甘蔗ScCRT1基因克隆及其应答SCMV侵染分子机制的研究

钙网蛋白(calreticulin,CRT)在真核生物中广泛表达,是重要的分子伴侣和钙离子结合蛋白,参与调控Ca2+稳态、钙依赖信号、内质网质量控制、植物生长发育、免疫反应和逆境应答等多种生物学过程。甘蔗(Saccharum spp.hybrid)中CRT应答甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)侵染尚未见报道。本研究从热带种Badila(S.officinarum)中克隆了1个CRT1/CRT2亚型的CRT编码基因,命名为ScCRT1。该基因开放读码框(open reading frame,ORF)长度为1281bp,编码长度为426aa的蛋白。生物信息学分析表明,ScCRT1具有典型的CRT蛋白结构域,为稳定的亲水性蛋白,其N端有一个信号肽,具有典型的跨膜结构域,C端有典型的内质网定位信号;二级结构多为无规则卷曲;系统进化树分析表明,该蛋白是典型的CRT蛋白,在单子叶和双子叶植物中具有明显的分化。亚细胞定位表明ScCRT1定位于内质网。实时荧光定量PCR分析发现,ScCRT1基因在甘蔗各组织中都有表达,在第8节间中的表达量最低,在心叶中的表达量较高;该基因在SCMV侵染早期表达量上调,后期下调表达。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,ScCRT1与SCMV-6K2蛋白互作。推测SCMV-6K2通过与ScCRT1互作调控钙离子稳态进而便于SCMV侵染。

Cloning of sugarcane ScCRT1 gene and its response to SCMV infection

Calreticulin(CRT)is widely expressed in eukaryotes.As a molecular chaperone and a Ca2+binding protein,CRT is involved in many biological pathways such as the regulation of calcium homeostasis,calcium-dependent signaling,endoplasmic reticulum quality control,plant growth and development,immunity and response to stress.However,the response of CRT of sugarcane(Saccharum spp.hybrid)challenged by Sugarcane mosaic virus(SCMV)has not been reported.In this study,a CRT gene was cloned from the noble cane cultivar Badila(S.officinarum)and designed as ScCRT1.ScCRT1 had an open reading frame(ORF)length of 1281 bp and encoded 426 amino acids.Bioinformatics analysis showed that ScCRT1 was a stable hydrophilic protein and possesses a signal peptide at the N-terminal,a typical transmembrane domain,and a typical endoplasmic reticulum location signal at the C-terminal.The secondary structure of ScCRT1 was composed of mostly random coils.Phylogenetic tree analysis indicated that ScCRT1 belonged to the CRT1/CRT2 subtype and was divergent between monocotyledons and dicotyledons.Subcellular location assays showed that ScCRT1 was mainly located in the endoplasmic reticulum.Real-time quantitative PCR analysis showed that ScCRT1 gene was extensively expressed in different tissues of sugarcane,with the highest expression in leaf roll and the lowest expression in the 8th internode.ScCRT1 gene was up regulated in the early stage of SCMV infection,but down regulated with time going.ScCRT1 interacted with the 6K2 from SCMV as confirmed by yeast two hybrid and bimolecular fluorescence complementation assays.Based on these foundlings,we speculated SCMV interfered the calcium homeostasis by the interaction of 6K2 with ScCRT1,thereby facilitating viral infection of sugarcane.