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

Abstract

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.