Homologous Evolutionary Analysis And Functional Studies Of The BZIP Genes In Brassica Oleracea, Brassica Rapa And Arabidopsis Thaliana

bZIP(Basic Leucine Zipper)transcription factors are involved in regulating the growth and development of plants and plays an important role in coping with environmental stress.ABI5(ABA Insensitive 5)and ABFs(Abcisic Acid Responsive Element Binding Factors)are transcription factors of the subfamily ABI5(ABI5 or ABFs)bZIP,have crucial roles to activate plant ABA(Abcisic Acid)signalling.They are undergone a variety of protein post-translational modifications,including phosphorylation and SUMOylation.Previously,the genome-wide annotation of the bZIP genes in Brassica rapa has been completed.However,the genome-wide annotation and biological function of the BobZIP transcription factor genes in Brassica oleracea remains elusive.In this study,genome-wide annotation of the bZIP family in Brassica oleracea and Brassica rapa was performed,and the structure and function of bZIP transcription factors of Brassica oleracea and Brassica rapa were studied.We obtained following results:(1)Genome-wide annotation analysis showed that 131 bZIP homologous genes were found in Brassica oleracea and 136 were found in Brassica rapa.The phylogentic analysis showed that all the bZIP subfamilies(A ~ S subfamily)were found in Brassica oleracea and Brassica rapa.The A subfamily in Brassica oleracea includes 22 BobZIP transcription factors.There are 23 BrbZIPs were categorized into the A subfamily in Brassica rapa.(2)The gene and protein structure of the A subfamily bZIP transcription factor of Brassica oleracea,Brassica rapa and Arabidopsis thaliana were highly conserved.(3)The expression levels of A subfamily BobZIPs and BrbZIPs were determined in different organs,and most of the genes were highly expressed in callus,root and silique.Further analysis showed ABA could induce the expression of most members of A subfamily BrbZIPs genes via qRT-PCR.(4)Sequence analysis of ABI5 homologous proteins in Brassica oleracea and Brassica rapa showed that they all contained the conserved domains as AtABI5 protein,including four conserved sites and NLS(Bipartite Nuclear Localization Signal),bZIP domain,and the conserved SUMOylation site.(5)The CRISPR-Cas9 vector attempt to knonk-out ABFs homologous gene in Brassica oleracea and Brassica rapa was successfully constructed,and the regenerated seedlings of Brassica napus were successfully obtained.Taken together,our results revealed that the bZIP homologous genes in Brassica oleracea and Brassica rapa was highly conserved.Moreover,the ABF-like bZIP transcription factors which may be involved in the regulation of plant ABA or drought responses were functionally analyzed via CRISPR-Cas9 mediated gene editing,which will help to determine their biological functions in response to ABA or drought in Brassica oleracea,Brassica rapa and Brassica napus.