优化尼罗红荧光染色法简便快速筛选高油脂裂殖壶菌  

Simplified and rapid lipid determination of Schizochytrium sp. by optimized Nile red fluorescence staining

作　　者：岳秀宏 李祥宇 刘鹏阳 陆姝欢 万霞[1,2,3] YUE Xiu-hong;LI Xiang-yu;LIU Peng-yang;LU Shu-huan;WAN Xia(Oil Crops Research Institute of Chinese Academy of Agricultural Sciences,Wuhan 430062,China;Key Laborato ry of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,China;National and Local Joint Engineering Laboratory of Oil Lipid Processing Technology,Wuhan 430062,China;CABIO Biotechnol ogy Company Limited,Wuhan 430073,China;College of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]中国农业科学院油料作物研究所,湖北武汉430062 [2]农业农村部油料作物生物学与遗传育种重点实验室,湖北武汉430062 [3]农业部油料加工重点实验室,湖北武汉430062 [4]嘉必优生物技术股份有限公司,湖北武汉430073 [5]湖北大学,生命科学学院,湖北武汉430062

出　　处：《中国油料作物学报》2019年第5期796-803,共8页Chinese Journal of Oil Crop Sciences

基　　金：“十三五”重点研发计划(2016YFD0501209-01);中国农业科学院科技创新工程(CAAS-ASTIP-2013-OCRI);中国光谷“创新人才”计划(K159)

摘　　要：裂殖弧菌是商业化生产二十二碳六烯酸(DHA)的重要菌株。为了快速检测裂殖壶菌细胞油脂含量,本文基于尼罗红荧光染色法,系统地筛选了激发光与发射光,并探究了简化细胞处理步骤,在不使用磷酸缓冲盐溶液洗涤细胞的条件下,优化二甲基亚砜(DMSO)体积分数、尼罗红用量、染色时间及细胞密度等因素对裂殖壶菌胞油脂检测的影响,并通过气相色谱法与荧光染色法的相关性分析,验证该方法的准确性。结果表明背景荧光未对荧光强度测定造成干扰;细胞密度在一定范围内,荧光强度与油脂含量的相关性良好,通过荧光强度可以较为准确地反映裂殖壶菌油脂含量。最佳染色及检测条件为:细胞密度0.7<OD600<1.3,尼罗红浓度1μg/mL,DMSO浓度20%,激发光515 nm,发射光576 nm,常温避光染色20 min。本研究优化后尼罗红荧光染色法可应用于高含油量的裂殖弧菌突变株或亲缘关系相近的高油脂菌株的高通量筛选。Schizochytrium sp.is an important strain for commercial production of docosahexaenoic acid(DHA).Improving DHA production by physicochemical mutagenesis is an important link in the development of microbial fermentation DHA industry.In order to establish a rapid optimal Nile red fluorescence staining method for determin ing the lipid content in Schizochytrium sp.,we systematically examined the effect of excitation wavelength,emission wavelength and explored the condition of cells without phosphate buffer solution washing.We also optimized the concentration of dimethyl sulfoxide(DMSO)and Nile red,staining time and cell density.The results showed that the culturing medium did not interfere with the determination of fluorescence intensity.In addition,we evaluated the correlation between the florescence intensity of Nile red staining and gas chromatographic method.The lipid content was positively correlated to the fluorescence intensity,when the cell density was within a certain range.We con clude that lipid content of Schizochytrium sp.can be effectively determined by fluorescence intensity.The optimal conditions for determining of lipid content in Schizochytrium sp were obtained as follows:cell density was 0.7<OD600<1.3,the concentration of Nile red was 1μg/mL,DSMO concentration was 20%(v/v),staining time was 20 min,and the excitation and emission wavelengths was 515 nm and 570 nm.The optimized Nile red fluorescence staining method could be used to rapidly and high-throughput screening of mutants with high lipid content in Schizochytri um or closely related strains.

关 键 词：裂殖壶菌 油脂含量 尼罗红荧光染色 

分 类 号：TK6[动力工程及工程热物理—生物能]