COMMUNITY STRUCTURES OF ENDOPHYTIC ACTINOBACTERIA FROM MEDICINAL PLANT CENTELLA ASIATICA L. URBAN-BASED ON METAGENOMIC APPROACH

摘要

Objective: This study aimed to assess the community structure of actinobacteria in rhizosphere and endophyte of a medicinal plant, Centella asiatica , based on a metagenomic approach using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of 16S rRNA gene. Methods: Total genomic DNA was extracted from the rhizosphere and plant tissue followed by PCR amplification of actinobacterial 16S rRNA gene using nested PCR. The community structure of actinobacteria was analyzed using the DGGE techniques on polyacrylamide gels. PCR products of excised bands result from polyacrylamide gel were sequenced and analyzed by bioinformatics software to construct a phylogenetic tree. Results: The results of separation in DGGE gel showed 16 major bands from rhizosphere and plant tissue. The bands distribution pattern showed that the community of actinobacteria in the plant tissue was slightly more diverse than rhizosphere, although it is not significantly different based on Shannon-Wiener analysis. The BLAST. N analysis showed that 7 bands related to Streptomycetaceae (83-100%), 5 bands related to Micromonosporaceae (99-100%), 1 bands related to Gordoniaceae (99%) and 3 bands still belonged to unculturable (87-99%). There were 6 genera under those 3 families, i.e. Streptomyce s, Micromonospora , Verrucosispora , Actinoplanes , Couchioplanes , and Gordonia . The percentage of strain similarity comparison to the database showed that there were 4 bands with<97% maximum identity which may be related to novel endophytic actinobacteria in C. asiatica . Conclusion: Diversity of endophytic actinobacteria based on a metagenomic approach using 16S rRNA gene-targeted PCR-DGGE analysis was found associated with C . asiatica . Several of them may have potency as novel actinobacteria and can be further explored for their medicinal function. Keywords: Centella asiatica , PCR-DGGE, Endophytic Actinobacteria, Metagenomic, 16S rRNA