| Brassica napus L.(B.napus)is one of the most important oil crops in China.Nowadays,the yield of B.napus is low in China,whereas increasing the yield per unit is an effective way to promote B.napus production.Root hair(RH)is an important agronomic trait that plays a key role in plant absorbing of water and mineral nutrients from the soil.As one of the largest TF families in plant,MYB transcription factors(TFs)have been proved to play crucial roles in the regulation of plant root development.Therefore,exploring MYB genes that are involved in regulating B.napus root hair development,and clarifying the molecular mechanisms of the candidates are theoretical and practical significance for promoting nutrient utilization efficient and high-yield molecular breeding of B.napus.Based on our previous bioinformatics analysis,the CDS and up-stream 1500 bp sequences of a MYB TF gene,BnaMYB019 that might regulate B.napus root hair development was identified and cloned.Then,the spatiotemporal expression profile,hormone-induced expression profile,and nutrient stress expression profile of BnaMYB019 gene were analyzed by RNA-Seq and q RT-PCR respectively.The precise expression position of BnaMYB019 was further clarified by fusing expression of its promoter sequence and the GUS and green fluorescent protein(GFP)reporter genes,respectively.Meanwhile,to explore the expression localization of BnaMYB019 protein,subcellular localization analysis was carried out by using BnaMYB019 gene fused with GFP reporter gene.Through the overexpression and functional complementation experiments in Arabidopsis and the overexpression experiment in B.napus,we proved that BnaMYB019 had a negative regulatory role in root hair development.Moreover,we screened the downstream target genes and target sites of BnaMYB019 by Dual luciferase reporter assay system and Electrophoretic mobility shift assay(EMSA)respectively.Furtherrmore,the interacting proteins of BnaMYB019 and its interaction mechanisms were illustrated through Yeast Two-Hybrid assay(Y2H)and Luciferase complementary imaging assay(LCI)methods.This study preliminarily elucidated the regulating mechanism of the candidate BnaMYB019 gene in root hair development.The main results obtained are as follows:1.Screening,cloning and expression analysis of BnaMYB019Based on the phylogenetic analyses,expression profile analyses and sequence alignment analyses,we identified a gene,BnaMYB019,that might be involved in root development of B.napus.Hence,we cloned the CDS sequence of BnaMYB019 from B.napus cultivar,Zhongshuang 11(ZS11).Its ORF full-length was 597 bp,encoding 198 amino acids.bioinformatics analysis showed that the N-terminal of BnaMYB019 protein contained two conserved MYB repeats and the C-terminal had a transcriptional activation region,indicating that it belonged to typical R2R3-MYB TF.BnaMYB019 is the most homologous to Arabidopsis At WER gene,with 93.6% similariy of their full-length protein sequences and 100% similarity of the MYB domains.This suggested that BnaMYB019 and At WER may have similar functions in regulating root hair development.RNA-Seq analysis showed that BnaMYB019 gene was preferentially expressed in roots and hypocotyl of B.napus at different developmental stages.RNA-Seq and q RT-PCR analysis showed that the expression of BnaMYB019 gene was obviously up-regulated under phosphorus and nitrogen deficiency conditions,especially under continuous phosphorus deficiency stress.2.Cloning and analysis of BnaMYB019 gene promoterWe cloned the 1500 bp upstream promoter sequence of BnaMYB019 gene(BnaMYB019p)from the genomic of ZS11.Subsequently,the BnaMYB019 promoter was used to drive GUS reporter gene expression(BnaMYB019p::GUS)in wild type Arabidopsis(Col 0),and the T3 homozygous generation of the transgenetic lines were obtained.And the GUS staining analyses revealed that BnaMYB019 p drived the specific expression of GUS gene in hypocotyl and epidermal cells of root apical.Meanwhile,the promoter was used to drive GFP reporter gene expression(BnaMYB019p::GFP)in Arabidopsis as well,and the results showed that the BnaMYB019-GFP infusion protein was specificly expressed in the cells of root apical meristem and elongation region by Laser confocal microscopy analysis.These results demonstrated the precise expression characteristics of BnaMYB019 p in root apex.In addition,the expression of BnaMYB019 p was up-reguated by low-nitrogen,low-phosphorus and low-potassium stress treatments,and phosphorus deficiency could continuously induce the up-regulation of BnaMYB019 p,based on the T3 transgenetic materials.Meanwhile,the expression of BnaMYB019 p was also induced by a variety of exogenous hormones(such as ABA,IAA,etc.),especially was negatively regulated by gibberellin(GA3)and salicylic acid(SA).3.BnaMYB019 gene negatively regulates root hair development in Brassica napusFirstly,the GFP reporter gene was fused in-frame with the BnaMYB019-coding region,and this fusion was expressed under the control of the Ca MV35 S promoter(35Sp::BnaMYB019-GFP)by transient expression in tobacco.The result showed that the BnaMYB019 protein was expressed in the nucleus,which confirmed the typical characteristic of TFs.Secondly,we performed the functional complementation and overexpression experiments of BnaMYB019 gene in Arabidopsis.The phenotypic analysis showed that the root hair density of the wer mutants was significantly increased,that of functionally complementary lines of BnaMYB019 recovered to the wild-type level,while the root hair density of its overexpression line significantly reduced,based on the T3 generation lines.These results indicated that BnaMYB019 gene had a negative regulatory role in root hair development in Arabidopsis.Similarly,based on the transgenic lines of BnaMYB019 over-expression in B.napus ecotype westar,the phenotypic analysis showed that the root hair density of transgenic B.napus was obviously lower than that of WT,which further comfirmed the negative role of BnaMYB019 gene in root hair development.4.Molecular mechanism of BnaMYB019 gene regulating root hair developmentPrevious studies have shown that the Arabidopsis WER gene regulated root hair development by regulating the downstream target gene At GL2.Therefore,a homologous gene BnaGL2 of At GL2 in ZS11 genome was identified by bioinformatics methods.Plant TFDB online software analysis showed that four potential MYB binding sites(P1-P4)were present in the promoter region of BnaGL2 gene.Then,the BnaMYB019 was further expressed in prokaryotic cells,and its recombinant protein was obtained after purification.EMSA analyses proved that BnaMYB019 protein could bind to three of the four binding sites(P1-P3).This result was further confirmed by Dual luciferase analysis system.Thus,it was suggesgted that the regulatory mechanism of WER and GL2 homologs was conserved in B.napus and Arabidopsis.Moreover,previous studies have shown that the regulatory mechanism of the MYB-b HLH-WD40 ternary complex(MBW)on root hair development is conserved in many species.Hence,we screened the homologs of this complex in ZS11 genome by bioinformatics methods.Subsequently,the Y2 H and LCI experiments proved that BnaGL3(b HLH TF)can interact with BnaMYB019(MYB TF)and BnaTTG1(WD40 protein),respectively,which suggested that the MBW protein complex existed in B.napus as well.In summary,BnaMYB019 protein negatively regulates root hair development by forming BnaMYB019-BnaGL3-BnaTTG1 complex to activate the expression of downstream BnaGL2 gene. |
