烟草 NtWRKY40在植物应答病毒侵染过程中的作用

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 Ⅱ a 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 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.