麻竹木醣轉移酶活性分析及基因選殖

Abstract

麻竹（Dendrocalamus latiflorus Munro, Dla)是臺灣的速生竹種，能於一個生長季由新筍成長至高於20 m的竹桿，生長速度極快。在麻竹細胞壁中，木聚醣（Xylan)是蘊藏量豐富的重要非纖維素多醣（Non-cellulosic polysaccharides)，含量可達細胞壁組成分之20%，然而目前對於其生合成的了解並不透徹。本研究比對實驗室先前自行組裝之麻竹Transcriptome資料庫，發現共有9個第43醣基轉移酶家族（Glycosyltransferase 43 family, GT43）（DlaGT43A - I）及5個GT47家族之酵素（DlaGT47A - E）可能與麻竹木聚醣主鏈之生合成有關。這些木聚糖主鏈生合成酶分屬於IRX9、IRX9L、IRX14/14L及IRX10/10L四個不同的親緣分支群（Clades），除了IRX9分支群外，其餘GT43家族之生合成酶均屬第二型膜蛋白；此外，大部分DlaGT43s與DlaGT47s序列中具有部分保守的DxD motifs與GT signature motifs可能提供重要的酵素功能，其序列之CTS區域（CTS, Cytoplasmic tail + Transmembrane domain + Stem region）具有類似的Arg-base motifs及訊息肽（Signal peptide），可能與酵素的派送（Sorting）有關。若分析主鏈生合成基因在麻竹各組織中mRNA之表現，可發現其具有動態的組織依賴性（Dynamically tissue-dependent expression）。而從麻竹微粒體分部（Microsomal fraction）中偵測木醣轉移酶（Xylosyltransferases, XylTs）酵素活性可看出，麻竹XylTs在20oC、pH 6.8並添加二價陽離子（Divalent cation）與外源受體（Exogenous acceptor）環境下具有最佳的酵素活性，且在竹筍中有較高的酵素比活性。除此之外，本試驗亦嘗試利用麻竹癒傷組織進行懸浮培養，初步建立原生質體（Protoplast）分離及基因短暫表現（Transient expression）之系統，藉此研究麻竹細胞中酵素運作之胞內表現位置（Subcellular localization）。利用此一系統發現，麻竹木聚醣主鏈生合成酶在單獨表現時並不完全表現於高爾基氏體（Golgi apparatus），其很可能需要形成蛋白質複合體才會具有完整表現功能。

Ma bamboo (Dendrocalamus latiflorus Munro, Dla) is one of the fast growing bamboo species in Taiwan. It can reach more than 20-m in height within one growing season. Xylan is the most abundant non-cellulosic polysaccharide in ma bamboo cell wall, and the content is nearly 20%. Althought it is well known that xylan plays an import role in cell wall structure, the mechanism of xylan biosynthesis of this species is still unknown. In this study, nine members of the glycosyltransferase 43 (GT43) family (DlaGT43A - I) and five members of the GT47 family (DlaGT47A - E) were identified by searching our in-house ma bamboo transcriptome database. These identified genes were potentially involved in the biosynthesis mechanism of xylan backbone biosynthesis, and could be classified into four phylogenetic clades named IRX9, IRX9L, IRX14/14L and IRX10/10L. All members of the GT43 family are predicted to be type II membrane proteins except for IRX9 clade. In addition to partially conserved DxD motifs and GT signature motifs that might be crucial for enzymatic function, several Arg-base motifs and signal peptides in CTS regions (CTS, Cytoplasmic tail + Transmembrane domain + Stem region) that might relate to enzyme sorting are also included in some amino acid sequences of DlaGT43s and DlaGT47s. Analysis of mRNA expression of these xylan backbone biosynthesis genes showed a clustered profile between different ma bamboo tissues. This result indicated the dynamically tissue-dependent expression pattern of xylan backbone biosynthesis mechanism in ma bamboo. Xylosyltransferases (XylTs) activities of microsomal fractions isolated from ma bamboo were also investigated, and the optimal activity is at 20oC, pH 6.8 with the addition of divalent cation and exogenous acceptor. Futhermore, higher specific activities were observed in the ma bamboo shoots over other ma bamboo tissues. We also culture the suspension cells from pre-established ma bamboo calli in our laboratory, and establish the protoplast isolation and transient expression system to study the enzyme subcellular localization in living ma bamboo cells. Using this system, XylTs of ma bamboo didn’t seem to be localized in Golgi apparatus perfectly when expressed alone. It is reasonable to speculate that forming a complex might be an essential process for complete localization and function in Golgi apparatus.