【作者】 王丽；

【导师】 原雪峰；

【作者基本信息】 山东农业大学 ， 植物保护， 2024， 硕士

【摘要】 大多数植物RNA病毒基因组5′末端没有帽子结构,需要通过不依赖帽子翻译机制起始翻译,实现病毒编码蛋白的表达。对于此类RNA病毒参与不依赖帽子翻译调控的关键元件的解析,有助于深化对不依赖帽子翻译调控的理解,也可以为植物病毒病的防控提供精准靶标。菜豆普通花叶病毒(bean common mosaic virus,BCMV)属于马铃薯Y病毒科马铃薯Y病毒属,基因组为一条单链正义RNA,基因组5′端没有帽子结构,有共价结合的Vpg蛋白,3′端有poly(A)尾。本研究首先定位BCMV的内部核糖体进入位点(IRES),进一步明确了IRES中的关键调控位点及结构元件,同时分析BCMV IRES元件与翻译起始因子eIF4E、eIF(iso)4E的分子互作关系,从不同方面解析BCMV不依赖帽子翻译调控的分子机制。具体研究结果如下:BCMV IRES的定位及核心区分析:利用荧光素酶(Fluc)载体和WGE体外翻译体系,分析BCMV 5′UTR及其下游紧邻ORF区序列对翻译的调控作用。BCMV 5′UTR表现出对翻译的正调控作用,其紧邻下游的90 nt ORF区序列在有无帽子时,均不影响BCMV 5′UTR对翻译的调控作用。若在5′UTR上游插入长茎环后,下游90 nt序列则明显增强BCMV 5′UTR对翻译的调控作用,而下游150 nt序列的增效作用与90 nt一致,表明BCMV完整的IRES活性区域位于5′UTR及紧邻下游的90 nt ORF序列。缺失突变分析表明,IRES的活性核心区域位于1-181位。BCMV IRES核心区的活性分析:RNA结构预测IRES元件包含5个茎环,发现5个茎环的缺失都导致BCMV IRES活性丧失超过30%。茎环SL1顶环对IRES活性起正调控作用。茎环SL2和茎环SL3间区域的碱基互补突变明显增强IRES活性,暗示BCMV IRES区域形成比较复杂的分子互作网络。BCMV IRES与eIF4E及eIF(iso)4E的互作分析:EMSA定性分析发现,BCMV IRES与花生和菜豆eIF4E、eIF(iso)4E存在互作,与番茄、玉米、辣椒的eIF4E、eIF(iso)4E及黄瓜eIF4E不存在互作。微量热泳动技术(MST)定量分析发现,BCMV IRES与花生、菜豆eIF4E和eIF(iso)4E结合的亲和力最强,初步暗示BCMV IRES在花生、菜豆中通过招募不同类型的eIF4E或eIF(iso)4E起始翻译。进一步研究发现对IRES每个茎环的单独缺失都不破坏BCMV IRES与花生eIF4E、eIF(iso)4E的相互作用,暗示BCMV IRES可能存在多个位点与花生eIF4E及eIF(iso)4E结合。MST、EMSA和体外翻译结合寡核苷酸反式竞争实验表明,BCMV IRES与花生eIF4E的结合区域有三个,分别是BCMV 5′端1-26 nt、茎环SL2的顶环区域72-78 nt和BCMV IRES中的100-125nt,且这3个区域的结合均与IRES活性有关。BCMV IRES与花生eIF(iso)4E结合区域有2个,分别是茎环SL1顶环区域39-49 nt及茎环SL3顶环区域89-96 nt,且这2个区域的结合同样与IRES活性相关。通过对几种植物eIF4E及eIF(iso)4E氨基酸比对以及缺失突变分析发现,花生eIF4E、eIF(iso)4E结合BCMV IRES的区域位于氨基酸序列保守区,而不是氨基酸高变区。本研究在定位BCMV IRES元件的基础上,分析了IRES核心活性区、关键结构元件以及与翻译起始因子eIF4E及eIF(iso)4E的互作关系,深化了对不依赖帽子翻译调控的理解,为BCMV病毒病的防治提供了新的理论依据及防治靶标。更多还原

