双河洞放线菌多样性和抗菌活性的初步研究——以阴河洞和杉林洞为例

Preliminary study on the diversity and antimicrobial activity of actinomycetes from Shuanghe Cave:A case study of Yinhedong and Shanlindong

放线菌是微生物中重要的药用菌,其产生的抗生素是20世纪医学皇冠上最耀眼的明珠,但随着抗生素滥用,抗生素耐药现象日趋严重,因此从特殊生态环境中发现新放线菌物种和新抗生素克服耐药是科研人员正在尝试的途径之一,文章初步探究双河洞系统中阴河洞和杉林洞两个支洞的放线菌多样性,并对分离菌株的抗菌活性进行筛选。以纯培养方法获得放线菌,以16SrRNA基因序列比对,分析菌株类别,获得的菌株进行液体发酵,纸片琼脂扩散法测定其发酵产物对真菌和细菌的抗菌活性,同时用PCR技术进行NRPS、PKS I和PKS II抗生素生物合成基因的探测。从两个支洞样品中分离到的放线菌分布于7个科10个属,两个洞共66株放线菌中有32株的发酵产物在至少一个抗菌活性筛选中显示阳性,总阳性率为48.5%。抗生素生物合成基因的初步探测结果显示,31株菌存在至少一个生物合成基因,NRPS 28株,PKS I17株,PKS II 21株,其中12株同时具有3种生物合成基因。初步显示了双河洞系统蕴含丰富的放线菌资源,值得深入研究。

Actinomycetes are important microorganisms in medicinal fungus, from which antibiotics inmedi- cation has been regarded as the most dazzling pearl in the 20th century. However, with the abuse of antibiot- ics, antibiotic resistance phenomenon has become increasingly serious. It is hence necessary to discover new actinomycete species from special ecological environment to overcome the problem of drug resistance to existing antibiotics. This study aims to analyze the diversity and antimicrobial activity of actinomycetes discovered from Yinhedong and Shanlindong caves, the branches of the Shuanghe cave system, by using cultivation-dependent method to isolate the actinomycetes from the samples collected in the two caves. The diversity was analyzed by comparison of 16S rRNA gene sequences. The antifungal and antimicrobial activity of the fermen- tation products of the isolates were tested by paper disk diffusion method. Screening of PKS I, PKS II and NRPS genes of the strains were carried out by PCR and agarose electrophoresis. Result shows that the iso lates obtained by pure culture distribute in 10 different genera affiliated with 7 families. Antimicrobial assay show that 32 among 66 strains exhibit positive results of antifungal or antibacterial response; and the total positive rate was 48.5%. PCR results show 31 strains have at least one biosynthetic gene including NRPS, PKS I and PKS II, of which 28 strains have NRPS gene, 17 strains have PKS I gene and 21 strains have PKS II gene. It is also noted that 12 strains have the all three biosynthetic genes. Conclusion： The study prelimi- narily indicates that the resource of actinomycetes in the Shuanghe cave system is abundant, which deserves for conducting further study.