转录因子NbMYB1R1通过促进活性氧积累抑制病毒侵染  

The Transcription Factor NbMYB1R1 Inhibits Viral Infection by Promoting ROS Accumulation

作　　者：姜兴林 于连伟 付涵 艾妞 崔荧钧 李好海 夏子豪 袁虹霞[1] 李洪连[1,3,4] 杨雪 施艳 JIANG XingLin;YU LianWei;FU Han;AI Niu;CUI YingJun;LI HaoHai;XIA ZiHao;YUAN HongXia;LI HongLian;YANG Xue;SHI Yan(College of Plant Protection,Henan Agricultural University,Zhengzhou 450002;Crop Science Postdoctoral Programme of Henan Agricultural University,Zhengzhou 450002;Collaborative Innovation Centre of Henan Grain Crops,Zhengzhou 450002;State Key Laboratory of Wheat and Maize Crop Science,Zhengzhou 450002;Henan Association for the Promotion of New Plant Protection Techniques,Zhengzhou 450002;Henan Plant Protection and Quarantine Station,Zhengzhou 450002;College of Plant Protection,Shenyang Agricultural University,Shenyang 110866)

机构地区：[1]河南农业大学植物保护学院,郑州450002 [2]河南农业大学作物学博士后流动站,郑州450002 [3]河南省粮食作物协同创新中心,郑州450002 [4]小麦玉米作物学国家重点实验室,郑州450002 [5]河南省植物保护新技术推广协会,郑州450002 [6]河南省植物保护检疫站,郑州450002 [7]沈阳农业大学植物保护学院,沈阳110866

出　　处：《中国农业科学》2024年第8期1490-1505,共16页Scientia Agricultura Sinica

基　　金：国家自然科学基金(3210170372);河南农业大学科技创新项目(KJCX2021A13)。

摘　　要：[背景]黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)是我国重要的检疫性植物病毒,严重降低了世界范围内蔬菜以及瓜类的产量。MYB蛋白家族庞大,功能多样,存在于所有真核生物中。大多数MYB作为转录因子控制植物的发育和代谢,并对植物应对生物和非生物胁迫反应起重要的调控作用。前期研究显示CGMMV侵染后可以显著上调寄主转录因子基因NbMYB1R1的表达。[目的]明确NbMYB1R1参与CGMMV侵染的机制,为CGMMV病害防控提供理论依据。[方法]运用MEGA 7.0构建系统进化树,对NbMYB1R1的氨基酸序列进行系统进化分析;通过构建NbMYB1R1的荧光表达载体,转化GV3101农杆菌后浸润本氏烟叶片,激光共聚焦显微镜观察其亚细胞定位;利用qRT-PCR技术分析NbMYB1R1在CGMMV侵染不同时期的转录水平及NbMYB1R1沉默烟草植株中活性氧(reactive oxygen species,ROS)相关基因的转录变化;通过沉默NbMYB1R1及下游调控基因NbAOX1a和NbAOX1b,分析NbMYB1R1及下游调控基因NbAOX1a和NbAOX1b在CGMMV侵染过程中的作用;瞬时过表达NbMYB1R1和NbMYB1R1关键氨基酸突变体,分析NbMYB1R1对CGMMV侵染的影响;使用台盼蓝染色以及DAB染色观察瞬时过表达NbMYB1R1造成的细胞死亡是否与程序性细胞死亡(programmed cell death,PCD)以及ROS的积累有关;运用酵母双杂交技术验证NbMYB1R1是否与CGMMV相关蛋白互作。[结果]系统进化树分析表明,NbMYB1R1属于1R MYB大类并与多种烟草的MYB转录因子同源性极高;亚细胞定位结果显示NbMYB1R1定位于细胞核;在CGMMV侵染的烟草植株中,NbMYB1R1的转录水平对比健康植株有明显变化,在CGMMV侵染8、12 d时NbMYB1R1的转录水平显著上调;在沉默内源基因NbMYB1R1的植株上接种CGMMV,3 d后NbMYB1R1沉默植株系统叶出现斑驳、卷曲症状,而对照植株在3.5 d时才出现症状;同时CGMMV CP mRNA水平和蛋白水平检测结果也表明沉默NbMYB1R1可以有效促进CGMMV的积累;在本[Background]Cucumber green mottle mosaic virus(CGMMV)is an important quarantine plant virus in China,which has seriously reduced the production of vegetables and melons worldwide.The MYB protein family is large,multifunctional and present in all eukaryotes.Most MYB proteins act as transcription factors that control development,and metabolism in plants,and regulate biotic and abiotic stress responses.Previous studies have shown that during CGMMV infection,the expression of transcription factor gene NbMYB1R1 was significantly up-regulated.[Objective]The objective of this study is to clarify the mechanism of NbMYB1R1 involved in CGMMV infection,and to provide a theoretical basis for controlling CGMMV infection.[Method]MEGA 7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbMYB1R1 protein.The expression vector NbMYB1R1-GFP was constructed,transformed into Agrobacterium GV3101 and infiltrated the tobacco leaves to observe the subcellular localization of NbMYB1R1 by confocal microscopy.The transcriptional levels of NbMYB1R1 at different stages of CGMMV infection and ROS related genes in silenced NbMYB1R1 plants were analyzed using qRT-PCR technology.TRV-mediated gene silencing(VIGS)was utilized to analyze the function of NbMYB1R1,NbAOX1a,and NbAOX1b during CGMMV infection.Transient overexpression of NbMYB1R1 and NbMYB1R1 mutant was conducted to analyze the effect of NbMYB1R1 on CGMMV infection.Trypan blue staining and DAB staining were used to observe whether the cell death caused by the transient overexpression of NbMYB1R1 was related to the programmed cell death(PCD)and the accumulation of ROS.Yeast two-hybrid system was used to verify whether NbMYB1R1 interacted with CGMMV related proteins.[Result]Phylogenetic tree analysis showed that NbMYB1R1 belonged to the 1R MYB category and had high homology with MYB transcription factors in a variety of tobaccos.The results of subcellular localization showed that NbMYB1R1 was localized in the nucleus.In CGMMV-infected tobacco plants,the tran

关 键 词：黄瓜绿斑驳花叶病毒 Nb MYB1R1 转录因子 致病机制 活性氧 

分 类 号：S432.41[农业科学—植物病理学]