臺灣杉生長發育基因－Mago nashi及Y14之研究

Abstract

臺灣杉 (Taiwania cryptomerioides Hayata)為臺灣原生針葉樹種，屬於孑遺植物並且為臺灣重要之經濟樹種。為了深入解析參與臺灣杉生長發育之基因，本研究首先建構臺灣杉發育中莖部組織之cDNA基因庫，共計獲得811個隨機挑選之表現序列標籤，經重組分析定義出322個單一基因。根據BLASTX分析結果得知，11.3%表現序列標籤與細胞防禦、死亡及老化有關，其中包括高度表現之類索馬甜蛋白；3.9%表現序列標籤與蛋白質合成及修飾有關，其中包括高度表現之熱休克蛋白。此外，0.9%表現序列標籤與細胞壁結構及代謝有關，0.7%表現序列標籤與二次代謝有關。然而，有40.9%表現序列標籤並無與基因資料庫中任何蛋白質具相似性，這些序列可能象徵著臺灣杉之獨特性。 Mago nashi (mago)及Y14蛋白於真核生物間具高度之保守性，其不僅於動物卵母細胞形成、胚形成以及生殖細胞性別決定上扮演重要之角色，並且參與細胞生長過程中mRNA之局部化及剪接。為了解臺灣杉生長分化之機制，本研究將臺灣杉cDNA基因庫中所獲得之mago nashi（Tcmago）及Y14（TcY14）之同源基因，進行分子特性及其於臺灣杉中生理功能之分析。Tcmago及TcY14之轉譯區分別可轉譯出149及216個胺基酸。由TcMago及TcY14之蛋白質結構預測顯示，其與果蠅及人類之mago-Y14蛋白質結晶構造相似。此外，TcMago可分別於體內及體外與TcY14進行交互作用，並且兩者皆主要局部化表現於細胞核內。整體式原位分析顯示，Tcmago及TcY14皆可於根毛上被偵測。另外，過量表現Tcmago於菸草轉殖株中，導致植株有較長之根及較複雜之根系。此結果顯示，Tcmago及TcY14可能具有與動物Mago及Y14相似之基礎調控細胞之功能，並且可能參與植物根系之發育。 為進一步了解Tcmago及TcY14之生理調節功能，本研究乃從分析它們的啟動子著手，所選殖獲得Tcmago及TcY14之啟動子序列，其分別包括2,496及1,780個鹼基對。Tcmago及TcY14之啟動子具有相似之調控基序，包括有賀爾蒙（植物生長素、離層酸、吉貝素及水楊酸）的調控，環境逆境（光、冷、創傷及乾旱）及特定組織（種子、葉、莖、根、胚、花粉及保護細胞）的表現。經北方雜合分析，Tcmago於生殖器官中表現較豐富，TcY14則高度表現於根部，且兩者皆於形成層中表現豐富。許多有關調控生殖器官發育及分生組織形成之賀爾蒙調控基序，亦與啟動子序列分析結果相一致。其中，植物生長素與形成層細胞分裂有關，而於Tcmago及TcY14之啟動子序列中分別擁有5個及2個相關基序。此外，藉由水楊酸及甲基茉莉酸可誘導Tcmago及TcY14表現之結果，可推測其可能與植物之防禦機制有關。 Mago及Y14可構成一穩定之異質二元體，其為外顯子連接複合體之核心要素。為進一步研究Mago-Y14異質二元體參與之植物發育過程及與其具交互作用之蛋白質，本研究利用TcMago-TcY14異質二元體做為誘餌蛋白進行酵母菌雙雜合系統分析，篩選於三年生臺灣杉苗木cDNA基因庫中與其具交互作用之蛋白質。共獲得3個與TcMago-TcY14異質二元體具交互作用之蛋白質，其分別為未知蛋白（Tc61）、果膠甲基脂解酶類似蛋白（Tc62）及TPR類似蛋白（Tc72）。Tc61與TcMago-TcY14異質二元體具很強之交互作用，其表現於三年生臺灣杉苗木之莖及葉中，且微量表現於雄毬果及已受粉之雌毬果中。果膠甲基脂解酶類似蛋白（Tc62）豐富表現於三年生臺灣杉苗木之根及已受粉之雌毬果中，顯示其與TcMago-TcY14異質二元體結合，可能參與根之發育及受粉過程中花粉之生長。獲得之Tc72可轉譯完整之TPR類似蛋白，並且於植物中具高度之保守性。Tc72蛋白由10個α-螺旋構成，其中3個保守之TPR基序各包含5個保守之胺基酸殘基，此用於形成穩定之內螺旋構造。組織表現及光/暗處理試驗結果顯示，Tc72基因豐富表現於苗木之葉中，並且對光及暗具敏感性，此結果與Tcmago及TcY14基因研究之結果相一致。此意味TcMago-TcY14異質二元體與Tc72進行交互作用，可能參與光合作用或葉綠體蛋白之傳遞。

