|Abstract: ||矽藻是海洋中重要的初級生產者，且對於減緩全球溫室效應具有相當的重要性。矽藻除了被攝食者捕食外，其消長狀態也會隨著環境因子而改變，如何進一步誘發細胞產生相對應的生理反應，亦是一個有趣的課題。當矽藻細胞發生損傷時或矽藻藻華發生末期，會誘發矽藻本身代謝產生「多元不飽和醛類」 (polyunsaturated aldehyde, PUA) 這類的化學物質。許多研究指出，PUA 不僅導致橈足類這類攝食者的受精卵無法孵化或是孵化後幼生發生畸型死亡的現象外，也會促使矽藻細胞本身發生類似多細胞生物的「程式化細胞死亡機制」 (programmed cell death, PCD) 的自殺現象。但對於 PUA 如何誘發矽藻 PCD 自殺現象，當中的細胞生理調控機制目前所知仍十分有限。本研究以海洋圓形矽藻「骨藻」(Skeletonema tropicum, Kaohsiung strain) 作為模式藻種，「死亡特異蛋白基因」(DSP1與DSP2) 為指標基因，先測試出合宜的 PUA 添加濃度，作為建立全轉錄體基因庫的依據。結果顯示，添加 100 μM 2,4-heptadienal (HEPTA)、或 50 μM 2,4-octadienal (OCTA) 或 50 μM 2,4-Decadienal (DECA) 三種不同型式的 PUA於培養液中，HEPTA 與 OCTA 可誘發 DSP1 與 DSP2 mRNA 大量表現，DECA 則不會誘發 DSP1 與 DSP2 mRNA 表現。此外，若添加 200 μM H2O2提供細胞大量 ROS 時，也會誘發 DSP1 與 DSP2 mRNA大量表現。但若以Tempol (ROS清除劑) 或 c-PTIO (NO清除劑) 先行培養後，再添加 HEPTA 則發現 DSP1 與 DSP2 mRNA 基因表現有被抑制的情形。依據上述結果得知，NO與 ROS 應是 PUA 誘發骨藻細胞大量表現 DSP mRNA 的重要二次訊息傳遞媒介。以此，進一步分別建立不同 PUA 等藥劑處理的全轉錄基因庫後，經由Illumina NextSeq500 高通量基因定序平台以pair-end方式進行分析，總共得到780 Mega 條序列 (平均讀長150 bases，總共117 Giga bases)，覆蓋率 50 倍以上。以 DSP 表現量 (reads per kilobase per million reads, RPKM) 為指標，初步檢視 RNA-seq 實驗結果，如同預期，在處理組中 DSP 的表現量均遠大於控制組，證實這些全轉錄基因庫是具有代表性，可以進一步分析經由 PUA 為媒介，所誘發的矽藻基因表現全貌。|
Polyunsaturated aldehydes (PUAs) derived from wounded-diatoms are considered as secondary metabolites belonging to the oxylipins known to act mainly as teratogenic compounds in different marine organism. Moreover, PUAs can also be toxic to diatoms themselves, and were shown to induce programmed cell death (PCD), which is proposed as an important cellular mechanism responsible for massive cell loss during phytoplankton bloom succession. Previous studies have showed that environmental stresses (e.g. cell aging or light limitation) trigger DSPs expression to lead to PCD via NO signaling. However, information for the signal transduction and regulatory mechanisms eliciting PCD in PUA-stressed diatoms is still in its infancy. In this study, a marine diatom, (Skeletonema tropicum, Kaohsiung strain), was applied as the model for this purpose. The mRNA levels of DSP1 and DSP2 in S. tropicum cells were measured after the treatments of the PUAs, 2,4-heptadienal (HEPTA) and 2,4-octadienal (OCTA) and 2,4-Decadienal (DECA). Our results indicated that only HEPTA and OCTA, but not DECA, were able to induce DSP1 and DSP2 expression. In addition, cells pre-treated with ROS scavenger, Tempol, or NO scavenger, c-PTIO, for 30 min prior to HEPTA addition could obviously reduce the DSP1 and DSP2 expression. These results implied that ROS and NO were the secondary messengers to upregulate DSP1 and DSP2 expression via PUA signaling. Furthermore, cells under different treatments of PUAs (i.e. HEPTA and OCTA), H2O2 (ROS donor) and DEANO (NO donor) were harvested for RNA-seq libraries construction. The analysis have been performed using pair-end sequencing approach by Illumina NextSeq 500. A total of 117 Giga bases (150 bases per read length) with a coverage more than 50 times was obtained. As expected, all of the DSP1 and DSP2 expressions level in the PUAs, H2O2, and DEANO treatments were significantly greater than the no-treatment control. Therefore, these RNA-seq libraries are confident for further elucidating the molecular regulatory mechanisms in S. tropicum cells under the stresses mediated by PUA signaling.