臺灣杉 Dirigent protein之基因選殖及基因表現情形

木酚素（Lignan）是重要的植物酚類化合物，就生合成角度來看，其歸屬於植物二次代謝產物，具抗癌、抗微生物、抗真菌、抗病毒、抗發炎、抗氧化及免疫調節等等的生物活性，同時在植物對抗多樣性的病原或害蟲扮演了重要的防禦角色。已有研究指出木酚素生合成需要經過Dirigent protein（DIR）的參與，協助基質進行專一性的結合。臺灣杉（Taiwania cryptomerioides Hayata）是地質時代第三紀之孑遺植物，為臺灣原產珍貴的針葉樹種。本研究自2年生臺灣杉之葉部組織cDNA以反轉錄聚合酶連鎖反應（RT-PCR）技術獲得DIR部分片段，續以Rapid Amplification of cDNA Ends（RACE）技術獲得基因頭尾序列，以及Genome Walking技術釣取promoter序列，共得到2563 bp，包括Promoter序列1570 bp、5’端73 bp、轉錄區（ORF）576 bp、3’端341 bp，並命名為TcDIR。經分析預測其蛋白質大小約為21.6 kDa。將所得基因構築於大腸桿菌（Escherichia coli BL21）用的載體pET-28a(+)與酵母菌（Pichia pastoris SMD1168）表現用載體pGAPZαA。由大腸桿菌得到可溶部重組蛋白約22 kDa；而由酵母菌表現之重組蛋白約27 kDa，且經分析具有醣基化現象。以反轉錄聚合酶連鎖反應分析不同組織，發現樹皮、雄毬花、雌毬花、毬果、老葉與幼葉均有TcDIR基因表現。以茉莉酸（Jasmonic acid, JA）600 μM與水楊酸（Salicylic acid, SA）400 μM處理小苗6小時後發現TcDIR表現量均較對照組為多。並且無論老枝葉或幼枝葉在創傷處理過後TcDIR之表現量均有上升之趨勢。另外，將TcDIR轉殖至阿拉伯芥（Arabidopsis thaliana）發現生長較野生型為快且葉部有較大的現象。本研究結果將可提供未來透過不同的基因表現層級，來推測TcDIR在臺灣杉面臨生物及物理逆境上所扮演的角色之重要參考。

關鍵字

Dirigent protein ；木酚素；抗性；酚類化合物；醣基化；臺灣杉

並列摘要

Lignan is one of important phenolics compounds, which is belonging to plant secondary metabolites. Numbers of literatures show that lignan possess anticancer, antioxidant, antimicrobial, antifungal, anti-inflammatory and immunosuppressive activities. In the meantime, it might contribute to defense and resistant mechanism against insects and pathogens in plant species. The previous reports have shown that the dirigent protein (DIR) is involved in biosynthesis of lignans in vivo, which contribute to the stereoselective linkage of radical-radical couplings. Taiwania (Taiwania cryptomerioides Hayata) is a relic plant from Tertiary period of geological age. It is an economical species and an endemic conifer in Taiwan. In our current study, a DIR gene in Taiwania cryptomerioides, TcDIR was cloned. By using reverse transcription-polymerase chain reaction from Taiwania 2-year-old leaf cDNA, 576 base pairs (bp) of coding region were obtained. Then the 5’ and 3’ RACE (Rapid Amplify of cDNA End) and Genome Walking were used to clone the full length of TcDIR. The promoter region is 1570 bp. The uncoding region on 5’-end and 3’-end is 73 bp and 341bp, respectively. The TcDIR gene was constructed into BL21 and SMD1168 to generate a great quantity of TcDIR recombinant protein. It’s found that TcDIR expresses in SMD1168 might be glycosylated. We found that TcDIR expresses in bark, male cone, female cone, cone, old leaf and young leaf. The TcDIR were up-regulated by 600 μM JA (Jasmonic acid) and 400 μM SA (Salicylic acid) after 6 hr treatments of 22-day-old seedings. The TcDIR were up-regulated by wounding in either old leaf or young leaf. We also found that transgenic Arabidopsis plants were growing faster and have bigger leaf than wildtypes. These results obtained in this study might provide a useful reference for the future studies in more gene expression level experiments, which are correlated with the rule of TcDIR with defense system and/or physiology in Taiwania.

並列關鍵字

Dirigent protein ； Glycosylation ； Lignan ； Phenoic compound ； Resistance ； Taiwania cryptomerioides Hayata

參考文獻

Alvarez, M.E. (2000). Salicylic acid in the machinery of hypersensitive cell death and disease resistance. Plant Molecular Biology 44, 429-442.

Bar-Nun, N., and Mayer, A.M. (2008). Methyl jasmonate and methyl salicylate, but not cis-jasmone, evoke defenses against infection of Arabidopsis thaliana by Orobanche aegyptiaca. Weed Biology and Management 8, 91-96.

Chang, S., Puryear, J., and Cairney, J. (1993). A simple and efficient method for isolating RNA from Pine Trees. Plant Molecular Biology Reporter 11, 113-116.

Charlton, J.L. (1998). Antiviral activity of lignans. Journal of Natural Products 61, 1447-1451.

Chen, Y.R., Lee, Y.R., Wang, S.Y., Chang, S.T., Shaw, J.F., and Chu, F.H. (2004). Establishment of expressed sequence tags from Taiwania (Taiwania cryptomerioides Hayata) seedling cDNA. Plant Science 167, 955-957.

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臺灣杉 Dirigent protein之基因選殖及基因表現情形

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