Characterization Of BES1/BZR1 Transcription Factor Genes In Maize

Brassinosteroid(BR),a class of plant-specific steroid hormone,plays an important role in plant growth,development and stress response.BES1(BRI1-ethylmethylsulfone-suppressor 1)/BZR1(Brassinazole-resistant 1)is key transcription factor in BR signaling pathway and involved in regulating plant growth,development and stress response through regulating the expression of BR responsive genes.The large size of maize genome with many gene duplications and recombinant sequences increases its complexity,which may lead to the evolutional and functional diversification among the ZmBES1/BZR1 gene family members.In this study,we identified the ZmBES1/BZR1 gene family in maize genome by using bioinforatics and analyzed the gene structure,phylogenetic reslationship,chromosome localization,cis-acting elements and expression patterns of the ZmBES1/BZR1 genes.Subsquently,we detectted the expression of the ZmBES1/BZR1 genes under induction of absisic acid(ABA)and different wavelengths of light through real-time quantitative PCR.Finally,the ZmBES1/BZR1 genes were cloned and used for subcellular localization.It will provide new insights into the function study and involved sinaling pawthway of the ZmBES1/BZR1 genes.The results were showed as below:(1)Eleven members of the ZmBES1/BZR1 gene family were identified by genome-wide analysis.These genes are unevenly distributed on seven of the ten chromosomes in maize.There are three pairs of paralogs in this gene family.Every ZmBES1/BZR1 gene possessed different introns,including phase-0,-1 and-2 introns.The frequence of phase-0 intron is 84.8% and the highest in the three types,indicating the low diversity of gene structure.(2)The N terminus of the ZmBES1/BRZ1 members cotains one basic helix loop helix(bHLH)domain,which is specific and highly conserved for BES1/BRZ1 protein.But,besides bHLH domain,ZmBES1/BRZ1-4 and-5 contain one BAM domain in their C terminus.According to the similarity of amino acid sequences and phylogenetic analysis,the ZmBES1/BZR1 members were divided into two subfamily,ZmBES1/BRZ1-4 and-5 were located on same sub-clade and other nine ZmBES1/BZR1 members were divided into another subclass.(3)The results of ZmBES1/BRZ1 promoter analysis showed that there were many light response and stress response elements in the upstream of the ZmBES1/BZR1 genes.We analyzed the expression pattern of ZmBES1/BZR1 genes in different tissues and development stages using published transcriptome data.We found that the expression of ZmBES1/BRZ1-1 was consistently high in every sample,ZmBES1/BRZ1-5,-7 and-10 expressed highly in most tissues and stages,while ZmBES1/BRZ1-2,-3,-4 and-9 only expressed in a few organs and stages.These results suggested that the expression of the ZmBES1/BZR1 genes might be regulated by growth or environmental factors,and the ZmBES1/BZR1 genes functioned in different stages of growth and development.(4)The results of real-time quantitative PCR(qRT-PCR)showed that the expression of ZmBES1/BRZ1-1,-4 and-5 was significantly up-regulated and the expression of ZmBES1/BRZ1-7,-8,-10 and-11 was significantly down-regulated in the leaf under ABA induction,but the expression of ZmBES1/BRZ1-4 and-5 was significantly down-regulated,the expression of ZmBES1/BRZ1-1,-3,-10 and-11 was significantly up-regulated in root under ABA induction.Under dark treatment,except ZmBES1/BZR1-9 and-10,the expression of other genes was up-regulated in leaf and roots.Under red light treatment,the expression of ZmBES/BRZ1-1,-4,-7 in leaf and the expression of ZmBES1/BZR1-1,-11 in the roots was significantly up-regualted,while the expression of ZmBES1/BZR1-2,-5,-8 in leaf and root and the expression of ZmBES1/BZR1-3,-11 in leaf was significantly down-regulated.Under far red light treatment,except for ZmBES1/BZR1-9 and-10,the expression of other genes in leaf and root was significantly up-regulated.In addition,the repression of ZmBES1/BZR1-6 was not detected in all samples.(5)The result of PCR cloning showed that ten members of ZmBES1/BZR1 were successfully cloned and there was no any amplication of the ZmBES1/BZR1-6 gene.The open reading frame(ORF)of the ZmBES1/BZR1 amplified by PCR was used to generate the expression vector of ZmBES1/BZR1 fusion eGFP(35S-ZmBES1/BZR1-eGFP),and transformed into onion epidermis cells and maize protoplast for transient expression.The results exhibited that ZmBES1/BZR1-2 and 7 were localized on the chloroplast and other memberslocalized in nucleus,which wass consistent with the bioinformatics predicted results.The above results showed the diversity of evolution,gene structure,expression pattern and subcellular localization of ZmBES1/BZR1 genes,and ZmBES1/BZR1-6 may be a non-functional pseudogene.The study illustrated that the function difference among ZmBES1/BZR1 genes and contributed for further study function of the ZmBES1/BZR1 genes.