多个稻曲病菌效应因子的信号肽验证和表达分析  

Signal Peptide Validation and Expression Analysis of Multiple Effectors from Ustilaginoidea virens

作　　者：尹潇潇 张芷菡 颜绣莲 廖蓉 杨思葭 Beenish HASSAN 郭岱铭 樊晶[1] 赵志学 王文明[1] YIN Xiaoxiao;ZHANG Zhihan;YAN Xiulian;LIAO Rong;YANG Sijia;Beenish HASSAN;GUO Daiming;FAN Jing;ZHAO Zhixue;WANG Wenming(State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学西南作物基因资源发掘与利用国家重点实验,成都611130

出　　处：《中国水稻科学》2024年第3期256-265,共10页Chinese Journal of Rice Science

基　　金：国家自然科学基金资助项目(U19A2033,32101728);四川省青年科技创新研究团队资助项目(2022JDTD0023);四川省科技创新创业苗子工程项目(2022JDRC0111)。

摘　　要：【目的】信号肽对效应因子的分泌至关重要,影响病原微生物的致病进程。前期鉴定到在稻曲病菌侵染水稻花器官过程中显著上调表达的10个效应因子基因,但这些效应因子是否含有功能性信号肽及其表达模式尚不明确。【方法】利用SignaIP-5.0数据库预测、烟草瞬时表达系统和酵母信号肽分泌实验,分析并鉴定这些效应因子的信号肽。利用实时荧光定量PCR技术,检测效应因子基因的表达模式。【结果】UV_44、UV_1548、UV_1567等10个稻曲病菌效应因子均含有信号肽。烟草瞬时表达结果显示,含有信号肽的eYFP融合蛋白在细胞质壁分离后均能分泌到质外体空间。酵母菌信号肽分泌结果显示,分别携带这10个信号肽的酵母菌株可在以棉子糖为唯一碳源的培养基上生长,且与TTC发生显色反应。实时荧光定量结果表明,除了UV_6754,其余9个效应因子基因的表达模式不尽相同,其中UV_44、UV_1567、UV_3667、UV_4213、UV_4989、UV_5215和UV_8184的表达量在接种后显著升高。【结论】这10个稻曲病菌效应因子均含有分泌功能的信号肽,编码效应因子的基因在稻曲病菌侵染过程中具有不同的表达模式。该结果为深入研究以上稻曲病菌效应因子的功能奠定了基础。【Objective】Signal peptides are crucial for effector secretion and affect the pathogenic process of pathogenic microorganisms.Previously,ten effector genes were identified to be significantly up-regulated during Ustilaginoidea virens infection in rice spikelets.However,it remains unclear whether these effectors contain functional signal peptides and how they express.【Method】The signal peptides of these effectors were predicted and verified by using SignaIP-5.0 database,Agrobacterium-mediated transient expression in Nicotiana benthamiana,and yeast invertase assay.Besides,the expression patterns of these effectors were analyzed by using RT-qPCR.【Results】All the ten effectors,including UV_44,UV_1548,UV_1567,etc.,were predicted to contain a signal peptide.The transient expression method showed that eYFP fusion proteins containing signal peptides were successfully secreted into the extracellular space after plasmolysis in N.benthamiana.Yeast invertase assay showed that the yeast strains harboring signal peptides could grow on the medium with raffinose as the only carbon source with the occurrence of a chromogenic reaction using TTC.All the effector genes had distinct expression patterns,except for UV_6754.Seven of them showed significant up-regulation after inoculation,including UV_44,UV_1567,UV_3667,UV_4213,UV_4989,UV_5215,and UV_8184.【Conclusion】The signal peptides of these effectors had secretory functions,and the effector genes have different expression patterns during U.virens infection.These findings lay a foundation for further research on the functions of the above-mentioned effectors from U.virens.

关 键 词：水稻 稻曲病菌 效应因子 信号肽 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]