Construction of an Efficient Agrobacterium tumefaciens-Based Transformation System in the Entomopathogenic fungus Metahizium rileyi,Journal of Agricultural and Food Chemistry

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Construction of an Efficient Agrobacterium tumefaciens-Based Transformation System in the Entomopathogenic fungus Metahizium rileyi
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-09-17 , DOI: 10.1021/acs.jafc.4c05535
Guang Wang ₁ , Xu Zhang ₁ , Bin Chen ₁ , Yuejin Peng ₁

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Metarhizium rileyi is a filamentous entomopathogenic fungus that is highly pathogenic to lepidopteran insects. In our study, we constructed an Agrobacterium tumefaciens-mediated transgene system using the hygromycin resistance gene (Hyg R) as a selection marker in M. rileyi through homologous recombination. Binary knockout vectors for two genes (NOR_03501, longevity assurance gene, and NOR_03153, ATP-binding domain protein domain gene) in the M. rileyi strain SZCY201010 were successfully developed. We compared the genetic transformation efficiency using five kinds of asexual spores. The initial genetic transformation rates using a competent blastospore for NOR_03501 and NOR_03153 were 54.35 and 47.19%, respectively. Subsequently, both genes were successfully knocked out, and the transformed fungi were verified by PCR, RT-qPCR, and green fluorescent protein labeling. The biological phenotypes of the two genes were analyzed. The NOR_03501 gene plays a crucial role in carbon source utilization, stress resistance, and cuticle infection of fungal mycelium growth, while the NOR_03153 gene is significant for conidial production, stress resistance, and body wall infection. This study provides a promising tool for gene manipulation in M. rileyi, enhancing research in functional genomics and the exploration of fungal gene resources.

中文翻译：

基于根癌农杆菌的高效昆虫病原真菌 Metahizium rileyi 转化系统的构建

Metarhizium rileyi 是一种丝状昆虫病原真菌，对鳞翅目昆虫具有高度致病性。在我们的研究中，我们使用潮霉素抗性基因（Hyg R）作为 M. rileyi 中的选择标记，通过同源重组构建了根癌农杆菌介导的转基因系统。成功开发了 M. rileyi 菌株SZCY201010中两个基因（NOR_03501，长寿保证基因和 NOR_03153，ATP 结合域蛋白结构域基因）的二元敲除载体。我们比较了使用 5 种无性孢子的遗传转化效率。使用感受态芽生孢子进行 NOR_03501 和 NOR_03153 的初始遗传转化率分别为 54.35% 和 47.19%。随后，两个基因都被成功敲除，并通过 PCR、RT-qPCR 和绿色荧光蛋白标记验证转化的真菌。分析了 2 个基因的生物学表型。NOR_03501 基因在真菌菌丝体生长的碳源利用、抗逆性和角质层感染中起着至关重要的作用，而 NOR_03153 基因对分生孢子的产生、抗逆性和体壁感染具有重要意义。这项研究为 M. rileyi 的基因操作提供了一个有前途的工具，加强了功能基因组学的研究和真菌基因资源的探索。

更新日期：2024-09-17

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