转录因子NbERF RAP2-1在黄瓜绿斑驳花叶病毒侵染中的功能

Function of Transcription Factor NbERF RAP2-1 in Cucumber Green Mottle Mosaic Virus Infection

【背景】黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)是我国重要的检疫性植物病毒,对蔬菜和瓜类产业造成严重的经济损失。ERF转录因子可以调控植物生物和非生物胁迫,参与植物对多种病害的防御作用。前期研究显示CGMMV侵染后可以显著下调寄主转录因子Nb ERF RAP2-1的表达。【目的】明确ERF转录因子家族成员在CGMMV侵染过程中的作用,为CGMMV病害防治提供理论依据。【方法】运用MEGA7.0构建系统进化树,对基于Nb ERF RAP2-1蛋白的氨基酸序列进行系统进化分析;构建Nb ERF RAP2-1的荧光表达载体,并观察其亚细胞定位;利用q RT-PCR技术分析Nb ERF RAP2-1在CGMMV侵染不同时期的表达量;通过烟草脆裂病毒(tobacco rattle virus,TRV)介导的基因沉默(VIGS)和瞬时过表达Nb ERF RAP2-1分析其在CGMMV侵染过程中的作用。【结果】系统进化树分析表明,Nb ERF RAP2-1与多种烟草的ERF转录因子同源性极高,与拟南芥的ERF转录因子亲缘关系较远;亚细胞定位结果显示Nb ERF RAP2-1定位于细胞核,作为转录因子行驶功能;CGMMV侵染对本氏烟内源基因Nb ERF RAP2-1的转录水平影响结果显示,在CGMMV侵染6、9、12 d时Nb ERF RAP2-1的表达量无明显变化,在CGMMV侵染15和18 d时Nb ERF RAP2-1表达量显著下调;TRV介导的VIGS可以将植物内源基因Nb ERF RAP2-1有效沉默,在沉默的植株系统叶上接种CGMMV,8 d对照植株系统叶出现斑驳、卷曲症状而Nb ERF RAP2-1沉默植株无症状,同时CGMMV RNA水平和蛋白水平检测结果也表明TRV:Nb ERF RAP2-1可以有效抑制CGMMV的积累;同样,分别在本氏烟叶片瞬时过表达Nb ERF RAP2-124、48和72 h时检测CGMMV RNA水平和蛋白水平表达量,结果显示在病毒侵染早期,即复制阶段就抑制CGMMV的表达。【结论】Nb ERF RAP2-1可以有效抑制CGMMV侵染初期病毒复制,即病毒RNA复制阶段;随着CGMMV不断侵染,在CGMMV细胞间运动和系统运动时期,CGMMV识别防御相关基因Nb ERF RAP2-1并抑制该基因的转录;当Nb ERF RAP2-1缺失后可能抑制了下游蛋白的转录或表达,从而抑制病毒侵染后期的积累。由此可见,Nb ERF RAP2-1在CGMMV侵染过程中发挥了重要作用。

【Background】Cucumber green mottle mosaic virus(CGMMV)is an important quarantine plant virus in China,which has caused serious economic losses to vegetable and melon industry.The ERF transcription factor is involved in biotic and abiotic stress and the defense response against a variety of plant pathogens.In previous study,it was demonstrated that the host transcription factor Nb ERF RAP2-1 was significantly down-regulated after CGMMV infection.【Objective】The objective of this study is to clarify the function of the ERF transcription factor family members in CGMMV infection,and to provide a theoretical basis for disease control caused by CGMMV.【Method】MEGA7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbERF RAP2-1.The expression vector NbERF RAP2-1-GFP was constructed to observe the subcellular localization of NbERF RAP2-1.The transcript level of NbERF RAP2-1 at different stages of CGMMV infection was analyzed by qRT-PCR.VIGS and transient overexpression of Nb ERF RAP2-1 were conducted to analyze the function of NbERF RAP2-1 during CGMMV infection.【Result】Phylogenetic tree analysis showed that NbERF RAP2-1 was highly homologous to ERF transcription factors in tobacco,and was far from ERF transcription factors in Arabidopsis thaliana.The results of subcellular localization showed that NbERF RAP2-1 was localized in the nucleus and acted as a transcription factor.The effect of CGMMV infection on the transcript level of the NbERF RAP2-1 showed that the expression level of NbERF RAP2-1 did not significantly change at the 6,9,and 12 d after inoculation,but at the 15 and 18 d after inoculation,the expression level of NbERF RAP2-1 was significantly down-regulated.Tobacco rattle virus(TRV)mediated VIGS was used to silence the NbERF RAP2-1,and CGMMV was inoculated in TRV:NbERF RAP2-1 and TRV:00plants.At 8 d after inoculation,the leaves of TRV:00 plants showed mottling and curling,while the TRV:NbERF RAP2-1 plants showed no symptoms.At the same time,the detection results of CGMMV RNA and protein levels showed that TRV:NbERF RAP2-1could effectively inhibit the accumulation of CGMMV.Similarly,transient overexpression of Nb ERF RAP2-1 inhibited the accumulation of CGMMV at 24,48,and 72 h,respectively.【Conclusion】Nb ERF RAP2-1 effectively inhibit the initial CGMMV replication,i.e.the viral RNA replication stage.With the invasion of CGMMV,i.e.the intercellular and systemic movements of CGMMV,CGMMV recognizes and inhibits the transcript level of NbERF RAP2-1.When NbERF RAP2-1 is knocked down,that may inhibit the transcription of downstream proteins,thereby inhibiting the accumulation of CGMMV at a later stage.Nb ERF RAP2-1 plays an important role during the CGMMV infection.