[1]丁林云,万可,杨丹,等.转基因在菊花叶肉原生质体瞬时表达及亚细胞定位分析[J].江苏农业学报,2023,(02):518-524.[doi:doi:10.3969/j.issn.1000-4440.2023.02.025]
　DING Lin-yun,WAN Ke,YANG Dan,et al.Analysis of transient expression of chrysanthemum mesophyll protoplasts and subcellular localization[J].,2023,(02):518-524.[doi:doi:10.3969/j.issn.1000-4440.2023.02.025]
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转基因在菊花叶肉原生质体瞬时表达及亚细胞定位分析()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年02期
页码:
518-524
栏目:
园艺
出版日期:
2023-04-30

文章信息/Info

Title:
Analysis of transient expression of chrysanthemum mesophyll protoplasts and subcellular localization
作者:
丁林云1; 2; 万可3; 杨丹1; 2; 王同1; 2; 王春梅1; 2; 郭书巧1; 2; 杨霞1; 2
（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）
Author(s):
DING Lin-yun1; 2; WAN Ke3; YANG Dan1; 2; WANG Tong1; 2; WANG Chun-mei1; 2; GUO Shu-qiao1; 2; YANG Xia1; 2
(1.Central Laboratory of Jiangsu Academy of Agricultural Sciences/Institute of Crop Germplasm and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2.Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;3.College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China）
关键词:
菊花; 原生质体; 瞬时表达; 亚细胞定位
Keywords:
chrysanthemum; protoplast; transient expression; subcellular localization
分类号:
S682.1+1
DOI:
doi:10.3969/j.issn.1000-4440.2023.02.025
文献标志码:
A
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。
Abstract:
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.

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备注/Memo

备注/Memo:
收稿日期：2022-05-09 基金项目：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)] 作者简介：丁林云(1980-)，女，江苏连云港人，硕士，助理研究员，从事植物转基因功能研究。（E-mail）20210991@jaas.ac.cn。 万可为共同第一作者。 通讯作者：杨霞，(E-mail)xiayang_njau@hotmail.com
更新日期/Last Update: 2023-05-12