Taiwania (Taiwania cryptomerioides Hayata) is an indigenous conifer tree in Taiwan. It is not only a relict plant but also one of the most economically important tree species in Taiwan. To investigate the genes involved in Taiwania development in depth, a developing stem tissue cDNA library of Taiwania was firstly constructed in this study. 811 randomly selected expressed sequence tags (ESTs) clones which were identified a total of 322 unigene sets by contig analysis were obtained. According to the results of BLASTX analysis, 11.3% of these ESTs, which included the highly expressed thaumatin-like protein, were found to be related to the cell rescue, defense, death and aging; 3.9% of the ESTs, which included the highly expressed heat shock proteins, were involved in protein synthesis and processing. Moreover, 0.9% of the ESTs were related to cell wall structure and metabolism while 0.7% of these were related to secondary metabolism. However, 40.9% of ESTs has showed no significant similarity to any other protein sequences in public databases. These sequences in turn may indicate the uniqueness of Taiwania. Mago nashi (mago) and Y14 proteins are highly conserved among eukaryotes, and not only play important roles in oogenesis, embryogenesis and ger-line sex determination during animal developmemt, but also participate in mRNA localization and splicing in cell prowth. In this study, the mago nashi (Tcmago) and Y14 (TcY14) homologues derived from cDNA library of Taiwania were obtained. To understand the molecular characteristics and physiological functions of Tcmago and TcY14 in Taiwania, the full-length of them were obtained for further analyses. Tcmago and TcY14 contained coding regions encoding 149 and 216 amino acids respectively. The predicted protein structures of TcMago and TcY14 were similar to those crystal structures of Drosophila melanogaster and human mago-Y14 proteins. Moreover, TcMago can interact with TcY14 in vivo and in vitro, and they were both predominately localized in the nucleus. Whole mount assays revealed that expression of Tcmago and TcY14 were both detected in root hairs. Additionally, the overexpession of Tcmago in transgenic tobacco plants resulted in longer roots and a more complex root system. It suggested that Tcmago and TcY14 may have essential cellular functions similar to those in animals, and also might be involved in root development of plants. In order to further understand the physiological regulatory functions of Tcmago and TcY14, their promoters were researched in this study. A 2,496 bp promoter of Tcmago and a 1,780 bp promoter of TcY14 from Taiwania were cloned and characterized. The Tcmago promoter and TcY14 promoter had similar motif regulation patterns which were regulated by hormones (auxin, ABA, GA, SA), environmental stresses (light, cold, wounding, drought), and specific tissues (seed, leaf, shoot, root, embryo, pollen, guard cell). According to the analyses of northern blots, Tcmago was expressed more abundantly in reproductive organs, TcY14 was highly expressed in roots, and they were both expressed abundantly in cambium. Consistent with the results of promoter analysis, there were many motifs concerning hormone regulations that are important to reproductive organ development and meristem formation. More importantly, auxin, which is associated with cambial cell division, revealed five and two motifs in the Tcmago and the TcY14 promoters, respectively. Additionally, Tcmago and TcY14 may be involved in plant defense as supported by the observations of their expressions induced by SA and MeJA treatment. Mago and Y14 can form a stable heterodimer which is the core component of the exon-exon junction complex (EJC). To further survey the proteins and the plant developmental processes that the Mago-Y14 heterodimer interacts with and is involved in, the TcMago-TcY14 heterodimer was used as the bait protein in a yeast two-hybrid screen in this study to search for its binding partners in a three-year-old Taiwania seedlings cDNA library. Three interacting clones, an unknown protein (Tc61), a PME (pectin methylesterase)-like protein (Tc62), and a TPR (tetratricopeptide repeat)-like protein (Tc72), were obtained. The Tc61 protein interacted strongly with the Mago-Y14 heterodimer, and its transcript was expressed in the stems and leaves of the three-year-old Taiwania seedlings, and slightly expressed in the microsporangiate cones and pollinated ovulate cones. The PME-like protein (Tc62) was expressed abundantly in the roots of the three-year-old Taiwania seedlings and the pollinated ovulate cones, indicating that binding with the TcMago-TcY14 heterodimer may be involved in the root development and growth of pollen tubes during pollination. The obtained Tc72 protein encoded a complete TPR (tetratricopeptide repeat)-like protein and was highly conserved among plants. The Tc72 protein had 10 α-helices and three conserved TPR motifs containing five consensus residues for forming a stable inter-helix packing. The results of a tissue expression assay and light and dark treatments showed that the Tc72 gene was expressed abundantly in the leaves of seedlings and was sensitive to light and darkness, which were the same with the investigation in Tcmago and TcY14 genes. It implied that the TcMago-TcY14 heterodimer interacting with the Tc72 may be related to photosynthesis or chloroplast protein transport.