本氏烟NbMBF1c的克隆、表达及在TMV侵染过程中的功能

Cloning,Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c

【目的】烟草花叶病毒(tobacco mosaic virus,TMV)是危害茄科、十字花科、葫芦科等农作物的重要病毒,给农业生产造成了巨大损失。通过分子克隆获得本氏烟(Nicotiana benthamiana)多蛋白桥梁因子1c(multiprotein bridging factor 1c,MBF1c),综合应用生物信息学、细胞生物学以及分子生物学手段,明确NbMBF1c的抗病毒功能和机制,为作物的抗病毒育种提供理论依据。【方法】根据Sol Genomics Network中报道的NbMBF1c序列全长,设计引物克隆NbMBF1c全长序列;使用GeneDoc及MEGA X对NbMBF1c蛋白和其他物种中的同源蛋白序列进行比对并构建系统进化树;利用生物信息学分析NbMBF1c的基因特征和蛋白结构;使用实时荧光定量PCR检测其组织表达及其在TMV侵染中的表达;利用烟草脆裂病毒(tobacco rattle virus,TRV)诱导的基因沉默(virus induced gene silencing,VIGS)技术沉默NbMBF1c后通过摩擦接种TMV-GFP,明确NbMBF1c对病毒侵染的影响;构建pART27-GFP-NbMBF1c和pART27-Myc-NbMBF1c瞬时过表达载体,在本氏烟叶片中表达融合蛋白GFP-NbMBF1c后在共聚焦显微镜下观察亚细胞定位,表达融合蛋白Myc-NbMBF1c后接种病毒观察TMV-GFP侵染情况;采用实时荧光定量PCR检测沉默NbMBF1c后相关激素基因的表达,分析NbMBF1c影响病毒侵染的机制。【结果】NbMBF1c全长441 bp,编码146个氨基酸,包含一个保守结构域HTH(helix-turn-helix);系统发育分析表明,NbMBF1c与绒毛状烟草(Nicotiana tomentosiformis)MBF1c(XP_009614458.1)亲缘关系最近;NbMBF1c的表达具有特异性,在根中的表达量最高,其次是茎、叶和花;NbMBF1c定位于细胞质和细胞核中;沉默NbMBF1c会显著影响本氏烟株高,出现矮化等症状,接种TMV-GFP第5天时,沉默处理相较于对照组植株新叶病毒含量更高;瞬时过表达Myc-NbMBF1c融合蛋白,在注射叶接种TMV-GFP后,病毒侵染减少,但NbMBF1c并不与TMV组分互作;沉默NbMBF1c后,脱落酸(abscisic acid,ABA)合成关键酶基因NCED3、脱落酸受体PYL1、ABA信号转导因子ABAI1和蛋白激酶SnRK2E的表达量显著上调;茉莉酸(jasmonic acid,JA)合成途径AOS1上调表达,茉莉酸信号通路上的受体COI1下调表达。【结论】本氏烟MBF1家族NbMBF1c的表达受TMV侵染诱导,且主要定位于细胞质和细胞核。NbMBF1c作为植物抗TMV的正调控因子抑制病毒在本氏烟上的侵染。NbMBF1c不通过与TMV组分互作来抑制病毒侵染,而是通过调控植物激素的产生和信号传导来抑制病毒侵染。

【Objective】Tobacco mosaic virus(TMV)is an important virus that harms crops such as Solanaceae,Cruciferae and Cucurbitaceae,causing great losses to agricultural production.The objective of this study is to obtain Nicotiana benthamiana multiprotein bridging factor 1c(MBF1c)by molecular cloning,clarify the antiviral function and mechanism of NbMBF1c by bioinformatics,cell biology and molecular biology methods,and to provide a theoretical basis for the antiviral breeding of crops.【Method】Based on the full-length NbMBF1c sequence reported in Sol Genomics Network,primers were designed to clone the full-length sequence of NbMBF1c.GeneDoc and MEGA X were employed to align the homologous protein sequences of NbMBF1c and other species.The gene characteristics and protein structure of NbMBF1c were analyzed by bioinformatics.The tissue expression and its expression after TMV infection were detected by real-time fluorescence quantitative PCR(qRT-PCR).Tobacco rattle virus(TRV)-induced gene silencing technology was used to silence NbMBF1c and determine its effect on TMV-GFP infection.The transient overexpression vectors of pART27-GFP-NbMBF1c and pART27-Myc-NbMBF1c were constructed and the fusion protein GFP-NbMBF1c was expressed in N.benthamiana leaf to observe its subcellular localization under confocal microscope.Myc-NbMBF1c was expressed and the changes of TMV-GFP infection after NbMBF1c expressed were observed.qRT-PCR was employed to detect the expression of hormone related genes after NbMBF1c silencing,and the mechanism of NbMBF1c affecting virus infection was analyzed.【Result】Nb MBF1c,which is 441 bp in full length,encodes 146 amino acids and contains a conserved domain HTH(helix-turn-helix).Phylogenetic analysis showed that NbMBF1c had the most closely relationship with Nicotiana tomentosiformis MBF1c(XP_009614458.1).NbMBF1c was localized in the cytoplasm and nucleus and had specific tissue expression,with the highest expression in roots,followed by stems,leaves and flowers.Silencing of NbMBF1c in N.benthamiana significantly reduced the tobacco plant height and promoted TMV-GFP infection,on the 5th day after TMV-GFP inoculation,the virus content of new leaf in the silencing treatment was higher than that in the control group.Transient overexpression of Myc-NbMBF1c suppressed TMV-GFP infection,after inoculation with TMV-GFP,virus infection was reduced.However,NbMBF1c did not interact with TMV components,but silencing NbMBF1c up-regulated the expression of abscisic acid(ABA)related gene NCED3,PYL1,ABAI,and SnRK2E and jasmonic acid(JA)synthesis pathway AOS1,while down-regulated the expression of JA signaling pathway receptor COI1.【Conclusion】NbMBF1c is mainly located in cytoplasm and nucleus,and acts as a positive regulator to inhibit TMV infection in N.benthamiana.NbMBF1c does not inhibit viral infection by directly interacting with TMV components,but by regulating phytohormone production and signal transduction.