阿拉伯芥TMAC2基因(two or more ABREs containing gene 2)受ABA及鹽誘導表現，轉譯成一個含有319個氨基酸且功能未知的蛋白質。前人研究發現，過量表現TMAC2的阿拉伯芥轉殖株，會抑制開花及根部生長。接上報導基因sGFP (synthetic green fluorescent protein gene)的TMAC2-sGFP融合蛋白，經由短暫性轉染法(transient transfection)於阿拉伯芥葉肉原生細胞(protoplasts)表現，利用定位分析結果得知TMAC2-sGFP位於細胞核，推測TMAC2具有核定位訊號(nuclear localization signal, NLS)。為了進一步找出影響TMAC2入核的重要訊號，利用PSORT分析TMAC2氨基酸序列，發現位於N端氨基酸82-86 (NLS1)屬於典型monopartite NLS，以NetNES分析發現氨基酸65-71為leucine-rich功能區，即所謂核輸出訊號(nuclear export signal, NES)。而分別從TMAC2蛋白兩端逐一刪除部分氨基酸並接上sGFP的融合蛋白定位結果，顯示TMAC2氨基酸中69-108及194-216區域是促使TMAC2蛋白進入細胞核所必須的序列。除了在69-108片段之中已知的NLS1，TMAC2 氨基酸194-216區域中尚有一段兩端富含離氨酸(K)，中間由一群低保守性氨基酸組成，屬於新穎的bipartite NLS (氨基酸194-214，NLS2)。而缺少NLS1或NLS2會影響TMAC2入核能力，顯示其為功能性NLS。經酵母菌雙雜合實驗(yeast two-hybrid)分析發現TMAC2與阿拉伯芥importin α (At-Imp α)蛋白之間具有交互作用，證實TMAC2藉由importin α-dependent路徑傳送到細胞核。最後，分別刪除TMAC2兩端部分氨基酸[TMAC2 (△1-68)與TMAC2 (△271-319)]的突變型阿拉伯芥轉殖株，晚開花及短根性狀即消失，顯示TMAC2兩端氨基酸序列會影響開花及根部生長。綜合以上研究，TMAC2蛋白含兩段功能性NLS (NLS1和NLS2)及一個NES，且TMAC2兩端氨基酸序列可能與調控開花及根部的生長有關聯。

TMAC2 (two or more ABREs containing gene 2) encodes a novel protein of 319 amino acids and is up-regulated under ABA and salt treatments in Arabidopsis. In previous study, constitutive overexpression of TMAC2 in Arabidopsis caused the inhibition of flowering and root elongation. In frame fusion of TMAC2 to the synthetic green fluorescent protein (sGFP) directed the sGFP to the nucleus in transiently transfected Arabidopsis mesophyll protoplasts, suggesting that TMAC2 may possess nuclear localization signals (NLSs). To clarify the specific sequence responsible for its nuclear localization, PSORT analysis shows that one classic monopartite NLS motif (NLS1) is found in the N-terminal amino acid 82-86, and NetNES analysis reveals the presence of a putative leucine-rich NES (nuclear export signal) in amino acid 65-71. However, subcellular localization of fusion proteins comprising sGFP and deletion constructs of TMAC2 expressed in protoplasts indicates that the regions aa 69-108 and 194-216 are required for TMAC2 nuclear localization. In addition to NLS1 in the region aa 69-108, another NLS (NLS2) is found in the region aa 194-214 containing two clusters of basic residues (lysine) separated by an unconserved linker region. Hence, NLS2 belongs to a novel bipartitle NLS. Furthermore, deletion of either NLS1/NLS2 or both would result in cytoplasmic localization, indicating that TMAC2 has two functional NLSs. Yeast two-hybrid analysis revealed that TMAC2 interacts with NLS receptor-Arabidopsis importin α (At-Imp α) and demonstrated that TMAC2 is targeted into the nucleus via importin α-dependent pathway. However, deletion constructs of either aa 1-68 or 271-319 and individually expressed in wild-type Arabidopsis plants did not show the late flowering and short root phenotypes, implying that these fragments may have essential roles for flowering and root growth. In conclusion, two functional NLSs and one putative NES were identified within the Arabidopsis TMAC2 protein and in which two regions, amino acids 1-68 and 271-319, appear to be associated with flowering and root growth.

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