| 陈健鑫1, 朱幼娇1, 吴峰婧琳1, 魏玉倩1, 马焕成2, 杨红玉3*, 伍建榕1, 2*.油茶暹罗炭疽菌的遗传转化及其侵染进程研究[J].植物保护,2025,51(4):275-284. |
| 油茶暹罗炭疽菌的遗传转化及其侵染进程研究 |
| Genetic transformation and infection process of Colletotrichum siamense in Camellia oleifera |
| 投稿时间：2024-07-02 修订日期：2024-09-30 |
| DOI：10.16688/j.zwbh.2024360 |
| 中文关键词: 油茶暹罗炭疽菌 遗传转化体系 绿色荧光蛋白 侵染进程 |
| 英文关键词:Colletotrichum siamense genetic transformation system green fluorescent protein infection process |
| 基金项目:国家自然科学基金(31860208); 国家重点研发计划(2019YFD100200X); 云南省教育厅科学研究基金(2023Y0720, 2106150086Y615) |
| 作者 | 单位 | E-mail | | 陈健鑫1, 朱幼娇1, 吴峰婧琳1, 魏玉倩1, 马焕成2, 杨红玉3*, 伍建榕1, 2* | 1. 云南省高校森林灾害预警控制重点实验室, 西南林业大学林学院, 昆明 650224
2. 西南地区生物多样性保育国家林业局重点实验室, 西南林业大学林学院, 昆明 650224
3. 昆明学院农学与生命科学学院, 昆明 650214 | 杨红玉Yanghongyukm@126.com; 伍建榕1176279044@qq.com |
|
| 摘要点击次数: 70 |
| 全文下载次数: 440 |
| 中文摘要: |
| 暹罗炭疽菌Colletotrichum siamense(菌株CA17)是引起云南省油茶炭疽病的强致病力菌株, 但其与油茶互作的机制尚不十分清楚。本研究建立了高效的遗传转化体系并利用荧光显微镜探究其分生孢子发育和侵染进程。首先构建了含有潮霉素抗性基因HygR和绿色荧光蛋白基因GFP的表达载体pCAMBIA1301∷GFP。同时优化了CA17原生质体的制备方法, 并通过CaCl2/PEG介导的转化法, 将表达载体转化CA17原生质体。本研究共筛选出6株稳定的阳性转化子, 转化子在菌落形态、产孢量和致病力上与野生型菌株无显著差异。阳性转化子CA17-GFP6的分生孢子培养4 h开始萌发, 6 h附着胞开始发育并逐渐黑化, 整个阶段均可见绿色荧光信号。CA17-GFP6接种拟南芥24 h可见分生孢子和少量菌丝的荧光信号, 接种后48~72 h在叶片内部可见大量菌丝的绿色荧光, 接种油茶叶片后24~48 h绿色荧光信号较弱, 主要分布在表皮和气孔附近, 接种72 h后, 随着菌丝的扩张, 绿色荧光信号逐渐增强。综上所述, 本研究成功建立了PEG介导的油茶暹罗炭疽菌原生质体的遗传转化体系, 并利用阳性转化子CA17-GFP6探究了暹罗炭疽菌侵染拟南芥和油茶的途径与进程, 为油茶炭疽菌致病的分子机制研究奠定了基础。 |
| 英文摘要: |
| Colletotrichum siamense (strain CA17) is a highly virulent pathogen causing anthracnose in Camellia oleifera in Yunnan province, but its interaction mechanism with the host remains poorly understood. In this study, an efficient genetic transformation system was developed to facilitate visualization of conidial development and infection process using fluorescence microscopy. An expression vector pCAMBIA1301∷GFP, harboring the hygromycin resistance gene HygR and the green fluorescent protein gene GFP, was constructed. The protoplast preparation protocol for CA17 was optimized, and transformation was achieved via CaCl2/PEG-mediated delivery. Six stable GFP-positive transformants were obtained, exhibiting no significant differences from the wild-type strain in colony morphology, sporulation or pathogenicity. In the selected transformant CA17-GFP6, conidia began germinating 4 h post-inoculation, with appressoria forming and darkening by 6 h, and persistent green fluorescence observed throughout development. Green fluorescence from conidia and limited hyphae was visible 24 hours after inoculating CA17-GFP6 into Arabidopsis thaliana, while extensive hyphal fluorescence was observed within leaf tissue 48-72 h after inoculation. In contrast, on C.oleifera leaves, weak GFP signals were localized near the epidermis and stomata between 24-48 hours, intensifying by 72 h as hyphae expanded. This study successfully established a PEG-mediated protoplast transformation system for C. siamense, and utilized the GFP-tagged transformant CA17-GFP6 to track infection progression in A.thaliana and C. oleifera, providing a basis for elucidating the molecular mechanism of anthracnose pathogenicity in oil tea. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
