Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomeand a versatile peroxidase gene from poroid white-rot fungus Ganoderma lucidum
Date Issued
2008
Date
2008
Author(s)
Huang, Shyue-Tsong
Abstract
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.
Subjects
Antrodia cinnamomea
Ganoderma lucidum
ligninolytic peroxidase
versatile peroxidase
gene cloning
heterologous expression
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