烟草NtWRKY40在植物应答病毒侵染过程中的作用  

Roles of Nicotiana tobacum NtWRKY40 in Plant Responding to Virus Infection

作　　者：刘晶晶[1] 程春玲[1] 席玉珍 魏书[1] 

机构地区：[1]安徽农业大学茶树生物学与资源利用国家重点实验室,合肥230036

出　　处：《生物技术通报》2016年第10期188-198,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31070614;31370687);教育部博士学科点专项科研基金项目(20123418110002)

摘　　要：WRKY转录因子家族在植物的抗病、抗逆反应中具有重要功能。已有研究表明,烟草花叶病毒(TMV)的侵染显著地诱导烟草Nt WRKY的表达,有必要进一步探明该基因在植物应答病毒侵染过程中的作用。采用PCR的方法克隆获得Nt WRKY cDNA,生物信息学分析结果显示,该基因属于WRKYⅡa亚族成员,与绒毛状烟草NtoWRKY40高度同源,命名为NtWRKY40。以此建立了过表达该基因的转基因烟草,并以TMV为毒源进行了转基因烟草和野生烟草的侵染实验,以观察NtWRKY40在烟草应答病毒侵染过程中的作用。实验结果表明,野生烟草在TMV侵染后9 d,NtWRKY40的表达量显著升高,而NtWRKY40过表达转基因烟草在病毒侵染后,病毒相关基因的表达高于野生型对照,与染病程度成正相关,说明过表达NtWRKY40增加了植株对病毒的敏感性,该基因为负调控因子。此外,为探索应用人工miRNA的抗病毒技术,以烟草天然miR167前体为骨架、马铃薯Y病毒(PVY)外壳蛋白基因的一段反向互补序列为成熟序列,构建了amiR167-PVY植物表达载体并转化烟草,以抑制PVY。对amiRNA转基因植株进行抗病毒实验的结果显示,amiR167-PVY能够部分抑制病毒基因的表达,转基因植株具有一定的抗病毒能力。Some of WRKY transcription factors have involved in plant abiotic defense. A WRKY gene,a homologue of Arabidopsis WRKY40,was found being dramatically induced by plant virus and further characterized in this study. To verify its function,NtWRKY40 over-expressed tobacco plants were established. After virus infection,the transcription levels of NtWRKY40 and virus related genes were quantified using quantitative real time PCR at three stages（6 h,3 d,9 d after infection）. Results showed that tobacco mosaic virus infection led to 150 fold higher of NtWRKY40 transcript in non-transgenic wild type tobacco than non-infected control. No significant change in NtWRKY40 transcription levels was noted in the NtWRKY40 over-expressed tobacco after virus infection. NtWRKY40 over-expressed tobaccos were more sensitive to TMV infection than non-transgenic wild type. The data suggested that NtWRKY40 was a negative regulator in responding to TMV. In addition,an artificial miRNA vector amiR167-PVY was constructed,for silencing the coat protein gene of Potato virus Y,using the miR167 precursor of Nicotiana tabacum as backbone and cauliflower mosaic virus 35 S promoter. Over-expression of ami R167-PVY exhibited inhibitory effect on the virus. The results provide the basis for the further research on amiRNA-mediated virus resistance mechanism,and contributes to antiviral transgenic plants cultivation with the optimal target sequence.

关 键 词：烟草花叶病毒 马铃薯Y病毒 WRKY转录因子 人工miRNA 抗病毒 

分 类 号：S432.41[农业科学—植物病理学]