海域細枝鹿角珊瑚的分子藍圖已進一步發展可適用的分子分析試驗。目前各國研究團隊所發展的分子分析試驗中，僅有大約55%可用於台灣的細枝鹿角珊瑚。舉例來說，由摩納哥細枝鹿角珊瑚所發展的即時聚合酶鏈鎖反應試驗（real-time PCR assay）並不適用於臺灣的細枝鹿角珊瑚。三，使用基因轉錄體序列研究辨識與適應高溫環境有關的基因。目前總共有28組基因（14組珊瑚基因以及14組共生藻基因）己可利用即時聚合酶鏈鎖反應準確地測量其基因表現並廣泛運用於印度太平洋區域。未來將利用上述基因分子指標研究臺灣海域珊瑚，並更進一步用於參與蘇爾坦海洋基金會的全球珊瑚考察團進行廣泛的分子指標調查。

關鍵字

珊瑚 ； 雙鞭毛藻 ； 胞內共生 ； 基因表現 ； 滲透壓調節 ； 細枝鹿角珊瑚 ； 即時聚合酶鏈鎖反應 ； 鹽度壓力

並列摘要

＂Pocillopora damicornis＂ has emerged as the model coral for studying global climate change given that it is found in the waters of nearly all nations that invest significantly in coral reef research. To further the development of a molecular blueprint for understanding the sub-cellular response of this coral to changing environmental conditions, three approaches were taken herein. First, a salinity challenge study was conducted with corals from Kaneohe Bay, Hawaii (United States). Specimens were exposed to 24, 28, 32 (control), 36, or 40 partial salinity units (psu) for 1, 2, or 3 hrs, and the expression of three mitogen-activated protein kinase (mapk) genes; stress-activated protein kinase 2 (p38), extracellular signal-regulated kinase 2 (erk2), and big mapk 1 (bmk1), was measured. p38 and erk2 both exhibited large increases in expression in corals exposed for 1 hr to 28 psu, potentially suggesting that the associated proteins are involved in the initial stages of the hypo-osmotic stress response and may be important molecular targets for understanding this coral's response to environmental perturbations. As a second effort to better develop the molecular infrastructure of P. damicornis populations in Southern Taiwan in particular, real-time PCR assays from previously published works were applied to cDNA generated from RNA derived from ＂P. damicornis＂ populations of Nanwan Bay in Southern Taiwan. In many cases, assays developed from ＂P. damicornis＂ cultures reared in Monaco were found to be unsuitable for use with Taiwanese specimens, and, overall, only ~55% of assays developed by other laboratories could be utilized with confidence with Taiwanese ＂P. damicornis＂ specimens. Finally, a next generation sequencing-based transcriptome sequencing study was used as a platform for the identification of genes involved in acclimation to high temperature, and 13 gene expression assays were developed for use with ＂P. damicornis＂. In total, there are now 28 genes (14 in each of the host and ＂Symbiodinium＂ compartments) whose expression levels can be measured accurately with real-time PCR in this ecologically important reef builder that exists in many regions of the Indo-Pacific. It is hoped that these assays will not only be employed in Taiwan-based studies, but in molecular biomarker surveys conducted as part of the Living Ocean Foundation's Global Reef Expedition aboard the research vessel ＂Golden Shadow＂.

並列關鍵字

coral ； dinoflagellate ； endosymbiosis ； gene expression ； mitogen-activated protein kinase (MAPK) ； osmoregulation ； ＂Pocillopora damicornis＂ ； real-time PCR ； salinity stress