川滇四区森林土壤纯培养放线菌多样性及生物活性

【目的】为了从放线菌发现新的药物先导化合物,研究了川滇4个地区的放线菌多样性及其生物活性。【方法】采集250份土样,用4种培养基分离放线菌;从中选择98株代表菌进行了初步分类鉴定;采用琼脂扩散法,检测了169株放线菌对4种细菌和7种真菌的抑菌活性;利用特异性引物扩增法,测定了它们产生的聚酮合酶(PKSI、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因。【结果】黄荆老林的放线菌有13个属,峨眉山、青城山仅5个属,九寨沟9个属,西双版纳达20个属;不同地区的放线菌具有抗菌活性的菌株平均约占10%;有27%-36%的菌株产生PKSI、II、NRPS、CPY化合物合成基因。【结论】在采集样品的地区中,人类干扰越少,放线菌的多样性越高。分离放线菌时,使用"极端"条件,虽然分离到的放线菌数量可能不多,但获得未知菌的比例较大。添加抑制剂可减少革兰氏阴性细菌和真菌,有利于分离放线菌。

Diversity and bioactivity of cultured actinomycetes in Sichuan and Yunnan area

Abstract: Objective] In order to discover new leader compounds, the diversity and some bioactivities of cultured actinomycetes from Sichuan and Yunnan were studied. Methods] In total 250 soil samples were collected from forest of Huangjin, Emei and Qingcheng Mountains, Jiuzhaigou in Sichuan, and Xishuangbanna in Yunnan. Actinomycetes in these samples were isolated and identified. Bioactivities of isolated strains were determined. Results] In total 2676 strains of actinomycetes were isolated from these samples. The 16S rRNA gene sequences of 98 selected strains were determined, and the phylogenetic analysis was carried out. 13, 5, 9 and 20 genera were identified from Forest of Huangjin, Emei and Qingcheng Mountains (one sample area), Jiuzhaigou, and Xishuangbanna respectively. The diversity of Xishuangbanna was the richest. That of Emei and Qingcheng Mountains was monotone, and only five genera were isolated. Antimicrobial activities of 169 selected strains against 11 bacteria and fungi were tested using agar well diffusion method, and genes of type I and II polyketide synthases (PKS I, PKS II), non-ribosomal peptide synthase (NRPS) and polygene cytochrome P450 hydroxylase (CYP) were detected by PCR. High rate of antimicrobial activity and the synthesis genes of four antibiotic existed in these actinomyctes. Conclusion] In sample-collecting areas, the poorer human beings disturbance, the richer the diversity of actinomycetes. For isolation of actinomycetes, author advocate using of "extreme" conditions, although it may got small number of actinomycetes, but the proportion of unknown actinomycetes was greater. Gram-negative bacteria and fungi could be inhibited by adding inhibitors in media.