| Lateral roots(LRs)play a key role in plant anchorage,acquisition of water and nutrient uptake etc,thereby is important for root architecture,nutrient utilization efficiency(NUE),stress resistance(such as resistance to root-lodging,drought stress,and nutrient deficiency),and yield.Brassica napus L.(B.napus)is the main source of edible vegetable oil in China.In general,the root system of B.napus is un-developed,about～20 cm in length in the soil,causing the low absorption and utilization rate of soil nutrients and poor stress resistance,that is not conducive to production and mechanized operations in crop production.MYB transcription factors(TFs)constitutes one of the largest transcription factor gene families in plants.Members of MYB family are widely involved in the regulation of plant growth and development,stress resistance and secondary metabolism,etc,and have essential regulatory roles in LRs development.Therefore,screening the candidate MYB gene(s)from B.napus genome,and exploring the mechanism of candidate(s)in regulating LRs development are important for theoretical research of B.napus root biology,high NUE and molecular breeding of stress resistance.In this study,based on bioinformatics analyses of MYB gene family in B.napus genome,a MYB gene,named BnaMYB311,that might regulate LRs development was screened and cloned.RNA-Seq and qRT-PCR methods were used to analyze the temporal and spatial expression profiles of BnaMYB311 in the whole growth stage of B.napus,and qRT-PCR was further applied to analyze its expression profiles in seedling roots under hormone induction.The precise expression pattern of BnaMYB311 was explored by clong its promoter sequence and then driving the GUS reporter gene expression in Arabidopsis.The molecular function of BnaMYB311 was verified by overexpression(in both Arabidopsis and B.napus)and gene editing methods.The downstream target genes and target sites were identified by CHIP-Seq,CHIP-qPCR,EMSA and Dual-luciferase analyses.The main results of this study are as follows:1.The expression characteristics of BnaMYB311 geneRNA-seq data including 110 samples and qRT-PCR analysis showed that BnaMYB311 was highly expressed in B.napus roots at seedling stage,while has very low expression levels in other tissues and organs investigated.Moreover,qRT-PCR analysis showed that the expression levels of BnaMYB311 were obviously up-regulated by exogenous auxin(IAA),abscisic acid(ABA),cytokinin(6-ba)and gibberellin(GA3)treatments,which were significantly increased after 12h treatment in all of these hormone inductions.This indicates that the biological function of BnaMYB311 might be induced by these hormones.2.The expression characteristics of BnaMYB311 gene promoterThe promoter sequence of BnaMYB311(1653bp upstream of the start codon)was amplified from B.napus genomic by PCR method,which was used to drive GUS reporter gene expression in Arabidopsis subsequently.The results showed that GUS gene was highly expressed in the cells at the initial stage of LRs development,and then decreased gradually from the formation of LR primordiums to the breakthrough of cortex,suggesting that BnaMYB311 might be involved in the germination and formation of LR primordiums.3.BnaMYB311 plays an important role in LRs developmentThe full-length cDNA sequence(1089bp cDNA)of BnaMYB311 was cloned,encoding 362 amino acids.Sequence analysis showed that BnaMYB311 protein contained two MYB repeat domains at the N-terminus and a transcriptional activation region at the C-terminus,suggesting that it_is a typical plant R2R3-MYB transcription factor.Phylogenetic tree analysis showed that BnaMYB311 gene was a member of S24 subfamily of the R2R3-MYB gene family.BnaMYB311 and Arabidopsis AtMYB93 proteins shared 89.86%sequence identity,with 100%and 85.20%sequence identity in their DNA binding domain and transcriptional activation domain,respectively.The transcriptional activation activity of BnaMYB311 protein was verified as well by yeast system.Subcellular localization analysis proved that the BnaMYB311 protein was located in cell nucleus.In B.napus,the LR density of the BnaMYB311 mutant(by CRISPR/Cas9)was significantly higher than that of the wild-type(WT),while the LR density of BnaMYB311 overexpression lines was significantly lower than WT.These resluts proved that BnaMYB311 negatively regulated the LR density in B.napus.4.Molecular mechanism of BnaMYB311 in regulating LRs developmentIn this study,transgenic Arabidopsis and B.napus materials of BnMYB311 fusion expression with FLAG-tag were created.ChIP-Seq and EMSA analyses showed that the downstream target genes of BnaMYB311 were four highly homologous genes BnaCYCD3;1s encoding the D type cell cyclin proteins.The binding site was’T[R]GTTA’ motif located at～500bp upstream promoter of the target genes.EMSA and Dual-luciferase analysis further confirmed the binding properties of BnaMYB311 protein to the target motif.Site-directed mutation analysis showed that the flanking sequence of the target motif affected the binding properties of BnaMYB311 protein,and the-1bp ’G’ base upstream of the target motif was the key site for BnaMYB311 binding to the target motif.Sequence analysis showed that the target motif of CYCD3;1 promoter in Brassica rape,Brassica oleracea,Arabidopsis and B.napus were highly conserved,suggesting that the regulation mechanism of BnaMYB311 on the target gene may be conserved in these species.At the same time,the LR density of Atcycd3;1 mutant in Arabidopsis was significantly higher than that of WT,further demonstrating that BnaMYB311 regulates downstream BnaCYCD3;1s genes to affect cell differentiation and division process,thus affecting the initiation and density of LRs.PlantCARE analyses revealed a ’GTCGTT’ sequence that was the binding motif of Arabdopsis AtARF7/19 was observed in the pormoter region of BnaMYB311.Then,we confirmed that the homologous protein of ARF7/19,BnaARF22/29,can bind to the’GTCGTT’ sequence by Y1H and Dual-luciferase analysis,explaining that BnaMYB311 involved in an auxin mediated gene noetwork by AtARF7/19 homologs in LT development. |
