麻竹稈之木質素特性與其Caffeic acid O-methyltransferase酵素動力學分析

Abstract

麻竹（Dendrocalamus latiflorus, Dla）是臺灣分布範圍最廣的竹類，生長速度快，單一生長季高度可達25 m。在麻竹稈生長過程中，細胞壁會快速累積木質素（Lignin）。麻竹稈細胞壁中具有可觀的p-Coumaric acid及Ferulic acid；而麻竹稈木質素為H-G-S type lignin，其中S lignin隨著生長而累積的量最為顯著。被子植物（Angiosperm）中S lignin主要由Caffeic acid O-methyltransferases （COMTs）進行5-O-Methylation催化生成。從麻竹稈選殖出6條DlaCOMTs，DlaCOMTs全長為1083 bp，可轉譯（Translation）出361個胺基酸。而完整DlaCOMTs皆具有COMT蛋白的特殊結構。由演化樹狀圖可知DlaCOMTs親源關係與孟宗竹（Phyllostachys pubescens）最為相近。利用qRT-PCR測得DlaCOMTs的組織特異性，在木質化程度高之麻竹稈下部的表現量最高。體外表現並純化出的DlaCOMTs重組蛋白，利用酵素動力學測得對5-HCAld （5-Hydroxyconiferaldehyde）在in vitro狀態下專一性最高，同時對5-HFA（5-Hydroxyferulic acid）為競爭型抑制。表示DlaCOMTs在整個木質素生合成途徑中，偏好走途徑下方5-HCAld催化生成SAld（Sinapaldehyde），顯示麻竹S lignin甲基化生合成途徑可能與雙子葉被子植物的S lignin生合成途徑相似。

Ma bamboo (Dendrocalamus latiflorus, Dla) is the most widely distributed bamboo species in Taiwan. It grows extremely fast and can reach about 25 meter in height within one growing season. During its growth, ma bamboo’s cell walls accumulate lignin rapidly. There is a considerable amount of p-coumaric acid and ferulic acid in ma bamboo’s cell walls. And ma bamboo’s lignin is a H-G-S type lignin, and it accumulates S lignin rapidly during bamboo’s growth. The 5-O-methylation of angiosperm S lignin had long been proposed to be catalyzed by the caffeic acid O-methyltransferases (COMTs). 6 DlaCOMTs were further cloned from ma bamboo culm. The full length of DlaCOMTs is composed of a 1083 bp open reading frame encoding 361 amino acids with the highest similarity to that of Phyllostachys pubescens. All DlaCOMTs have the specific protein structures of COMT family. Gene expression profiles revealed by qRT-PCR indicated that DlaCOMTs had tissue specificity and expressed in the lignification stage during culm development. Enzyme activity assays also revealed that DlaCOMTs have in vitro substrate preference for 5-HCAld (5-Hydroxyconiferaldehyde) of the monolignol biosynthetic pathway. The O-methylation activities of DlaCOMTs on 5-HFA (5-Hydroxyferulic acid) were strongly inhibited by 5-HCAld (competitive inhibition). The results implicated that DlaCOMTs would catalyze 5-HCAld to SAld (Sinapaldehyde), and the biosynthesis pathway of S lignin methylation in ma bamboo might be similar to that in dicotyledons.