臺灣杉核質間穿梭蛋白TcNMD3之基因表現圖譜與分子特性之研究

Abstract

NMD3 (nonsense-mediated mRNA decay protein 3)為一細胞核質間的穿梭蛋白，將60 S核糖體從細胞核運送至細胞質，再回到細胞核。為了了解NMD3在演化及臺灣杉中所具之生理現象，我們從臺灣杉苗木選殖出的假定NMD3基因命名為TcNMD3。TcNMD3的轉譯區共有1548個核苷酸，此基因在臺灣杉苗木的基因體中存在三個套數。經由組織專一性的表現分析結果顯示TcNMD3基因在70年生臺灣杉的樹皮和頂端分生組織較其雄花與雌花中為多；而在四年生的臺灣杉苗木中，則是在葉和莖中較其在根中為多。 根據TcNMD3啟動子序列分析，我們對22天大的臺灣杉小苗做了不同的物理逆境、植物賀爾蒙濃度梯度及鹽逆境處理。TcNMD3的RNA表現量在6小時的400 μM 吲哚乙酸，600 μM 茉莉酸，400 μM 水楊酸，10 μM 離層酸和200 mM 氯化鈉處理下及72 hr光照和37 ℃處理下，比起控制組為增多。這個現象也許提示了TcNMD3和植物的生長發育(吲哚乙酸)、防禦機制(茉莉酸和水楊酸)與植物賀爾蒙所牽涉的訊息傳導(離層酸)有著直接或間接的關連性。 NMD3蛋白質主要特徵為在其胺基酸N端序列中四個Cys-X2-Cys結構，且在其C端中發現NLS和NES序列，此結果也符合NMD3在細胞核質間穿梭的角色。利用酵母菌雙雜合方法，以TcNMD3做釣餌，釣取獲得交互作用之蛋白質TcSRC2。TcSRC2蛋白質中的N端具C2功能區塊(C2 domain)屬於膜蛋白的一個特徵，因此，也使TcSRC2和穿梭於細胞核質間的TcNMD3有了合理的關連性。

NMD3 (nonsense-mediated mRNA decay protein 3) is a shuttling protein between nucleus and cytoplasm. It helps export 60S ribosomal subunit from nucleus to cytoplasm and then is recycled to the nucleus. Putative NMD3 gene cloned from Taiwania seedling in this research was named TcNMD3 to elucidate the evolutional role and the physiological phenomenon in Taiwania. There are 1548 nucleotides in the coding region of TcNMD3. TcNMD3s are multiple copies in the Taiwania genome. By tissue-specific expression analysis, the TcNMD3 transcripts were more abundant in bark and apical meristem than in male and female flowers in mature trees. In seedlings, TcNMD3 transcripts are more abundant in leaves and stems than in roots. According to the cis-elements analysis of TcNMD3, several physiological stress, pytohormone gradient concentration and salinity stress treatments were treated to Taiwania 22-d-old seedlings to profile the pattern of the TcNMD3 transcripts. The TcNMD3 transcripts were up-regulated by 6 hr of 400 μM IAA, 600 μM JA, 400 μM SA, 10 μM ABA, 200 mM NaCl treatments, 72hr light condition and treatment of 37℃. The results might mean that TcNMD3 has a direct or indirect connection with growth development (IAA), defense mechanism (JA and SA), and signal transduction (ABA). The main characteristic in NMD3 is four Cys-X2-Cys motifs in its N terminal region which is the zinc-binding motif, and the NLS and NES, also found in the C terminus of TcNMD3. These results are consistent with which NMD3 is a shuttling protein between nucleus and cytoplasm. By using the yeast two-hybrid method, TcSRC2 was the interacting protein with the TcNMD3 as a bait. The C2 domain in SRC2 is a feature belonged to membrane proteins, and this makes a possible connection to NMD3, a shuttling protein between nucleus and cytoplasm. [Keywords] NMD3, Taiwania, stress, phytohormone, yeast two-hybrid, C2 domain.