Selective Isolation Of Mangrove Strptomyces Violaceoruber Clade From Mangrove Soil

Soil samples were collected from different mangrove of Hainan province including Qinglan Harbors, Dongzai Harbors, Sanya and Danzhou as well as those in Futian, Shenzhen and Techeng Island, Zhanjiang, Guangdong province, Jiulong Estuary, Xiamen, Fujian province, and Beihai, Guangxi province. The PCR amplification of Streptomyces phylogenetic probe from metagenomic DNA extracted from the soil samples were employed for bioprospecting the existence of streyptomycetes in these samples. Samples of positive PCR results were pretreated with air-dry, wet-heat and DDC (Dispersion And Differential Centrifugation) and then applied to selective isolation of Streptomyces and Streptomyces violaceoruber clade with media(DH,RH,YE,Gause) adding with different antibiotics. Two hundred and sixteen isolates were selected and detected for their bioactivities. Fourty-six strains showed activity against B16 cell, which was 21.30% of total isolates, the isolates from Fujian mangrove showed not only a largest number but also had a high ratio of bioactivity; Twenty five strains were active against MRSA, which was 9.72% of total strains. Strains isolated from Shenzhen mangroves showed higher anti-MRSA percentage of bioactivity than other sites, which was 27.27 %.All of the isolates were primarily classified by their morphology on the YE medium agar plates. Twenty eight isolates were selected out as representative strains by their activity against murine melanoma cells B16 and MRSA within the color group. They were putatively identified to the genus level based on 16S rDNA sequence analysis and cell wall composition analysis by TLC. Neighbor-joining tree based on almost-complete 16S rRNA gene sequences showing the systematic relationship between the 28 strains.The 28 representative strains were classified to 3 genus of Actinomadura, Nonomura and Streptomyces.The effects of different pretreatements and media show that the mostly isolates of DDC pretreatement from section A; wet-heated at 55℃for 20 min is better than other wet-heated pretreatements; the most number, the highest diversity of isolates were from RH medium and there were few yellow-white clones on the isolating medium; DH medium is effective to selectively isolate Streptomyces violaceoruber clade. The members of Streptomyces violaceoruber clade was only found in acidic soil other than alkalescent sample.Bioactivity related genes of synthases of ribonucleoside antibiotics, aminoglycoside antibiotics and polyketone antibiotics were detected by PCR amplification of their conservative sequence from DNA extracted from representative isolates. Some positive isolates from the results of PCR amplification have not found activity while detecting the bioactivity of their metabolites againt murine melanoma cells B16 and MRSA.