麻竹初生壁纖維素合成酶選殖及特性分析

Abstract

麻竹（Dendrocalamus latiflorus）為臺灣分布最廣且數量最多的竹類，其生長快速，又竹筍階段便能累積多量的結晶型纖維素，使得麻竹有潛力成為木質纖維材料的來源。本試驗先測定麻竹筍及竹稈之結晶度，得知麻竹筍基部即具有相當高的結晶度。 從麻竹筍中選殖出5個DlaCESAs，並與負責初生壁生合成的CESAs親源性較接近。5個CESAs可分成3個群組，分別代表負責初生壁纖維素合成酶的3個蛋白質異構物（isoform）：DlaCESA1、DlaCESA3及 DlaCESA6。而完整DlaCESAs皆具有CESAs的特殊蛋白結構。再透過組織表現差異分析可以確認所選殖到3個完整的DlaCESAs於竹筍及竹籜表現量最高，證實3個完整的DlaCESAs為主要參與初生壁生合成相關。 將DlaCESAs轉殖到相對應的阿拉伯芥突變株中，並比較野生型、各互補轉殖株與突變株小苗表型的不同。外源DlaCESAs改變互補轉殖株小苗的外表型趨近於野生型，證實所選殖的DlaCESAs具有初生壁纖維素生合成的功能性。接著探討外源DlaCESAs是否對次生壁加厚的花序莖有所影響。結果顯示僅在rsw1-2組別中， DlaCESA1互補轉殖株的結晶度及結晶型纖維素含量與野生型相同並高於突變株。在ixr1-1及prc1-1組別中，突變株與DlaCESAs互補轉殖株無差異。而野生型、突變株及DlaCESAs互補轉殖株的α-纖維素含量彼此並無差異。至於細胞壁的醣類含量，野生型、突變株及DlaCESAs互補轉殖株在一些成分上有差異，而主要生成纖維素的glucan含量則差異不大。 由以上的試驗結果，顯示所選殖出之DlaCESA1、3及6有發揮其功能，皆能補足初生壁AtCESAs突變株的缺失。而在次生壁加厚的組織中影響有限，推測初生壁纖維素合成酶於次生壁加厚的組織中作用較不明顯。

Ma bamboo (Dendrocalamus latiflorus) is the most widely distributed and abundant bamboo in Taiwan. Ma bamboo can growth rapidly and accumulate large amount of crystalline cellulose during bamboo shoot phase. And hence, ma bamboo has the potential to become a large source of lignocellulosic materials. In this study, the crystallinities of ma bamboo shoot and culm were determined, and the results indicated that there were higher crystallinities in base shoots. 5 CESAs were further cloned from ma bamboo shoot. These 5 DlaCESAs are related to primary cell wall biosynthesis. These 5 DlaCESAs were divided into three groups, corresponding to 3 major primary wall CESA isoforms: DlaCESA1, DlaCESA3, and DlaCESA6. All 3 complete DlaCESA sequences contain special protein structures of CESAs. Expression analysis showed that the relative expression level of these 3 DlaCESAs were highest in the bamboo shoot and bamboo sheath. This result supported that these 3 DlaCESAs might participate in the primary cell wall biosynthesis. DlaCESAs were complemented into the corresponding CESAs mutants of Arabidopsis, and the phenotypic traits of wild-type, the mutants, and complemented mutants were compared. DlaCESAs successfully rescued mutants in its seedling phenotypes, and the phenotypes of the complemented mutants were similar with that of wild-type. Overall, the cloned DlaCESAs in this study are functional in cellulose biosynthesis of primary cell walls. To further understand whether the cloned DlaCESAs had impacts on Arabidopsis inflorescence stems which were abundant with thickened secondary cell walls, the cell wall characteristics of inflorescence stems were analyzed. Only in rsw1-2 group, the degree of crystallinity and the content of crystallinity cellulose of DlaCESA1 complemented mutants were similar with that of wild-type and higher than that of rsw1-2. There were no differences in the the degree of crystallinity and the content of crystallinity cellulose within mutants and complemented mutants of ixr1-1 and prc1-1 groups. The α-cellulose content of wild-type was the same as that of mutants and DlaCESAs complemented mutants. There were some polysaccharide composition differences in the cell walls between wild-types, mutants, and DlaCESAs complemented mutants. However, the glucan content, the major composition of cellulose, was not different in wild-types, mutants, and DlaCESAs complemented mutants. Overall, our cloned DlaCESA1, 3, and 6 all are functional and can rescue corresponding primary cell wall CESA mutants but there is less effect in secondary thickened tissue. It is speculated that the functional roles of primary cell wall CESA were less significant in the secondary thickened tissues.