甘蔗NAD(P)H脱氢酶复合体O亚基基因克隆及其与甘蔗花叶病毒VPg互作

Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of Sugarcane mosaic virus

NAD(P)H脱氢酶(NDH)复合体介导循环电子传递,对于维持叶绿体高效的光合作用具有重要作用。甘蔗(Saccharum spp. hybrid)中NDH复合体应答及参与甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)的侵染尚未见报道。本研究克隆了甘蔗NDH复合体的O亚基,命名为ScNdhO,其开放读码框(open reading frame,ORF)长度为471 bp,编码长度为156 aa的蛋白。生物信息学分析表明,ScNdhO为稳定的亲水性蛋白,不存在信号肽,无跨膜结构域;蛋白二级结构元件多为无规则卷曲,具有典型的NDH复合体O亚基结构域;系统进化树分析表明,该蛋白属于NDH复合体O亚基蛋白家族。实时荧光定量 PCR分析发现,ScNdhO基因的表达具有明显的组织特异性,在成熟甘蔗叶片中的表达量最高,在茎中的表达量最低,在根中几乎不表达;ScNdhO基因在SCMV侵染早期上调表达,后期下调表达。亚细胞定位分析表明,ScNdhO定位于叶绿体。酵母双杂交(yeast two hybrid,Y2H)和双分子荧光互补(bimolecular fluorescence complementation,BiFC)实验表明,ScNdhO与SCMV-VPg互作。推测ScNdhO被SCMV选择性利用,可能参与SCMV基因组复制及花叶病症状的产生。

NAD(P)H dehydrogenase (NDH) complex mediates cyclic electron transports,playing key role in efficient photosynthesis in chloroplast. The involvement of NDH complex in Sugarcane mosaic virus (SCMV) infection of sugarcane (Saccharum spp. hybrid) has not been reported. In this study,we isolated the coding sequence of the subunit of the NAD(P)H dehydrogenase complex from sugarcane and designated it as ScNdhO. The open reading frame (ORF) of ScNdhO is 471 bp and encodes a 156 aa length protein. Bioinformatics analysis showed that ScNdhO is a stable hydrophilic protein with no signal peptide and transmembrane domain. The secondary structure of ScNdhO is composed of mostly random coilα-helices,with a typical domain of NDH complex O subunit. Phylogenetic tree analysis showed that ScNdhO belongs to the NDHO supperfamily. Real-time quantitative PCR analysis showed that ScNdhO gene was tissue specific in sugarcane,with the lowest expression level in roots or stem,and the highest in leaf. The expression of ScNdhO was upregulated in the early stage of SCMV infection,but downregulated with time going. Subcellular location assays showed that ScNdhO was located in chloroplast. ScNdhO interacted with the VPg from SCMV as demonstrated by yeast two hybrid and bimolecular fluorescence complementation assays. We proposed that ScNdhO should be selectively employed by SCMV and involved in the mosaic symptom.