Isolation And Identification Of Mangrove Actinomycetes And Its Resuscitation Activity Of Rpf

When bacteria are exposed to a stressful environment,they enter VBNC,a state which viable but non-culturable,causes our strains isolated in the laboratory to account for only 0.01%-4% of the environment.Resuscitation-promoting factor(Rpf)is a protein that promotes bacterial resuscitation.It promotes the recovery of non-culturable bacteria at very low concentrations and promotes growth of normally growing bacteria.Fragments encoding the resuscitation promoting factor gene are widely present in actinomycetes.In mangroves,it is rich in Actinomycetes resources,and has abundant enzymatic activity,antitumor activity,antiviral activity and antagonism against some pathogenic bacteria and nematodes.In order to explore more actinomycetes resources in mangroves,the rpf gene of Actinomycetes was cloned,expressed,and tested for activity of expressed proteins.42 strains of Actinomycetes were isolated from the rhizosphere soil samples from mangrove sources.The 16 S rRNA gene amplification and identification results indicated that 42 strains of Actinomycetes belonged to 5 orders of 6 under Actinomycetes.8 genus.These five orders are Micrococcineae,Streptomycetales,Micromonosporineae,Corynebacterineae,and Sphingomonadales.In this study,Streptomyces is the dominant actinomycete population,accounting for 52.38% of all strains,including 2 genus Kitasatospora 1 and Streptomyces 22 strains.The new strains of the potential 3 strains under Actinomycetes were found to be GXN 032,GXN 112,GXN 114,and their 16 S rRNA gene amplification and identification were similar to Streptomyces avermitilis,Streptomyces similanensis and Nocardia speluncae,respectively.Using amylase,protease,and cellulase as substrates to screen for enzymatic activity,a total of 16 strains with enzymatic activity were screened,and the active strain producing amylase accounted for 50% of the enzyme-producing strain,and 50% of the strains producing enzyme activity were from Streptomyces,and 31.25% were from the genus Micromonas.A total of 13 strains with rpf were screened from Actinomycetes,and only one strain of Rpf-E and RhRpf actinomycetes were selected.Srpf-1 and Srpf-7 were screened for the rpf of three actinomycetes,Srpf.The rpf of 13 actinomycetes was amplified by-3 primers,accounting for 30.95% of all strains,accounting for 100% of the strains amplified by rpf,and five strains could amplify two or more rpf;The strain capable of amplifying the actinomycete rpf gene was mainly 76.92% of the strains which were selected by Streptomyces.The biological information of the proteins was predicted by online software.The results showed that the Actinomycetes Rpf is a stable hydrophilic protein composed of 20 kinds of amino acids.There is a dissociation site of the signal peptide,and both have a lysozyme-like domain and the domain of LysM.Homology analysis showed that Rpf-3 is evolutionarily closer to Rpf of M.tuberculosis.The amplified Actinomycetes were constructed by cloning vectors and expression vectors,and the expression vectors were transformed into host cells for expression,and a protein of about 32 kDa was isolated.By flow cytometry,it was confirmed that the expressed protein has the activity of resuscitation of M.luteus in VBNC state.