内质网应激因子NAC089突变体的创建及其对病毒侵染胁迫的响应

Mutagenesis of endoplasmic reticulum stress sensor NAC089 and its response to viral infection in Nicotiana benthamiana

有研究表明烟草花叶病毒(Tobacco mosaic virus,TMV)或黄瓜花叶病毒(Cucumber mosaic virus,CMV)侵染烟草能够激活转录因子Nb NAC089,从ER膜移至细胞核。为进一步阐释内质网应激因子Nb NAC089对病毒侵染胁迫的响应机制,利用CRISPR/Cas9技术构建敲除载体,经烟草遗传转染获得Nb NAC089基因突变植株。植株接种病毒后采用qRTPCR检测病毒CP基因和寄主UPR基因的表达。结果表明:CRISPR/Cas9系统定点敲除Nb NAC089基因后,目的基因靶位点序列有碱基的置换与缺失。正常生长条件下,转基因植株与野生型无差异。植株接种TMV-GFP后24~96 h,突变体中UPR基因(Bi P、b ZIP28、b ZIP60)的表达量显著高于野生型;接种TM V-GFP后2~6 d突变体中病毒的积累量和扩展速度显著高于野生型。表明Nb NAC089为UPR的抑制因子,对病毒增殖具有负调控作用。

Previous study showed that TMV or CMV infection in tobacco activated transcription factor NAC089,translocated from ER to nucleus.To further study its stress response to viral infection,CRISPR/Cas9 plant expression vector was constructed and the NAC089 knockout mutations were obtained.The virus accumulation and plants UPR genes in mutations were compared with that in wide type by qRT-PCR at 24-96 hours post inoculation （hpi） TMV-GFP.The results showed that DNA deletion and displacement had been found in the target sites of NAC089.Compared to wide type,mutant plants showed no different phenotypes under normal condition.Upon inoculation with TMV-GFP, mutant plants showed more virus accumulation than wt plants,in addition,UPR genes （BiP,bZIP28,bZIP60） expression in mutant plants was higher than that in wt at 24-96 hpi.These results show that NAC089 was a negative regulator of UPR and plays a role in suppression of viral infection.