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

Molecular cloning of Caffeoy-CoA 3-O-methyltransferase gene from Chamaecyparis formosensis and lignin analyzing in its transgenic tobacco

林彥良(Yan-Liang Lin)

指導教授 ： 曲芳華

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02565

摘要

木質素為植物體內含量僅次於纖維素之重要成分，於木質素生合成過程中，甲基轉移酵素扮演相當重要的角色。本研究依據基因資料庫中數個已發表之草本和木本植物Caffeoyl CoA 3-O-methyltransferase (CCoAOMT)基因序列，在具有高保守性的序列中設計引子對，並於紅檜試材中，選殖出526個鹼基對大小之片段，再經由5’和3’ RACE (Rapid Amplify of cDNA End)及Genome walking後共計獲得1724個鹼基對，所得之基因則命名為CfCCoAOMT，其中蛋白質轉譯區有750個鹼基對，可產生249個胺基酸。根據親緣關係圖顯示，CfCCoAOMT與台灣杉的CCoAOMT基因的親緣關係最為相近，並與火炬松和挪威雲杉之針葉樹同屬一分支。本研究並針對紅檜進行南方雜合分析顯示CfCCoAOMT在紅檜的基因體核酸中應該具有2個拷貝數，而在北方雜合分析結果發現，CfCCoAOMT以發育中木質部的表現量最高。而由紅檜之CfCCoAOMT基因轉入大腸桿菌中大量表現的重組蛋白，則製作成多株抗體進行免疫雜合反應，當紅檜受物理彎曲之處理時，藉由Tissue printing觀察到CfCCoAOMT基因在受壓面具有較高的表現量。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在正義股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煙草轉殖株在G lignin/S lignin的比值上皆有明顯增加的趨勢。

關鍵字

紅檜 ； 木質素 ； 甲基轉移酵素 ； 轉基因 ； 煙草

並列摘要

Lignin is the second abundant biopolymer and less then cellulose present in the cell wall of plant. The previous reports have shown that O-methyltransferase (OMT) plays as a key enzyme for stepwise biosynthesis of lignin. Our current study focused on the Caffeoyl CoA 3-O-methyltransferase, abbreviated as CCoAOMT, in Chamaecyparis formosensis. By using polymerase chain reaction with designed degenerated primers from highly conserved region of many herbaceous and woody plants were taken as references, 526 base pairs (bp) of DNA fragment were obtained. 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. After combining the sequence data, there was a total of 1724 bps in full length. The coding region of this DNA fragment was 750 bps, which can be translated to 249 amino acids. The gene was named as CfCCoAOMT. The result of phylogenetic tree shows that C. formosensis is most similar to the CCoAOMT of Taiwania cryptomerioides and is categorized to the same branch with Pinus taeda and Picea abies. Analyzing C. formosensis with Southern blotting, the results show that there are two copies of CfCCoAOMT in C. formosensis’ genome. Moreover, the developing xylem from C. formosensis has the most abundance expression pattern by using the Northern blotting analysis. The CfCCoAOMT gene was constructed into the E. coli to generate a great quantity of CfCCoAOMT recombinant protein and the polyclonal antibody was produced by purifying recombinant protein. Using tissue printing to observe the expression pattern in C. formosensis stem with mechanical bending and found there is a greater expression in the compression site. Because the lignin biosynthesis pathway in woody plant and herbaceous plant are quite different, the CfCCoAOMT’s coding region of C. formosensis was taken to transform into tobacco, a type of herbaceous plant, using Agrobacterium mediated transgenic method. Klason’s lignin method was then used to monitor the total lignin content in transgenic tobacco. The resulting tobacco did not present much difference in sense transgenic line but the content in antisense transgenic line had slightly increased. The pyrolysis GC-MS monitored each G and S lignin in transgenic line and found that the G/S ratio increased dramatically in all the transgenic line.

並列關鍵字

Chamaecyparis formosensis ； lignin ； Caffeoyl CoA 3-O-methyltransferase ； CCoAOMT ； transgenic ； tobacco

參考文獻

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