万寿菊花冠高效瞬时转化体系的建立及TeCYC2c基因启动子活性分析
收稿日期: 2024-10-02
录用日期: 2025-01-20
网络出版日期: 2025-01-21
基金资助
国家自然科学基金(32172616)
Establishment of an Efficient Transient Transformation System for Tagetes erecta Corollas and Analysis on the Promoter Activity of TeCYC2c Gene
Received date: 2024-10-02
Accepted date: 2025-01-20
Online published: 2025-01-21
为建立万寿菊(Tagetes erecta)花冠高效瞬时转化体系并探究花器官对称性形成关键调控基因TeCYC2c的启动子活性, 将CaMV35S启动子与GUS基因的融合表达载体瞬时转化万寿菊花冠, 探究菌株类型、菌液浓度、侵染时间和共培养时间对GUS基因瞬时转化效率的影响。结果显示, GV3101菌株的侵染效率最高; 菌液浓度OD600=1.0时转化效率最高; 侵染时间对瞬时转化效率无显著影响; 共培养1天为最佳培养时间。基于建立的万寿菊花冠瞬时转化体系, 对TeCYC2c基因的启动子活性进行探究。克隆了TeCYC2c基因上游1 735 bp序列, 并通过PlantCARE预测的元件分布位置及数量构建了4个启动子缺失表达载体, 以GUS基因为报告基因, 瞬时转化万寿菊花冠进行不同长度启动子活性研究。结果显示, 启动子核心区域位于ATG上游-650 - -1 bp, 推测该区域内的光响应元件正调控下游基因的表达, 而pTeCYC2c (-1 735)和pTeCYC2c (-1 406)所特有的植物激素响应和逆境响应元件可能具有抑制启动子驱动下游基因表达的功能。结合万寿菊花冠瞬时转化体系的建立和TeCYC2c启动子活性分析, 可为进一步快速解析花发育相关基因的功能奠定技术基础。
窦淋琳 , 朱钰 , 刘翠翠 , 臧运平 , 陶正国 , 包满珠 , 何燕红 . 万寿菊花冠高效瞬时转化体系的建立及TeCYC2c基因启动子活性分析[J]. 植物学报, 2025 , 60(6) : 875 -887 . DOI: 10.11983/CBB24150
INTRODUCTION: Marigold (Tagetes erecta), an important ornamental and medicinal plant, has a unique capitulum characteristic of the Asteraceae family with distinct ray and disc florets. However, the lack of efficient genetic transformation system has limited the research on the mechanism of floral organ development of marigold. Floral transient transformation system offers a rapid approach to study the function of genes expressed specifically in floral organs. This study aimed to establish an efficient transient transformation system for marigold corollas and to analyze the promoter activity of TeCYC2c, which highly expressed in corollas, thereby laying the technical foundation for the rapid verification of floral gene function.
RATIONALE: A fusion expression vector, incorporating the CaMV35S promoter and the GUS reporter gene, was constructed to facilitate the transient transformation in marigold corollas. The study delved into the effects of bacterial strain type (GV3101, LBA4404, EHA105), bacterial suspension concentration (OD600 values 0.5-2.0), infection duration (10- 40 min), and co-culture time (1-4 d) on the transient transformation efficiency of the GUS gene. Based on this transient transformation system for the marigold corollas, the promoter activity of the TeCYC2c gene was investigated. A 1 735 bp upstream sequence of the TeCYC2c gene was cloned and four promoter deletion expression vectors, with the GUS gene as the reporter gene, were constructed based on the distribution and quantity of elements predicted by PlantCARE. Subsequently, these vectors were employed for transient transformation of marigold corollas to facilitate an in-depth analysis of promoter activity.
RESULTS: The transient transformation efficiency in marigold corollas demonstrated that the GV3101 strain achieved the highest infection efficiency; the bacterial suspension concentration, quantified at an OD600 value of 1.0, yielded the most robust transformation efficiency; the infection time was observed to exert no substantial influence on transient transformation efficacy; moreover, a co-culture time of 24 hours was identified as the optimal condition for the process. The results of GUS staining and GUS activity assay revealed that the core region of the promoter was located at -650 to -1 bp. It was speculated that the light-responsive elements within this region positively up-regulated the expression of downstream genes, while the hormone-responsive and stress-responsive elements unique to pTeCYC2c (-1 735) and pTeCYC2c (-1 406) might have an inhibitory effect on promoter-driven downstream gene expression.
CONCLUSION: This study established an efficient transient transformation system for marigold corollas, optimized through strain selection and parameter tuning. The identification of the TeCYC2c promoter core region (-650 to -1 bp) and its regulatory elements provides critical insights into the regulatory mechanism of the TeCYC2c gene. The transient transformation system and promoter analysis method lay a technical foundation for accelerating functional studies of floral development genes in marigold, with potential applications in the genetic improvement of ornamental plants.
GUS staining of marigold (Tagetes erecta) corollas under different transient transformation conditions. (A) GUS staining of marigold corollas infected by three different bacterial strains; (B) GUS staining under four different concentrations (OD600) of bacterial suspension; (C) GUS staining under four different infection duration; (D) GUS staining under four different co-culture time. (A)-(D) Bars=1 cm
Key words： marigold (Tagetes erecta); TeCYC2c; promoter; transient transformation; GUS activity
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