甘蔗VAMP相关蛋白ScPVA12与甘蔗花叶病毒P3N-PIPO的互作研究

Interaction of sugarcane VAMP associated protein ScPVA12 with SCMV P3N-PIPO

植物囊泡膜蛋白相关蛋白PVA12 (plant vesicle-associated membrane protein (VAMP)-associated proteins homolog12)属于VAP27家族蛋白,在细胞中介导内质网囊泡运输以及膜融合。甘蔗(Saccharumspp.hybrid)PVA12应答甘蔗花叶病毒(Sugarcane mosaic virus, SCMV)侵染尚未见报道。本研究从栽培种新台糖22号(ROC22)中克隆了PVA12基因,命名为ScPVA12。该基因开放读码框(open reading frame, ORF)的长度为735 bp,其编码长度为244 aa的蛋白。生物信息学分析表明,ScPVA12是一种不稳定的亲水性脂溶蛋白,C端具有跨膜结构域;二级结构中无规则卷曲占比最高;进化树分析表明,该蛋白在单子叶和双子叶植物中存在明显分化。酵母双杂交(yeast two-hybrid, Y2H)和双分子荧光互补(bimolecular fluorescence complementation, BiFC)试验表明, ScPVA12与SCMV-P3N-PIPO蛋白互作。亚细胞定位试验表明ScPVA12定位于内质网。共定位试验表明ScPVA12与SCMV-P3N-PIPO共定位于内质网。实时荧光定量PCR分析发现,ScPVA12基因在甘蔗各组织中均有表达,在第8节间中的表达量最低,在正七叶中的表达量最高;SCMV侵染对ScPVA12基因表达影响显著,在SCMV胁迫下ScPVA12基因先下调表达,后期恢复正常水平。

PVA12(plant vesicle-associated membrane protein(VAMP)-associated proteins homolog 12)is a member of the VAP27 family of proteins that mediate endoplasmic reticulum(ER)vesicle transport and membrane fusion in cells.Sugarcane(Saccharum spp.hybrid)PVA12 responding to Sugarcane mosaic virus(SCMV)infection has not been reported.In the present study,the coding gene of PVA12 was cloned from sugarcane cultivar ROC22 and designated as ScPVA12.The open reading frame(ORF)of ScPVA12 was 735 bp in length,which encoded a protein with 244 amino acids.Bioinformatics analysis showed that ScPVA12 was an unstable hydrophilic liposoluble protein with a transmembrane at the C-terminal domain.The ratio of the random coil ranked the highest in the secondary structure.Phylogenetic tree analysis revealed that the ScPVA12 was differentiated in monocotyledons and dicotyledons.Yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)assays showed that ScPVA12 interacted with SCMV-P3N-PIPO.Subcellular localization experiments indicated that ScPVA12 was localized in the ER.Co-localization experiments demonstrated that ScPVA12 and SCMV-P3N-PIPO co-localized in the ER.Real-time quantitative PCR analysis showed that ScPVA12 gene was expressed in all sugarcane tissues,with the lowest expression level in the eighth internode and the highest expression level in the 7th leaves.The relative expression level of ScPVA12 gene was significantly affected under the stress of SCMV.ScPVA12 was down-regulated upon SCMV infection and then recovered to the regular level compared with the control.