Genome-wide Isolation And Expression Analysis Of Stress-responsive ERF Genes In Citrus Sinensis

It has been reported that AP2/EREBP genes are involved in the plant development and abiotic stresses and exhibited great potential in molecular breeding of resistance in Citrus. However, the researches on isolation and identification of stress-responsive AP2/EREBP genes in sweet orange(Citrus sinensis) are limited. Therefore, in this study some stress-responsive AP2/EREBP genes were isolated, which could enrich the gene resources in genetic improvement of sweet orange.In this study, 126 AP2/EREBP gene sequences were identified from the sweet orange genome database. Based on their structures and sequences, these AP2/EREBP genes can be divided into 5 subfamilies, including AP2 subfamily(18 members), RAV subfamily(4 members), ERF subfamily(60 members), DREB subfamily(38 members) and soloist(6 members). These ERF and DREB subfamily were further divided into 10 groups; MEME motif analysis predicted the defining AP2/ERF domain and other conserved domains.We isolated 19 and 15 ERF genes that are responsive to cold stress in sweet orange and trifoliate orange(Poncirus trifoliata), respectively. In addition, CsERF#10, CsERF#36, PtERF#10 and PtERF#36 which were induced by various stresses were cloned. Their gene sequences between sweet orange and trifoliate orange do not show much difference, and their amino acid sequences on the AP2/ERF conservative area are basically identical. Subcellular localization assay confirmed that they are located in the nucleus.In this research, we overexpressed CsERF#36 and PtERF#36 gene in tobacco(Nicotiana nudicaulis). According to the gene expression analysis, two CsERF#36 transgenic lines(#3 and #32) and two PtERF#36 transgenic lines(#37 and #38) of T1 generation were chose for cold treatment(2 ℃ for 36 h). Transgenic plants were shown to be more tolerant to cold stress than wild type(WT), as indicated by better morphological phenotypes of leaf or whole plant and less ROS levels(shown by histochemical staining with DAB and NBT). Taken together, our data suggest that the overexpression of CsERF#36 and PtERF#36 may confer enhanced tolerance under cold stress.