剑叶龙血树内生放线菌活性菌株的筛选和鉴定

Bioactivity and identification of endophytic actinomycetes from Dracaena cochinchinensis

为筛选具有抗菌抗肿瘤活性的药用植物内生菌资源,该文对300余株分离自中国云南西双版纳及越南地区剑叶龙血树的内生放线菌采用琼脂块扩散法进行抗病原细菌活性筛选、平板对峙法进行抗真菌活性筛选、SRB法测定菌株的细胞毒活性及PCR扩增方法筛选非核糖体肽合成酶NRPS基因及聚酮合酶PKS-I、PKS-Ⅱ基因;对得到的优势菌株经8种培养基进行发酵,采用10种病原细菌、3种病原真菌及2种人肿瘤细胞株测试其发酵粗提物的抗菌及抗肿瘤活性以确定最佳发酵培养基;用16S rRNA基因测序方法初步鉴定5株优势菌株的分类地位。结果表明:初筛得到5株抗菌活性、体外细胞毒活性及NRPS、PKS相关基因表达阳性的菌株S01-S05,其中4株(S01-S04)属于链霉菌属、1株(S05)属于类诺卡氏菌属;菌株经不同培养基发酵后产物的抗菌和抗肿瘤活性不同,其中Streptomyces sp.S04在7种培养基中的发酵提取物对8种测试病原菌均有较强抑制作用,且该菌株用Medium C的发酵提取物对人肝癌Hep G2细胞株抑制率达到100%,为剑叶龙血树内生菌活性成分挖掘及新型抗菌药物筛选奠定了基础。

In order to screen endophytic bacteria resources with antitumor activities from medicinal plant,300 endophytic actinomycetes isolated from Dracaena cochinchinensis collected from Xishuangbanna,China and Vietnam were studied.SRB method was used to detect the activity of antitumor cells,agar diffusion and plate confrontation methods were used to test antimicrobial activities,Polymerase Chain Reaction was used to detect NRPS,PKS-I and PKS-Ⅱgenes,and the taxonomic studies of the five selected active strains were performed using 16S rRNA gene analysis.Eight media were designed to cultivate five selected active strains and the antimicrobial activities of their fermented extraction were detected by oxford-cup test against ten pathogenic bacteria and three pathogenic fungi,the antitumor activities were measured by MTT assay in two tumor cell lines.As a result,four of the five active strains were classified as Streptomyces spp.and the remaining one as Nocardioides sp.Different media resulted in different activities,most media of strain S04 showed strong activities against eight pathogens and the inhibition rate of the fermentation product of S04 in Medium C on Hep G2 cell line was up to 100%,which could be a novel resource for the mining of bioactive compounds for further studies.