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农业生物技术学报  2023, Vol. 31 Issue (12): 2654-2664    DOI: 10.3969/j.issn.1674-7968.2023.12.019
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
基于农杆菌真空渗透法的菊花瞬时表达系统的优化
俞瑶, 程华, 陈素梅, 陈发棣, 蒋甲福*
南京农业大学 作物遗传与种质创新国家重点实验室/农业农村部 景观农业重点实验室/华东地区花卉生物学国家林业和草原局重点实验室/园艺学院,南京 210095
Optimization of Transient Expression System in Chrysanthemum morifolium based on Agrobacterium Vacuum Infiltration Method
YU Yao, CHENG Hua, CHEN Su-Mei, CHEN Fa-Di, JIANG Jia-Fu*
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs/Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
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摘要 菊花(Chrysanthemum morifolium)多是六倍体,遗传背景复杂,稳定遗传转化效率低。本研究旨在优化菊花中的瞬时表达系统,提高瞬转效率、目的基因表达活性,为开展菊花相关基因的功能研究提供有效手段。利用真空渗透法对'神马' ('Jinba')菊花插穗进行瞬时转化,通过选择不同种类的悬浮液、不同悬浮液pH、是否添加乙酰丁香酮、不同悬浮终浓度、农杆菌(Agrobacterium)菌液暗培养时间以及植物暗培养温度6个方面进行研究,对所得侵染效率进行统计分析,同时通过测定分析荧光素酶(luciferase, LUC)平均荧光强度进一步判断表达效率,探索这6种条件对菊花瞬时转化效率及目标基因表达活性的影响程度。结果表明,侵染菊花时,2-(N-吗啡啉)乙磺酸(2-(N-morpholino) ethanesulfonic acid hydrate, MES)缓冲液为最适宜的悬浮液;且悬浮液pH=6.0时,荧光值表达水平最高;其次,加入乙酰丁香酮(100 μmol/L)能够提高瞬时侵染的转化效率和荧光值的表达水平;当悬浮菌液的OD600值为1.8时,荧光值表达量显著上升(P<0.05);菌液悬浮后置于28 ℃暗培养4 h更有利于荧光值的表达;另外,10 ℃低温暗培养植物时侵染效率和荧光值表达量均有所提升。本研究通过更改悬浮液的成分、悬浮菌液的浓度、悬浮菌液和瞬时转化后植株的培养条件显著提高了菊花荧光值表达量,在较短的实验周期内做到了有效的瞬时表达,可为菊花基因功能研究提供有效手段。
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俞瑶
程华
陈素梅
陈发棣
蒋甲福
关键词 菊花根癌农杆菌瞬时表达系统荧光值    
AbstractChrysanthemums morifolium are mostly hexaploid, with complex genetic backgrounds and low efficiency in stable genetic transformation. This study aimed to optimize the transient expression system in C. morifolium and improved the transient transformation efficiency as well as target gene expression activit, to provide an effective way to carry out functional studies of Chrysanthemum-related genes. C. morifolium 'Jinba' cuttings were transient transformed by using the vacuum infiltration method, and the infiltration efficiency was statistically analyzed by 6 aspects: Type of suspensions, pH of suspensions, whether to add acetosyringone, final concentration of Agrobacterium suspension, dark incubation time of Agrobacterium and dark incubation temperature of plants. And the expression efficiency was determined by measuring and analyzing the expression of fluorescence values of luciferase (LUC) to explore the influence degree of 6 conditions on the transient transformation efficiency and target gene expression activity of C. morifolium. The experimental results showed that 2-morpholinoethanesulphonic acid (MES) buffer was more suitable as suspension when infiltrating chrysanthemum, and the highest level of fluorescence expression was obtained when the pH of suspension was 6.0; next, the addition of acetosyringone (100 μmol/L) could improve the transformation efficiency of transient infection and the expression level of fluorescence value; the expression of fluorescence increased significantly when the final OD600 value of suspension was 1.8; the expression of fluorescence value was more favorable when the suspension was placed in dark culture at 28 ℃ for 4 h. In addition, the efficiency and the expression of fluorescence value were increased when the plants were cultured in the low temperature condition (at 10 ℃). This study significantly improved the expression of fluorescence value of C. morifolium and achieved effective transient expression in a short experimental period by changing the conditions of the composition of the suspension, the concentration of the suspension, the suspension, and the culture conditions of the plant after instantaneous transformation, which provides efficient method for the functional study of C. morifolium genes.
Key wordsChrysanthemum morifolium    Agrobacterium tumefaciens    Transient expression system    Fluorescence value
收稿日期: 2022-12-07     
ZTFLH:  S682.1+1  
基金资助:国家自然科学基金重点项目(31930100)
通讯作者: *jiangjiafu@njau.edu.cn   
引用本文:   
俞瑶, 程华, 陈素梅, 陈发棣, 蒋甲福. 基于农杆菌真空渗透法的菊花瞬时表达系统的优化[J]. 农业生物技术学报, 2023, 31(12): 2654-2664.
YU Yao, CHENG Hua, CHEN Su-Mei, CHEN Fa-Di, JIANG Jia-Fu. Optimization of Transient Expression System in Chrysanthemum morifolium based on Agrobacterium Vacuum Infiltration Method. 农业生物技术学报, 2023, 31(12): 2654-2664.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.12.019     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I12/2654
 
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