木材腐朽菌新型基礎過氧化酶基因特性及親緣分析

Demonstrating of novel basal peroxidases (BaPs) in wood brown and white rot fungi based on gene characterization and phylogenetic analysis

賴瑞亞(Jui-Ya Lai)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10446

摘要

木材腐朽菌依其具有降解木質素之能力與否可分為白腐菌與褐腐菌。前人研究指出在白腐菌之外，屬於褐腐菌之牛樟芝 (Antrodia cinnamomea) 亦具有木質過氧化酶之部份活性。而木質過氧化酶之有無為白腐菌及褐腐菌分類依據之一，牛樟芝具有木質過氧化酶此一事實強烈挑戰此論點。本研究以此為出發點，嘗試選殖出它種褐腐菌之木質素過氧化酶。藉由簡併式引子對自數種木材腐朽菌中增幅出具相當保守性之木質素過氧化酶基因片段。此外以末端選殖技術 (Rapid amplification of cDNA Ends, RACE) 選殖出 Laetiporus sulphureus 及香衫芝 Antrodia salmonea 之 cDNA 全長，分別為 1,278 及 1,378 個鹼基對。選定 L. sulphureus 做進一步之分析發現，其基因長度為 1,920 個鹼基對並帶有十個內插子 (intron)，拷貝數 (copy number) 為一。並以熱不對稱交錯 PCR (tail-PCR) 獲取其啟動子 (promoter) 區域並進行分析，發現有兩個非成對之金屬調節元件 (metal regulatory elements, MREs) 之轉錄因子結合位點於此木質素過氧化酶基因上游，此為錳依賴型過氧化酶 (Mn-dependent peroxidase, MnP) 之特徵，此外也其具有與 MnP 較高之序列相似性。為探討其與基因表現之關聯，進行即時定量 PCR (quatitative real-time PCR) 發現於高濃度錳離子 (180 μM ) 處理下基因之表現量較控制組高 3.5 倍，但此結果與 MnP 基因受錳離子催化結果相較較不明顯。為進一步探討此基因之屬性，從譜系分析上切入，將自褐腐菌與白腐菌以及子囊菌上選殖之過氧化酶基因進行分析，採用最大似然法 (Maximum likelihood)，鄰近相接法 (neighbor-joining) 及貝葉氏導出式分析 (Bayesian inference) 建構第二類過氧化酶 (class II peroxidase) 之親緣樹。結果指出基礎過氧化酶 ( basal peroxidase, BaP) 成在第二類過氧化酶中成一單系群 (monophyletic group)，此類過氧化酶是否參與木質素降解功能尚未知。三種方法建構之演化樹樹型並不完全相同但分群近似，皆支持 BaP 相較於其餘類型之第二類過氧化酶較為原始，可能為現有木質分解過氧化酶之起緣酶。由於 BaP 不僅存在於擔子菌 (Basidiomycetes)，也在子囊菌 (Ascomycetes) 中被發現，說明木質素過氧化酶之特化應在擔子菌自子囊菌中演化之後發生。

關鍵字

木材腐朽菌 ； 第二類過氧化酶基礎過氧化酶同源模擬 ； 最大似然法 ； 鄰近相接法 ； 貝葉氏導出法

並列摘要

In light of our previous discovery indicating the existence of a novel lignolytic peroxidase in several wood brown rot fungi (Huang et al. 2009, Huang and Tzean, data unpublished), study on the novel basal peroxidases (BaPs) of additional brown rot and white rot Basidiomycetes was initiated. By degenerate primers, gene encoding the putative novel peroxidase was cloned from Antrodia salmonea and Laetiporus sulphureus. The coding sequences harbored 1,363 and 1,278-bp in length, respectively, and both were interrupted by 10 introns. Further analysis of the BaP amino acid sequence of L. sulphureus showed the high similarity to manganese peroxidase (MnP), and also the presence of unpaired metal regulatory elements (MREs) on the promoter region. The result of quantitative RT-PCR (qRT-PCR) revealed 3.5 times more BaP transcripts while augmented with 180 μM Mn2+ in the initial 24 hr compared with the control. One copy of BaP was shown by Southern blot. Further phylogenetic analysis of the class II fungal peroxidases of 153 taxa represent in Agaricomycetes or Ascomycete by maximum-likelihood (ML), Neighbor-joining (NJ) and Bayesian inference (BI), indicated that BaP, which clearly separated from MnP, lignin peroxidase (LiP) and versatile peroxidase (VP) clades, not only present in the tested brown rot- (A. cinnamomea, A. salmonea, L. sulphureus, Gloeophyllum trabeum and Fomitopsis pinicola), but in a small group of white rot (Ganoderma lucidum, G. australe, Phellinus noxius, Trametes versicolor, Agaricus bisporus and Pycnoporus sanguineus) Agaricomycetes and even Ascomycete (Ophiostoma quercus). The phylogenetic tree topology implicated the possible evolutionary route of BaP diverged toward MnP, LiP and VP.

並列關鍵字

wood rot fungi ； class II peroxidase ； basal peroxidase ； homologous modeling ； Maximum-likelihood ； Neighbor-joining ； Bayesian inference

參考文獻

Abbasi, T. and Abbasi, S. 2010. Biomass energy and the environmental impacts associated with its production and utilization. Renewable and Sustainable Energy Reviews 14:919-937.

Alic, M., Akileswaran, L., and Gold, M.H. 1997. Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium 1. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1338:1-7.

Andrawis, A., Pease, E.A., Kuan, I., Holzbaur, E., and Tien, M. 1989. Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 162:673-680.

Asada, Y., Watanabe, A., Irie, T., Nakayama, T., and Kuwahara, M. 1995. Structures of genomic and complementary DNAs coding for Pleurotus ostreatus manganese (II) peroxidase. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1251:205-209.

Baunsgaard, L., DALBOGE, H., Houen, G., RASMUSSEN, E.M., and Welinder, K.G. 1993. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. Eur. J. Biochem. 213:605-611.