紅檜Caffeoyl-CoA 3-O-methyltransferase基因之選殖及其轉基因菸草木質素分析

Molecular Cloning of Caffeoyl-CoA 3-O-Methyltransferase Gene from Chamaecyparis Formosensis and Its Effects on Lignin Content in Transgenic Tobacco Plants

林彥良(Yan-Liang Lin) ； 王升陽(Sheng-Yang Wang) ； 曲芳華(Fang-Hua Chu)

《中華林學季刊》 41卷3期 (2008/09) Pp. 323-338

https://doi.org/10.30064/QJCF.200809.0003

摘要

木質素為植物細胞壁內含量僅次於纖維素之生物聚合物，而甲基轉移酵素(O-methyltransferase, OMT)在其生合成反應中扮演了相當重要的角色。本研究於紅檜試材中，選殖出526個鹼基對之Caffeoyl CoA 3-O-methyltransferase (CCoAOMT)片段基因序列，再經由5'和3' RACE (Rapid Amplify of cDNA End)及Genome walking，共獲得計有1721個鹼基對，包含啟動子及基因全長，命名為CfCCoAOMT。其中，蛋白質轉譯區有750個鹼基對，可產生具有249個胺基酸之蛋白質。利用北方雜合分析結果發現，CCoAOMT以發育中木質部的表現量最高。續將CfCCoAOMT基因轉入大腸桿菌大量表現其重組蛋白，並製作成多株抗體以進行免疫雜合反應。由於木本植物與草本植物具有不盡相同的木質素生合成機制，在轉基因的試驗當中發現木質素生成的總量於正義股CfCCoAOMT的轉殖株中並無太大改變，而反義股的轉殖株中有些微升高的趨勢；然而無論正義股或反義股的菸草轉殖株，在木質素單體G/S的比值上皆有明顯增加的趨勢。

關鍵字

紅檜 ； G/S比率 ； 木質素 ； 甲基轉移酵素 ； 轉基因菸草

並列摘要

Lignin is the second abundant biopolymer and less then cellulose present in the plant cell wall. O-Methyltransferase (OMT) plays as a key enzyme for stepwise biosynthesis of lignin. In this study, 526 base pair DNA fragment of OMT were obtained from Chamaecyparis formosensis. Then the 5' and 3' RACE (Rapid Amplify of cDNA End) coupled with the Genome walking were used to clone the full length of CCoAOMT, named CfCCoAOMT. After combining the sequence data, there is a total of 1721 bps in full length including promoter sequence. The coding region of this DNA fragment was 750 bps, which encodes a protein of 249 amino acids. Moreover, the developing xylem from C. formosensis has the most abundance expression pattern by Northern blotting analysis. The CfCCoAOMT was constructed into the E. coli to produce a CfCCoAOMT recombinant protein and then the polyclonal antibody was prepared. Finally, the CfCCoAOMT's coding region of C. formosensis was transformed into tobacco, a type of herbaceous plant, by using Agrobacterium-mediated transgenic method. Due to the biosynthesis pathways of lignin in woody plant and herbaceous plant are quite different, there is no significant difference in the total lignin content in sense transgenic tobacco, but in antisense transgenic line had slightly increased. On the other hand, the G/S ratio increased dramatically in both transgenic lines.

並列關鍵字

Chamaecyparis formosensis ； G/S ratio ； Lignin ； O-methyltransferase ； Transgenic tobacco