Analysis of endophytic actinobacteria species diversity in the stem of Gynura cusimbua by 16S rRNA gene clone library

Endophytic actinobacteria that live in any associations with plant tissues represents a rather unexplored area of actinobacteria compared with soils. Gynura cusimbua is a kind of medicinal plant which has prevention effects for high blood pressure, coronary heart disease, alzheimer's disease, atherosclerosis, etc. Endophytic actinobacteria of G. cusimbua might produce some secondary metabolites which has the same function as their host. Stem samples of G. cusimbua collected from Hainan Province were used to study their endophytic actinobacteria species diversity by constructing a metagenomic library and help us to understand and use their endophytic actinobacteria to find some new compound. In order to avoid vast proportions of the host plant DNA in the metagenomic library, enrichment for the constituent microorganisms from plant tissues was achieved by coupling SDS with NaCl and DNA was extracted. At the same time, 16S rRNA genes were amplified by PCR and then digested with PvuII which most eubacterial 16S rRNA gene are devoid of the recognition site for PvuII and G. cusimbua plastid rRNA gene is sensitive to PvuII. Two sets of actinobacteria specific primers were used for targeting endophytic actinobacteria from metagenomic library. 63 actinobacteria clones were selected for sequencing and constructing 16S rRNA gene phylogenetic tree and 59 clones were higher similar to the recent pattern of 16S rRNA gene sequence and belonged to Microbacterium, Arthrobacter, Micrococcus, Curtobacterium, Okibacterium, Quadrisphaera and Kineococcus, respectively. Four clones were  in low similarity and one belonged to unclassified Micrococcineae, one to unclassified Intrasporangiaceae and three to unclassified Microbacteriaceae.