Research On The Mechanisms Of IPT5b Gene Expression And Dwarfing Regulation In Root Of M9 Rootstock

The rootstock is important for fruit crop's cultivation,since yield and quality for fruit production have been great affected.Dwarfing rootstock is widely used in major countries for apple production all over the world.Unfortunately,the molecular mechanism of dwarfing in the apple rootstock is poorly understood.In this study,root zeatin contents were detected in different apple rootstocks.The results showed root zeatin contents in dwarfing rootstock were significantly lower than that in vigorous rootstock.Then,screening for gene members in CKs metabolic pathways provided a candidate being the difference of gene expression in apple rootstocks named IPT5b.Further studies found abundant with SNP and InDel differences in IPT5b gene promoter region between M9 rootstock(dwarfing)and Robusta rootstock(vigorous).With Arabidopsis protoplast transformation,vector construction for segmental deletion and site-directed mutagenesis of IPT5b gene promoter helped to determine Deletion variant as the core site for promoter activity,which located at 1172bp of ATG upstream sequences.Based on developed detection for Deletion variant,correlation with dwarfing was confirmed in germplasm resources for apple rootstock and Robusta×M9 hybrid population.Zeatin biosynthesis in root was certified to be affected with Deletion variant in IPT5b heterologous transformation,which implied zeatin levels in root to be the intermediate link between Deletion variant and dwarfing.In addition,bisulfite sequencing exhibited that two CpG islands(CpG2 and CpG4)in the IPT5b promoter region suffered higher methylation levels in M9 rootstock compared to Robusta rootstock.Furthermore,IPT5b expression increased when M9 rootstock was treated with 5-azaC,a methylation blocker,indicating that methylation levels influence IPT5b expression.It was reported,AUX/IAA family members regulated AtIPT5 for root development in Arabidopsis.AUX/IAA proteins would be suppressed to degrade in ubiquitination pathway when Domain ? mutant existed,thus inhibited AtIPT5 expression.CDS regions of AUX/IAA family members were cloned in M9 rootstock and Robusta rootstock,then deleted sequence for two amino acids was found in an AUX/IAA114 member of M9 rootstock.The preliminary mechanism of IPT5b regulation was discussed in this study.