樟芝與靈芝木質分解酵素基因之選殖與表現

Abstract

樟芝與靈芝在台灣均為十分著名的藥用真菌，關於其醫藥上的應用與生理活性的試驗研究一直未曾中斷，然而，關於樟芝與靈芝在侵染寄主的相關研究卻一直不多，本研究針對褐腐與白腐此兩種真菌，對其殖據侵染基因進行研究，主要著重在木質分解酵素的選殖與表現。研究結果顯示，樟芝之木質分解酵素基因為首次在褐腐菌中得到並加以表現，其基因全長為1183 bp，open reading frame 為 990 bp，而genomic DNA全長為2111 bp，並且有12個introns。蛋白質序列經3-D模擬比對後，發現其蛋白質結構較接近Pleurotus eryngii之versatile peroxidase，以pQE31表現載體表現後為38kDa，進一步純化後並與樟芝胞外蛋白初萃物進行酵素活性分析，發現此蛋白質可以氧化vetrayl alcohol，並且對bromophenol blue 與 2, 6-dimethoxyphenol 人工合成染劑進行脫色，顯示確有木質分解酵素的活性。若進一步設計專一引子，針對其他褐腐菌進行PCR分析並定序，結果顯示十種褐腐菌除了樟芝外，尚有A. salmonea 及 A. vaillantii與white rot fungi的木質分解酵素比對出同源性很高的基因序列，顯示尚有其他褐腐菌具有木質分解酵素基因，若以不同人工染劑進行其分解活性測試，A. vaillantii可對bromophenol blue 與 2, 6-dimethoxyphenol進行脫色，A. salmonea亦可對bromophenol blue進行脫色，同時，A. xantha更可分解2,2’-azino-bis-3-ethylbenzothiazline -6-sulphnic acid (ABTS)與guaiacol等，顯示其具有Laccase與木質分解酵素的活性。實驗並以樟芝接種於寄主牛樟木塊，以SEM進行顯微觀察，發現於接種初期樟芝會分泌草酸協助其侵入，侵入後破壞寄主牛樟細胞壁，造成許多入侵的小孔洞。研究的另一方向為靈芝的木質分解酵素，結果顯示靈芝木質分解酵素之基因全長為1340 bp，open reading frame 為 1092 bp，而genomic DNA全長為2830 bp，並且有11個introns。蛋白質序列經3-D模擬比對後，發現蛋白質結構較接近P. eryngii之versatile peroxidase，以pET21(a)表現載體表現後為43.9 kDa，若以靈芝對不同人工染劑進行其分解活性測試，發現靈芝在培養基中加入Mn離子或vetrayl alcohol時，均可分解guaiacol 及 bromophenol blue，若在培養基中加入vetrayl alcohol時，更可分解remazol brilliant blue R 及 2,6-dimethoxyphenol，顯示其木質分解酵素的性質為versatile peroxidase。綜合研究結果顯示，褐腐菌亦會分泌木質分解酵素，然而表現量遠較白腐菌弱，因此，白腐菌與褐腐菌再也無法以木質分解酵素的有無做二元的區隔，同時，其所對應的傳統形態分類也受到分子類緣演化的挑戰，因此對於多元基因資訊的加入，未來的分類體系經修正後勢必更加完善。

Antrodia cinnamomea and Ganoderma lucidum are two famous medicinal fungi in Taiwan and their medical therapies and biological activities have been studied for a long time. However, the researches of A. cinnamomea and G. lucidum in the invasion of the host plants are still unclear. In this study, we investigated these two fungi, A. cinnamomea and G. lucidum, on the colonization and infection genes especially on cloning and expression of their fungal peroxidase genes. This is the first report on the cloning and expression of the fungal peroxidase gene from A. cinnamomea. The peroxidase gene has not been reported from brown rot fungi. This gene was named ligninolytic peroxidase of A. cinnamomea (ACLnP). The full length of ACLnP gene is 1183 b.p. with 990 b.p. open reading frame and the full length of ACLnP genomic DNA is 2111 b.p. containing 12 introns. Analyzing ACLnP protein 3-D structure showed that its protein structure was closer to versatile peroxidase of Pleurotus eryngii. In addition, ACLnP was ca. 38 kDa and was proven to have ligninolytic activity by decoloration of bromophenol blue and 2, 6-dimethoxyphenol. In order to study the ligninolytic enzyme from 10 species of brown rot fungi, we designed specific primers to perform PCR analysis and then sequenced and annotated form NBCI database. The result revealed that except A. cinnamomea, certain genes from A. salmonea and A. vaillantii also had high homologous with the ligninolytic enzymatic gene from white rot fungi. Furthermore, A. vaillantii had the ability of decoloration of bromophenol blue and 2, 6-dimethoxyphenol; A. salmonea had the ability of decoloration of bromophenol blue, and meanwhile, A. xantha could degrade 2,2’-azino-bis-3-ethylbenzothiazline-6-sulphnic acid (ABTS) and guaiacol, etc. to reveal laccase and fungal peroxidase activities. In addition, the Cinnamomum kanehirai Hey was decayed by A. cinnamomea in the further research observation. In primary invasion, A. cinnamomea would secrete oxalic acid to help invasion to destroy host cell walls and cause many pores as observed with SEM. On the other hand, the full length of the fungal peroxidase gene from G. lucidum, which was named versatile peroxidase of G. lucidum (GLVP), was 1340 b.p. with 1092 b.p. open reading frame and the full length of GLVP genomic DNA was 2830 b.p. including 11 introns. The protein structure was also similar to versatile peroxidase from P. eryngii and the expression of GLVP protein size was 43.9 kDa. The fungal peroxidase from G. lucidum could degrade guaiacol and bromophenol blue under existence of Mn2+ or vetrayl alcohol, as well as could degrade remazol brilliant blue R and 2,6-dimethoxyphenol in the existence of vetrayl alcohol. The result of enzyme activity tests revealed that the fungal peroxidase from G. lucidum had the character of versatile peroxidase. In conclusion, brown rot fungi can secrete fungal peroxidases, no matter the fungal peroxidases expression from brown rot fungi was weaker than from white rot fungi. Therefore, it was inappropriate to distinguish brown rot and white rot fungi by their ability to secrete fungal peroxidase. The traditional taxonomy has been challenged by the evidences of genetic evidences and molecular evolution. In the future, the classification between white rot and brown rot fungi will be improved based on more and more molecular and genomic evidences.