农杆菌介导的香椿叶片瞬时转化体系的建立及优化

doi:10.13560/j.cnki.biotech.bull.1985.2025-0449

摘要/Abstract

摘要：

目的建立香椿中快速高效的遗传转化系统，为基因功能研究及分子育种提供技术支持。方法以香椿组培苗为材料，采用农杆菌介导真空渗透法进行瞬时转化植物叶片，并利用甜菜红素RUBY报告基因载体评估转化效率和目的基因表达水平，针对农杆菌菌株类型、侵染浓度、真空渗透时间等条件进行优化研究，并采用来自不同地区的5个香椿组培苗材料进行验证与评估。结果建立了适用于香椿的瞬时转化体系，筛选出了适用于香椿瞬时转化的农杆菌菌株为K599和GV3101菌株，通过比较转化效率获得的最适转化条件为150 μmol/L乙酰丁香酮、菌液浓度OD₆₀₀为0.5，真空渗透30 min，共培养3-4 d。结果表明该转化体系具有高效的转化效率。结论成功建立了适合香椿的瞬时基因转化体系，基于RUBY报告基因表达情况优化了转化条件，并在不同香椿材料转化验证，为分子育种奠定基础。

关键词: 香椿, 农杆菌, 瞬时转化, 体系建立, 体系优化

Abstract:

Objective Establishing a fast and efficient genetic transformation system in Toona sinensis may provide technical support for gene function research and molecular breeding. Method This study utilized tissue-cultured seedlings of T. sinensis as the experimental materials, employed Agrobacterium-mediated vacuum infiltration, and adopted the red RUBY reporter gene to monitor transformation events and gene expression in the plants. Research was conducted to optimize conditions such as the type of Agrobacterium strain, infection concentration, and vacuum infiltration duration. Finally, validation and evaluation were conducted using five tissue-cultured seedlings from different provenances of T. sinensis. Result Simultaneously, a transient transformation system suitable for T. sinensis has been established. The Agrobacterium strains suitable for transient transformation in T. sinensis were screened out, namely K599 and GV3101 strains. The optimal transformation conditions obtained by comparing transformation efficiency were 150 μmol/L acetosyringone, OD₆₀₀ at 0.5, vacuum infiltration for 30 min, and co-cultivation for 3–4 d. Ultimately, the results indicated that the transformation system has high transformation efficiency. Conclusion This system provides technical support for rapidly and effectively conducting functional identification of genes in T. sinensis.

Key words: Toona sinensis, Agrobacterium, transient transformation, system establishment, system optimization

张瑶毅, 殷恒福, 刘军, 韩小娇, 范艳如, 王民炎, 曹受金. 农杆菌介导的香椿叶片瞬时转化体系的建立及优化[J]. 生物技术通报, 2025, 41(11): 221-227.

ZHANG Yao-yi, YIN Heng-fu, LIU Jun, HAN Xiao-jiao, FAN Yan-ru, WANG Min-yan, CAO Shou-jin. Establishment and Optimization of Agrobacterium-mediated Transient Transformation System of Toona sinensis Leaves[J]. Biotechnology Bulletin, 2025, 41(11): 221-227.

图/表 6

表1 荧光定量PCR引物序列

Table 1 Fluorescence quantitative PCR primer sequences

引物名称 Primer name	引物序列 Primer sequence （5'-3'）	碱基数 Base pairs （bp）
Ts-acting-F	TGATTGGGATGGAAGCAGCA	20
Ts-acting-R	GAACATGGTTGAACCGCCAC	20
Ts-RUBY-F	AACAGCATCCTTGAGTCTCTTCG	23
Ts-RUBY-R	TTCTCTTTGGAGATCTCGCCTTC	23

图1 香椿瞬时转化体系建立A：未侵染的香椿组培苗，图中标尺为2 cm；B：瞬时转化RUBY的香椿苗，图中标尺为2 cm；C：RUBY基因表达水平分析，EV为空载侵染过的香椿苗，RUBY-OE为RUBY侵染的过表达香椿苗。***P<0.001

Fig. 1 Establishment of transient transformation system of T. sinensisA: Uninfected tissue-cultured seedlings of T. sinensis, scale bar=2 cm; B: transiently transformed T. sinensis seedlings with RUBY, scale bar=2 cm; C: analysis of RUBY gene expression, where EV indicates empty vector-infected T. sinensis seedlings, and RUBY-OE indciates RUBY-overexpressing T. sinensis seedlings infected with RUBY. *** P<0.001

图2 不同处理香椿转化效率的比较A：不同含RUBY质粒的农杆菌菌株侵染过的香椿植株，图中标尺为2 cm；B：不同农杆菌浓度处理的香椿植株，图中标尺为2 cm；C：不同真空渗透处理时长侵染的香椿植株，图中标尺为2 cm

Fig. 2 Comparison of transformation efficiency of T. sinensis bydifferent treatmentsA: T. sinensis seedlings infected with different Agrobacterium strains containing RUBY plasmids, scale bar=2 cm. B: T. sinensis seedlings treated with different concentrations of Agrobacterium, scale bar=2 cm. C: T. sinensis seedlings infected with different durations of vacuum infiltration treatment, scale bar=2 cm

图3 不同处理对香椿叶片转化效率的影响A：农杆菌菌株类型对香椿叶片瞬时转化效率的影响；B：农杆菌浓度对香椿叶片瞬时转化效率的影响；C：真空渗透时间对香椿叶片瞬时转化效率的影响；不同小写字母表示在0.05水平上差异显著；+为红色面积占变色叶总面积≤20%；++为红色面积占变色叶总面积20%-50%；+++为红色面积占变色叶总面积50%-80%

Fig. 3 Effects of different treatments on the transformation efficiency of T. sinensis leavesA: The effect of Agrobacterium strain types on the transient transformation efficiency of T. sinensis leaves. B: The effect of Agrobacterium concentration on the transient transformation efficiency of T. sinensis leaves. C: The effect of vacuum infiltration time on the transient transformation efficiency of T. sinensis leaves. Different letters indicate significant differences at the 0.05 level. + is red area ≤20% of total area of variegated leaves; ++ is red area 20%-50% of total area of variegated leaves; +++ is red area 50%-80% of total area of variegated leaves

图4 来自不同地区的5个香椿组培苗材料的侵染图中C-1和C-2为源自广西地区的香椿材料；C-3为源自英国的香椿材料；C-4为源自安徽的香椿材料；C-5为源自四川的香椿材料，所用材料均来自中国林业科学研究院亚热带林业研究所林木种质资源课题组组培室继代培养30 d，图中标尺为2 cm

Fig. 4 Infection of five T. sinensis tissue culture seedling materials from different regionsIn the figure， C-1 and C-2 are T. sinensis material originating from the Guangxi region. C-3 is a T. sinensis material originating from the United Kingdom. C-4 is a T. sinensis material originating from Anhui. C-5 is a T. sinensis material originating from Sichuan. All used materials were sub-cultured for 30 d in the tissue culture room of research group on forest tree germplasm resources of Research Institute of Tropical Forestry， Chinese Academy of Forestry. The scale bar in the figure is 2 cm

图5 不同地区的5个香椿组培苗材料转化效率不同小写字母表示在0.05水平上差异显著

Fig. 5 Transformation efficiency of five T. sinensis tissue culture seedling materials from different regionsDifferent letters indicate significant differences at the 0.05 level

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