转基因及相关亚品系鱼腥藻的纯化  

Separation and purification for transgenic Anabaena sp. PCC 7120 and its subspecies

作　　者：朱嘉诚 贺婵娟 贾晓会[1] 顾娟 施定基[1,2,3] 庄旻敏 冯思豫 贾睿 栾必荣[2] 何培民 ZHU Jiacheng;HE Chanjuan;JIA Xiaohui;GU Juan;SHI Dingji;ZHUANG Minmin;FENG Siyu;JIA Rui;LUAN Birong;HE Peimin(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Tianhe Biotec.Ltd.Zhenjiang 212200,Jiangsu,China;Institute of Botany,Chinese Academy of Sciences,Beijing 100093,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]镇江天和生物技术公司,江苏镇江212200 [3]中国科学院植物研究所,北京100093

出　　处：《上海海洋大学学报》2018年第5期645-655,共11页Journal of Shanghai Ocean University

基　　金：国家海洋863计划项目(2014AA093506);上海市科委项目(16391903500)

摘　　要：转基因蓝藻纯化是研究其生理、生化、分子生物学、营养学、药理学、毒理学等必不可少的环节,更是其作为生产原料的基础。传统纯化方法仅对不同物种间的分离进行了讨论,尚未涉及野生藻种和转基因突变株在实验和生产中相互污染情况下的鉴别和分离。本文的转基因藻纯化方法先后从种群、细胞、分子3个层面纯化分离了藻种。该方法使用平板划线的方法将杂藻杂菌与鱼腥藻分离,再使用核酸电泳、RT-qPCR、Western Blot将鱼腥藻野生种、转空载体突变株以及转目的基因突变株3个亚品系逐步分离。纯化分离后的藻株生长速率得到了提高,目的基因的表达效率增长,生产目的蛋白的产率自然增长。本研究对转基因藻在实验和生产中出现的一些问题提出了相应的解决方案,对转基因微藻的应用具有积极的作用。Separation and purification of transgenic cyanobacteria has became an indispensable step in the research of its physiology,biochemistry,molecular biology,nutritional value,etc.However,has been were no research on the separation and purification of transgenic cyanobacteria under the circumstance of cross contamination during the production of transgenic product.The method used in this article separated and purified Anabaena at three levels：species,cell and gene.In this study,the separation and purification between wild type,transgenic type and empty carrier type began at biochemical level via different culture solutions with antibiotics.Then,RT-qP CR and Western blot were applied to distinguish transgenic type and empty carrier type at molecular level.Our study not only can separate three substrain,but also obtain a result of product increase and higher growth rate.This study provides a solution to the cross contamination during the production of transgenic product and can be positive to the development of transgenic microalgae industry.

关 键 词：转基因 蓝藻 纯化 WESTERN BLOT 抗生素筛选 RT-QPCR 

分 类 号：S917[农业科学—水产科学]