美洲大蠊肠道内生微杆菌的分离鉴定及其抑菌活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-1103

摘要/Abstract

摘要： 对美洲大蠊肠道内生微杆菌进行形态学和分子生物学鉴定，对其抑菌活性进行初步研究，并从基因水平分析微杆菌产生卤化次级代谢产物的潜力。利用平板划线法对美洲大蠊肠道内生微杆菌进行分离和纯化，革兰氏染色后进行光学显微镜观察，并利用16S rDNA PCR、BLAST同源性比对和构建系统发育树等方法对分离菌株进行分子生物学鉴定以及部分抗生素合成关键酶基因的筛选。在此基础上，以8株常见致病菌为指示菌，利用牛津杯法对分离菌株的抑菌活性进行初步研究。分离得到的8株微杆菌其菌落颜色呈淡黄色或白色；16S rDNA基因测序和BLAST比对结果表明，该8株微杆菌与已知微杆菌同源性均达到98%以上，系统发育树结果表明8株微杆菌与土壤、海洋来源的微杆菌同源性较高。部分抗生素合成关键酶基因筛选结果表明8株微杆菌中有6株至少对FADH2-依赖性卤化酶、非核糖体多肽合成酶（NRPS）及I型聚酮合酶（PKSI）3种基因中的2种呈阳性表达。抑菌实验显示，8株微杆菌中有6株微杆菌对受试真菌和细菌的生长表现出了不同程度的选择性抑制作用，表明美洲大蠊肠道存在具有抑菌活性的内生微杆菌。

关键词: 美洲大蠊, 内生微杆菌, 16S rDNA 系统发育树, 抑菌活性

Abstract: This work is aimed to investigate the morphology and molecular biology of the endophytic microbacilli in the intestine of Periplaneta americana，to study its antibacterial activity，and to analyze its potential of producing halogenated secondary metabolites at the gene level. The endophytic microbacillus in the intestine of P. americana was isolated and purified by plate scribing. After Gram staining，the cells were observed by microscopy. The 16S rDNA PCR，BLAST homology alignment and phylogenetic tree were used to identify the isolated strains in molecular biology and to screen key enzyme genes for antibiotics synthesis. Eight strains of common pathogenic bacteria were used as indicator bacteria，and the antibacterial activities were primarily tested by the method of Oxford cup. The colony colors of 8 isolated endophytic microbacilli were light yellow or white. The results of 16S rDNA PCR and BLAST homology alignment showed that the eight microbacilli had high homology with known microbacilli at 98%；while the results of phylogenetic tree demonstrated that they had high homology with the microbacilli from soil and marine. Screening of key enzyme genes in antibiotic synthesis revealed that 6 strains of above 8 presented expressions to 2 of FADH2-dependent halogenase，NRPS，and PKSI. Bacteriostatic experiments showed that 6 strains of 8 presented selective inhibitions to the growth of tested fungi and bacteria at varied level，indicating that there exist endophytic microbacilli in the intestine of P. americana.

Key words: Periplaneta americana, endophytic microbacillus, 16S rDNA phylogenetic tree, antibacterial activity

刘凌燕, 陈志宇, 曾还雄, 林培彬, 金小宝. 美洲大蠊肠道内生微杆菌的分离鉴定及其抑菌活性研究[J]. 生物技术通报, 2018, 34(6): 172-177.

LIU Ling-yan ,CHEN Zhi-yu ,ZENG Huan-xiong, LIN Pei-bin, JIN Xiao-bao. Isolation and Identification of Endophytic Microbacilli in the Gut of Periplaneta americana and Their Antibacterial Activity[J]. Biotechnology Bulletin, 2018, 34(6): 172-177.

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