| Brassinosteroid(BR)is a steroid hormone peculiar to plants,which is commonly found in various tissues of plants.BR is an endogenous plant hormone discovered after the five hormones.Its physiological activity is much higher than other hormones,and the lower concentration can regulate all aspects of plant growth and development.Since the discovery of BR,its signaling pathway and transduction pathway have been deeply studied.BR was recognized and received by its receptor BRI1(Brassinosteroid Insensitive 1)and was transferred into the cells through a cascade reaction,which stimulated the co-receptor BAK1(Bri1-association receptor kinase1)to form dimer.The phosphorylation or dephosphorylation of BES1/BZR1 was regulated through a cascade reaction.Phosphorylated BES1/BZR1 enters the cytoplasm and is degraded by proteasome,while dephosphorylated BES1/BZR1 enters the nucleus to regulate the expression of downstream target genes,thus regulating plant growth and development.Studies have shown that BES1/BZR1 regulates the expression of these genes directly or by binding to E-box or BRRE elements in the promoter region of downstream related genes after forming complexes with other transcription factors.In our preliminary study,we identified 10 maize Zm BES1/BZR1 s members.Zm BES1/BZR1-5 differs from other Zm BES1/BZR1-5 members in that in addition to a highly conserved BES1 domain at the N-terminal,there is also a β-amylase domain(BAM)at the C-terminal,suggesting that the function and mechanism of Zm BES1/BZR1-5 members may be different from other Zm BES1/BZR1-5 members.Therefore,bioinformatics analysis,subcellular localization,protein structural characteristics,Real time quantative PCR(RT-q PCR),Arabidopsis mutant complementation,overexpression of rice and maize,and maize mutant analysis were used to analyze its function.Then,Through Yeast library screening,Yeast hybrid(Y2H),Bimolecular fluorescence complementation Bi FC,Chromatin immunoprecipitation sequencing(Chchip-SEQ),Yeast one hybrid,Y1H)and Luciferase assay were used to identify the upstream and downstream components of Zm BES1/BZR1 s in BR signaling.The main results were as follows:1.The coding sequence of Zm BES1/BZR1-5 gene is 1956 bp,encoding 651 amino acids,containing 10 exons and 9 introns.The promoter region contains multiple cis-acting elements.The n-terminal of Zm BES1/BZR1-5 protein has a typical b HLH domain(BES1 domain)and is highly conserved with BES1/BZR1 of Arabidopsis thaliana and rice.Meanwhile,the c-terminus contains a β-amylase(BAM)domain of glycoside hydrolase family 14,which is homologous to BAM7,BAM8 and BAM in Arabidopsis thaliana.2.Zm BES1/BZR1-5 was in the nucleus of onion epidermis,tobacco leaves and maize protoplasts,while b HLH domain was located in the nucleus and cytoplasm,and BAM domain was located in the cytoplasm.The full-length Zm BES1/BZR1-5,b HLH and BAM domains have no transcriptional activation activity,but Zm BES1/BZR1-5 can form a polymer by itself,and BAM is a necessary domain for the formation of the polymer.3.Transcriptomic data from Maize GDB database showed that Zm BES1/BZR1-5 was expressed at different levels in different tissues and developmental stages of maize.Meanwhile,Zm BES1/BZR1-5 gene expression was induced by osmosis,salt stress and ABA treatment.Under 100 m M Na Cl treatment,Zm BES1/BZR1-5 was significantly up-regulated in leaves at9 h,but not at other times.In roots,there was no significant difference except 9h,and the expression was downregulated at all other time points.However,under PEG6000 condition,Zm BES1/ bzr1-5 was down-regulated in general,with significant difference in leaves at 1,3,6and 9h,and significantly down-regulated in roots at all time points.Under Abscisic acid(ABA)treatment,Zm BES1/BZR1-5 was significantly up-regulated in leaves and down-regulated in roots.4.Arabidopsis thaliana mutant overexpressing Zm BES1/BZR1-5 is drought and salt tolerant and responds to ABA.There was no significant difference in the growth of untransformed mutant and transgenic lines,while the growth of transgenic lines was significantly better than that of untransformed mutant at Na Cl,mannitol,and ABA.The root length and fresh weight of transgenic lines were significantly higher than those of control.These results suggest that Zm BES1/BZR1-5 can enhance drought and salt resistance and reduce ABA sensitivity of Arabidopsis thaliana.The content of malondialdehyde(MDA)and relative electrical conductivity(REL)were significantly lower than those of the control under salt and drought stress,which indicated that the damage degree of mesangium was less.A total of 84 differentially expressed genes(DEGs)were obtained by transcriptional analysis of transgenic Arabidopsis thaliana.KEGG analysis showed that all differentially expressed genes were involved in secondary metabolism and biosynthesis,among which 30 genes directly responded to stress.These gene promoters contained 2-15 E-boxes,and a few contained BRRE.Y1 H confirmed that Zm BES1/BZR1-5 could directly bind to e-box,and directly bind to the promoter of AT3G05880(RCI2A),a gene that simultaneously responds to drought,salt stress and ABA.5.In addition to regulating abiotic stress,Zm BES1/BZR1-5 can also regulate grain size.Four Single nucleotide polymorphisms(SNPs)were found to be associated with 100-grain weight and three SNPS were found to be associated with grain width,among which one SNPS were located in the promoter region of Zm BES1/BZR1-5.In arabidopsis thaliana,rice and maize overexpression,grain length,grain width and 1000-grain weight of overexpression lines were significantly larger than those of control.The two types of maize mutants had significantly smaller grains than the wild type.These results indicated that Zm BES1/BZR1-5 could increase seed length,width and weight.At the same time,arabidopsis complementary and overexpressed rice also showed an early flowering phenotype,which made the plant advance from vegetative growth to reproductive growth.6.Zm CKIIβ4(Zm00001d03500)and Zm Fdx2(Zm00001d02155)are the upstream target protein of Zm BES1/BZR1-5.After Y2 H and Bi FC validation,Zm CKIIβ4 could only interact with Zm BES1/BZR1-5 full-length protein,while Zm Fdx2 could only interact with Zm BES1/BZR1-5 full-length protein and its b HLH domain,but not with the BAM domain alone.Meanwhile,Zm CKIIβ4 localized in the nucleus and cytoplasm,while Zm Fdx2 localized only in the cytoplasm.7.The AP2/EREBP genes Zm00001d010676 and Zm00001d032077 were the target gene of Zm BES1/BZR1-5.The yeast one hybridization experiment proved that Zm BES1/BZR1-5could bind the promoters of these two genes.Luciferase assay further confirmed that Zm BES1/BZR1-5 could directly bind the promoters of the two genes and inhibit their expression,thus regulating grain size... |
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