烟草NtWRKY40在植物应答病毒侵染过程中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2016.10.023

摘要/Abstract

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NtWRKY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NtWRKY cDNA,生物信息学分析结果显示,该基因属于WRKY Ⅱa 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。

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

Abstract: 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,NtWRKY40over-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 35S promoter. Over-expression of amiR167-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.

Key words: Tobacco mosaic virus, Potato virus Y, WRKY transcription factor, artificial microRNA, virus resistance

刘晶晶, 程春玲, 席玉珍, 魏书. 烟草NtWRKY40在植物应答病毒侵染过程中的作用[J]. 生物技术通报, 2016, 32(10): 188-198.

LIU Jing-jing, CHENG Chun-ling, XI Yu-zhen, WEI Shu. Roles of Nicotiana tobacum NtWRKY40 in Plant Responding to Virus Infection[J]. Biotechnology Bulletin, 2016, 32(10): 188-198.

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