【Abstract】 Most plant RNA viruses lack a cap structure at the 5′ end of their genome.And they need to necessitate initiation of translation through a cap-independent mechanism to express virusencoded proteins.The analysis of the essential components involved in cap-independent translation regulation of these RNA viruses will deepen understanding of this process and pinpoint precise targets for preventing and managing plant virus diseases.Bean common mosaic virus(BCMV),a member of the genus Potyvirus in the family Potyviridae,has a singlestranded sense RNA without a cap structure at its 5′ end of the genome,but with a 5′ genomelinked protein VPg and a poly(A)tail at the 3′ end.In this study,firstly,we mapped the internal ribosome entry site(IRES)of BCMV,and elucidated the key regulatory sites and structural elements within the IRES.Additionally,the molecular interactions between BCMV IRES elements and translation initiation factors eIF4 E and eIF(iso)4E were analyzed to provide insights into the molecular mechanism of BCMV cap-independent translation regulation.The specific research findings were outlined below:Localization and core region analysis of BCMV IRES: The study utilized the luciferase vector(Fluc)and in vitro translation system of wheat germ extract(WGE)to analyze the impact of the 5′ untranslated region(UTR)and adjacent open reading frame(ORF)sequences of BCMV on translation regulation.Results indicated that the BCMV 5′ UTR positively influenced translation,while the downstream 90 nt ORF sequence did not alter this regulatory effect in the presence or absence of caps.However,when a long stem loop was introduced upstream of the 5′ UTR,the downstream 90 nt sequence notably enhanced the regulatory effect of the BCMV 5′ UTR on translation.Furthermore,the combined effect of the downstream 150 nt sequence was similar to that of the 90 nt sequence,indicating that the complete IRES active region of BCMV was situated within the 5′ UTR and the downstream 90 nt ORF sequence.Additionally,the deletion mutation analysis showed that the active core region of IRES was located at 1-181 nt.Activity analysis of the core region of BCMV IRES: The RNA structure analysis indicated the presence of five stem loops within the IRES element,and subsequent experimentation demonstrated that deletion of these stem loops led to a reduction in BCMV IRES activity exceeding 30 %.The apical loop of stem-loop SL1 was identified as a positive regulator of IRES activity.Additionally,base complementary mutations in the region spanning stem-loop SL2 and SL3 were found to markedly increase IRES activity,implying the formation of a more intricate molecular interaction within the BCMV IRES region.Interaction analysis of BCMV IRES with eIF4 E and eIF(iso)4E: The qualitative analysis of EMSA revealed that BCMV IRES interacts with eIF4 E or eIF(iso)4E in peanut and kidney bean,while no interaction was observed with eIF4 E or eIF(iso)4E in tomato,maize and pepper,and eIF4 E in cucumber.Microscale thermophoresis(MST)quantitative analysis indicated that BCMV IRES exhibited the highest binding affinity with eIF4 E and eIF(iso)4E in peanut and kidney bean,suggesting that BCMV IRES triggers translation initiation by recruiting different types of eIF4 E or eIF(iso)4E in peanut and kidney bean.Subsequent investigation revealed that the individual deletion of each stem loop within the IRES did not disrupt the interaction between BCMV IRES and peanut eIF4 E and eIF(iso)4E,indicating the presence of multiple binding sites on BCMV IRES for these proteins.Utilizing MST,EMSA and in vitro translation binding oligonucleotide trans-competitive experiments,it was determined that there were three distinct binding regions between BCMV IRES and peanut eIF4 E,located at nucleotides 1-26 nt at the 5′ end of BCMV,72-78 nt in the apical loop region of stem loop SL2,and 100-125 nt within BCMV IRES.And the binding of these three regions was positively correlated with IRES activity.The interaction between the BCMV IRES and peanut eIF(iso)4E was mediated by two specific binding regions,located at 39-49 nt within the top ring region of stem loop SL1 and 89-96 nt within the top ring region of stem ring SL3.The binding of these regions has been shown to correlate with IRES activity.By conducting amino acid alignment and deletion mutation analysis of eIF4 E and eIF(iso)4E in multiple plant species,it determined that the binding region of eIF4 E and eIF(iso)4E to BCMV IRES in peanut was situated in the conserved amino acid sequence region rather than the hypervariable region.This study examined the localization of BCMV IRES elements,focusing on the core active region,key structural features of the IRES,and its interaction with translation initiation factors eIF4 E and eIF(iso)4E.These findings contribute to a deeper understanding of cap-independent translation regulation,offering a new theoretical framework and potential target for the prevention and treatment of BCMV virus diseases.更多还原