油樟内生细菌分离及α-松油醇高产菌株的筛选与诱变

Isolation of endophytic bacteria from Cinnamomum longepaniculatum and screening and mutagenesis of strain with high-yield α-terpineol

该研究以5株油樟产油率和1,8-桉叶油素成分相对含量存在差异的油樟植株叶片为研究材料,采用组织块法、研磨法、中和剂辅助法结合LB培养基、胰酪大豆胨琼脂培养基(tryptose soya agar,TSA)、卵磷脂吐温-80琼脂培养基分离油樟叶内生细菌,并采用气相色谱-质谱联用定量分析筛选α-松油醇高产菌株,对其进行紫外诱变选育。按照菌落形态特征排重后获得176株油樟叶内生细菌,经鉴定分属于4个门13个属34个种,芽孢杆菌属(Bacillus)为油樟叶内生细菌第一优势属。34株不同油樟叶内生细菌中,高产α-松油醇菌株T3-M-39于30 W紫外灯30 cm照射2 min时,致死率达74.07%,为最佳诱变条件。该条件下,与出发菌株T3-M-39相比,诱变菌株产α-松油醇能力提高了34.85%,并能保持稳定的遗传特性,为生物技术生产天然α-松油醇提供基础。

Screening for microorganisms from aromatic plants is an important way to produce natural fragrances using biotechnology.Endophytic bacteria were isolated from five different strains of Cinnamomum longepaniculatum(Gamble)N.Chao leaves with various oil production rates and 1,8-cineole content.Using tissue block,grinding,and neutralizer auxiliary methods with lysogeny broth culture medium(LB),tryptose soya agar medium(TSA),and lecithin Tween 80 nutrient agar medium,endophytic bacteria were isolated and grown.Gas Chromatography-Mass Spectrometer(GC-MS)were used as quantitative analysis tool to screen for high α-Terpineol-yield strains and the high-yield strains were further mutated by UV.After removing the duplicate strains according to the morphological characteristics of the colony,176 strains of endophytic bacteria in the leaves of Cinnamomum longepaniculatum(Gamble)N.Chao were obtained.These strains were divided into 4 phyla,13 genera,and 34 species.The Bacillus was the first dominant class.Among the 34 strains of endophytic bacteria,the optimal mutagenicity of the strain T3-M-39 with high α-Terpineol production was obtained using a 30 W UV lamp with 30 cm irradiation for 2 minutes,which had a mortality rate of 74.1%.The production of α-Terpineol was enhanced with the mutant strain,reaching 1.45 mg/L,which was increased by 34.9%compared with that of the wild strain.In addition,this mutant strain was also able to maintain its stable genetic characteristics.This finding provided the basis for the natural α-Terpineol production using biotechnology.