为探究湖北清江和尚洞岩壁放线菌物种组成及其与碳酸盐岩相互作用的关系,分别采集该洞穴无光区风化岩壁样品2份(W1和W2)和弱光区风化岩壁样品1份(W3),采用经改良的5种已知放线菌分离培养基及1种自主设计的风化岩壁浸汁培养基两种策略优化设计分离培养基.采用稀释涂布法分离放线菌,并选择放线菌特异引物进行16S rRNA基因扩增和序列测定并构建系统发育树.结果表明,从3份风化岩壁样品中共纯化到放线菌72株,分属于放线菌纲(Actinobacteria)的4个属:链霉菌属(Streptomyces,69株)、红球菌属(Rhodococcus,1株)、冢村氏菌属(Tsukamurella,1株)和微杆菌属(Microbacterium,1株),其中链霉菌属为优势菌属.无光区样品W1和W2分离到菌株较少,分别为9株和10株,而弱光区样品W3分离到菌株较多,为53株.研究结果为进一步研究放线菌与洞穴碳酸盐矿物相互作用的潜在关系奠定了基础.%The specific goal of this work was to investigate the actinobacterial diversity in the weathered rocks by culture-dependent approach in Heshang Cave,Hubei,China.Three weathered rocks samples (W1,W2 and W3) were collected from aphotic zone and twilight zone in Heshang Cave with sterile centrifuge tubes in August,2015.Five modified media and one self-designed medium of extracting of weathered rocks agar medium were used for isolation of actinomycetes in the weathered rocks of Heshang Cave.The isolates were identified by 16S rRNA gene sequencing using special primer for actinomycetes.Result shows that in total of 72 strains obtained by pure culture from 6 different media.The phylogenetic analysis showed that 72 actinomycetes sequences were classified into four genera in three orders of Actinobacteria with Streptomyces being dominative.The number of isolated strains in genera Streptomyces,Rhodococcus,Tsukamurella and Microbacterium was 69,1,1 and 1,respectively.Samples of W1 and W2 in aphotic zone were only isolated 9 and 10 strains respectively,while sample of W3 in twilight zone were obtained 53 strains.The results offer valuable information and isolates for the subsequent study on the interaction between carbonate and microbes.
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