转基因在菊花叶肉原生质体瞬时表达及亚细胞定位分析  

Analysis of transient expression of chrysanthemum mesophyll protoplasts and subcellular localization

作　　者：丁林云 万可 杨丹[1,2] 王同[1,2] 王春梅[1,2] 郭书巧[1,2] 杨霞[1,2] DING Lin-yun;WAN Ke;YANG Dan;WANG Tong;WANG Chun-mei;GUO Shu-qiao;YANG Xia(Central Laboratory of Jiangsu Academy of Agricultural Sciences/Institute of Crop Germplasm and Biotechnology,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院中心实验室/江苏省农业科学院种质资源与生物技术研究所,江苏南京210014 [2]江苏省农业科学院经济作物研究所,江苏南京210014 [3]南京农业大学农学院,江苏南京210095

出　　处：《江苏农业学报》2023年第2期518-524,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31772183);江苏省农业科技自主创新基金项目[CX17(3050)]。

摘　　要：为探究用于亚细胞定位研究的菊花叶肉原生质体分离的最适条件,本研究以菊花无菌苗叶片为受体材料,分析不同叶龄叶片和酶解时间对菊花原生质体细胞产量的影响,并利用外源质粒的导入验证了聚乙二醇(PEG)介导的菊花原生质体瞬时转化体系。结果表明,以组织培养14 d的菊花无菌苗叶片为外植体材料,在含1.5%纤维素酶、0.4%离析酶和0.8 mol/L甘露醇的酶解液中振荡酶解6 h,能获得高产的原生质体细胞。采用PEG介导的瞬时转化含绿色荧光蛋白(GFP)的表达载体,在菊花原生质体细胞中观察到强烈的绿色荧光信号。结果显示,稗草乙烯转录因子基因(EcERF)在菊花原生质体中与本氏烟草叶片中的瞬时表达一致,EcERF基因定位于细胞核中。To explore the optimal condition for the separation of mesophyll protoplasts of chrysanthemum for subcellular localization study,leaves of aseptic chrysanthemum seedlings were used as acceptor material in this study to analyze the effects of leaves with different culture times and different enzymatic hydrolysis time on the yield of chrysanthemum protoplast cells,and polyethylene glycol(PEG)-mediated transient transformation system of chrysanthemum protoplasts was verified by leading in exogenous plasmids.The results showed that,protoplasmic cells with high yield could be obtained by shaking enzymatic hydrolysis for six hours in enzymatic hydrolysis solution containing 1.5%cellulase,0.4%macerozyme and 0.8 mol/L mannitol,using leaves of aseptic chrysanthemum seedlings cultured for 14 days as explants material.A strong green fluorescent signal was observed in chrysanthemum protoplast cells by using PEG mediated expression vector for transient transformation containing green fluorescent protein(GFP).The results indicated that ethylene transcription factor gene in Echinochloa crusgalli(EcERF)were transiently expressed in chrysanthemum protoplasts and tobacco(Nicotiana benthamiana)leaves,which were both located in the nucleus.

关 键 词：菊花 原生质体 瞬时表达 亚细胞定位 

分 类 号：S682.11[农业科学—观赏园艺]