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

doi:10.11983/CBB24150

植物学报

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

窦淋琳¹, 朱钰¹, 刘翠翠¹, 臧运平², 陶正国³, 包满珠¹, 何燕红¹

¹华中农业大学园艺林学学院, 果蔬园艺作物种质创新与利用全国重点实验室, 武汉 430070; ²云南立达尔生物科技有限公司, 文山 663100; ³广州立达尔生物科技股份有限公司, 广州 510663

收稿日期:2024-10-02 修回日期:2025-01-07 出版日期:2025-01-21 发布日期:2025-01-21
通讯作者: 何燕红
基金资助:
国家自然科学基金(No.32172616)

Establishment of an Efficient Transient Transformation System for Tagetes erecta corollas and Promoter Activity Analysis of the TeCYC2c Gene

Dou Linlin¹, Zhu Yu¹, Liu Cuicui¹, Zang Yunping², Tao Zhengguo³, Bao Manzhu¹, He Yanhong¹

¹College of Horticulture and Forestry Sciences, Huazhong Agricultural University, National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Wuhan 430070, China; ²Yunnan Lidar Biological Technology Co. LTD, Wenshan 663100, China; ³Guangzhou Lidar Biotechnology Co. LTD, Guangzhou 510663, China

Received:2024-10-02 Revised:2025-01-07 Online:2025-01-21 Published:2025-01-21
Contact: He Yanhong

摘要/Abstract

摘要： 该研究旨在建立万寿菊(Tagetes erecta)花冠高效瞬时转化体系并探究调控花器官对称性形成关键基因TeCYC2c启动子活性。以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为1.0转化效率最高; 侵染时间对瞬转效率无显著影响; 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表达, 而pTeCYC2c (–1735)和pTeCYC2c (–140 6)所特有的植物激素响应元件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。通过结合万寿菊花冠瞬时转化体系的建立和TeCYC2c启动子的活性分析, 为进一步快速解析花发育相关基因的功能奠定了技术基础。

关键词: 万寿菊, TeCYC2c, 启动子, 瞬时表达, GUS活性

Abstract: The objective of this study was to establish an efficient transient transformation system for the corollas of Tagetes erecta and to investigate the promoter activity of TeCYC2c, a key gene regulating floral organ symmetry. A fusion expression vector, incorporating the CaMV 35S promoter and the GUS reporter gene, was constructed to facilitate the transient transformation in Tagetes erecta corollas. The study delved into the effects of bacterial strain type, bacterial suspension concentration, infection time, and co-culture time on the transient transformation efficiency of the GUS gene. The results revealed 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. Based on this transient transformation system for the Tagetes erecta 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 transiently transformation of Tagetes erecta corollas to facilitate an in-depth analysis of promoter activity. The results 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 upregulated the expression of downstream genes, while the hormone-responsive and stress-responsive elements unique to pTeCYC2c(-1735) and pTeCYC2c(-1406) might have an inhibitory effect on promoter-driven downstream gene expression. By combining the establishment of the transient transformation system for the marigold corollas and the promoter activity analysis of TeCYC2c gene, a technical foundation has been laid for further rapid elucidation of the functions of genes related to floral organ development.

Key words: Tagetes erecta, TeCYC2c, promoter, transient expression, GUS activity

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. 植物学报, DOI: 10.11983/CBB24150.

Dou Linlin, Zhu Yu, Liu Cuicui, Zang Yunping, Tao Zhengguo, Bao Manzhu, He Yanhong. Establishment of an Efficient Transient Transformation System for Tagetes erecta corollas and Promoter Activity Analysis of the TeCYC2c Gene. Chinese Bulletin of Botany, DOI: 10.11983/CBB24150.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24150

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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

Establishment of an Efficient Transient Transformation System for Tagetes erecta corollas and Promoter Activity Analysis of the TeCYC2c Gene

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