浮萍原生质体瞬时表达体系的建立及其应用研究  

Establishment and Application of Transient Expression System of Protoplast in Duckweed

作　　者：宋乐元 方扬[2] 田润 杜安平 王海燕[1] SONG Le-yuan;FANG Yang;TIAN Run;DU An-ping;WANG Hai-yan(Key Laboratory of Bio-resource and Eco-environment of Ministry of Education,College of Life Sciences,Sichuan University,Sichuan Chengdu 610065,China;Key Laboratory of Environmental and Applied Microbiology,Chengdu Institute of Biology,Chinese Academy of Sciences,Sichuan Chengdu 610041,China;Sichuan dimension chuangyan biotechnology co.,LTD,Sichuan Chengdu 610041,China)

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,四川成都610065 [2]中国科学院成都生物研究所,中国科学院环境与应用微生物重点实验室,四川成都610041 [3]四川维度创研生物科技有限公司,四川成都610041

出　　处：《西南农业学报》2020年第8期1792-1798,共7页Southwest China Journal of Agricultural Sciences

基　　金：中国科学院种子创新研究院、国家水生生物种质资源库、中国科学院“西部青年学者”项目(2018XBZG_XBQNXZ_B_007)。

摘　　要：【目的】建立浮萍原生质体瞬时表达体系,以加快浮萍生物反应器工程的开发进程。【方法】采用酶解法制备浮萍原生质体,通过对愈伤组织类型、渗透压、预处理方法等一系列参数的优化调整,建立了浮萍原生质体制备方法。利用农杆菌介导外源基因进入浮萍愈伤组织,并制备原生质体观察瞬时表达情况,通过对农杆菌种类、菌浓度、乙酰丁香酮浓度及共培养时间等参数进行调整,筛选最佳的浸染条件。最后利用该瞬时表达体系进行亚细胞定位研究和启动子功能验证。【结果】原生质体制备的最佳条件为:以4℃过夜培养的浮萍悬浮培养愈伤组织为制备材料,用酶液(2%纤维素酶、0.5%离析酶、0.5%果胶酶、0.1 mol/L CaCl2、0.2 mol/L KCl、1%BSA、20 mmol/L MES pH 5.8、0.5 mol/L甘露醇)于25℃避光消化16 h(40 r/min);农杆菌浸染最佳条件为:农杆菌GV301在菌浓度(OD600)为1、乙酰丁香酮浓度为100μmol/L时,浸染悬浮培养愈伤有最大转化率。【结论】成功建立了浮萍原生质体瞬时表达体系,并证明其可用于亚细胞定位和启动子功能验证等方面的研究。【Objective】The present paper aimed to establish transient expression system of duckweed protoplast to accelerate the development of duckweed bioreactor.【Method】The protoplast of duckweed was prepared by enzymatic hydrolysis,and a preparation method for the protoplast system of duckweed was established by optimizing and adjusting a series of parameters such as callus type,osmotic pressure and pretreatment method.Then,agrobacteria-mediated exogenous genes were used to enter the callus of duckweed,and the protoplast was prepared to observe the transient expression.By adjusting the parameters of agrobacteria-mediated species,bacterial concentration,acetosyringone concentration and co-culture time,the optimal leaching conditions were selected.Finally,subcellular localization and promoter function verification were carried out by using the transient expression system.【Result】The best conditions for the preparation of the protoplast were as following:the suspension cultured callus of duckweed cultured at 4℃overnight was used as the preparation material,and enzyme solution(2%cellulase,0.5%macerozyme,0.5%pectinase,0.1 mol/L CaCl2,0.2 mol/L KCl,1%BSA,20 mmol/L MES pH 5.8,0.5 mol/L mannitol)was used for digestion at 25℃for 16 hours(40 r/min).The optimum conditions of agrobacterium tumester culture were as follows:when the concentration of agrobacterium tumester GV301(OD600)was 1 and the concentration of acetosyringone was 100μmol/L,the callus of the suspension culture had the maximum transformation rate.【Conclusion】The transient expression system of duckweed protoplast was successfully established,which proved that it could be used for subcellular localization and promoter function verification.

关 键 词：浮萍Lemna gibba 原生质体 瞬时表达体系 亚细胞定位 启动子功能 

分 类 号：Q943[生物学—植物学]