Functional Characterization And Mechanism Analysis Of PbrMYB21 Gene From Pyrus Betulaefolia

Pyrus species originated in the mountainous regions of western and southwestern China.Since the reform and opening up,with the rapid development of pear industry,China has been the world's largest pear-producing country.However,the growth and development of pears are vulnerable to abiotic stresses,especially drought stress.How to develop new varieties of drought resistance have been becoming troublesome problems to be solved by breeders.The rapid development of genetic engineering has provided new ideas for solving this problem.The premise of genetic engineering is the selection of resistance genes.The study show that MYB comprises a large family of transcription factors that play significant roles in plant development and stress response in plants.However,knowledge concerning the functions of MYBs and the target genes remains poorly understood.We previously obtained a dehydration-induced MYB TF(Pbr028812.1)from a transcriptome of Pyrus betulaefolia.Homology search showed that the gene shared the highest identity with MdMYB21,so it was designated as PbrMYB21(Pyrus betulaefolia MYB21).PbrMYB21 was reported rarely in the literature.In this study,a new stress-responsive gene PbrMYB21 was cloned from a Pyrus betulaefolia accession,we verified its anti-reversal function and found its drought resistance mechanism.The main results were as follows:1?The MYB gene,designated as PbrMYB21,belongs to the R2R3-type.Sequence analysis demonstrated that it was a full-length sequence with a complete open reading frame(ORF),which encodes a protein of 296 amino acid residues with a calculated molecular mass of 33.2 kDa and an isoelectric point of 5.98,and shares high degree of sequence similarity to MdMYB21.PbrMYB21 was localized to nucleus and had transactivation activity.The transcript levels of PbrMYB21 were up-regulated under various abiotic stresses,especially dehydration.2?Overexpression vector and gene silencing vector of PbrMYB21 were constructed.The overexpression vector was used to transform tobacco(Nicotiana tabacum),and VIGS was performed by infiltration into the leaves of 30-day-old Pyrus betulaefolia.Overexpression of PbrMYB21 in tobacco enhanced tolerance to dehydration and drought stresses,whereas down-regulation of PbrMYB21 in Pyrus betulaefolia by virus-induced gene silencing(VIGS)resulted in elevated drought sensitivity.Compared with wild type,transgenetic tobacco had higher survival rate,lower conductivity content,lower malondialdehyde content,higher chlorophyll content,lower accumulation of H2O2 and O2-after dehydration and drought treatment.Transgenetic tobacco exhibited higher antioxidase activitives(SOD,CAT and POD),higher expression of drought-responsive genes especially level of ADC(arginine decarboxylase)and accumulated larger amount of polyamine before and after drought treatment.On the contrary,the gene was silenced in the pear by VIGS technology,which decreased resistance under drought conditions.After dehydration and drought treatment,compared with wild type,silencing lines had lower survival rate,higher conductivity content,higher MDA content,higher chlorophyll content,higher H2O2 and O2-accumulation.Silencing plants had lower antioxidant enzyme activitives(SOD,CAT,and POD),lower accumulation of drought-responsive genes especially level of ADC(arginine decarboxylase)and lower polyamine level before and after drought.3?The elevation or decrease of ADC genes in PbrMYB21-overexpressing or silencing plants,respectively,implied that ADC gene might be one of the potential target genes that are regulated by PbrMYB21.To determine this hypothesis,we used the yeast one-hybrid assay.The results indicate that PbrMYB21 interacted with the promoter of PbrADC in yeast.To confirm the results of the yeast one-hybrid assay,transient expression assay was further performed to confirm the interaction between PbrMYB21 and the promoter of PbrADC.The promoter activities,expressed as LUC/REN ratio,of the PbrMYB21 were significantly higher than those in the WT,and the results were consistent with the yeast one-hybrid assay results.To further determine whether PbrMYB21 specifically binds to the MYB recognition site in the PbrADC promoter,electrophoretic mobility shift assay(EMSA)was performed usingprokaryon-expressed and purified PbrMYB21-His fusion proteins.The results showed that PbrMYB21 recognized and bound specifically to the sequences of PbrADC promoter.We used the GAL4/UAS basic system to study transcriptional activation or protein expression.Histochemical staining showed that GUS expression was prominently activated in the N.benthamiana leaves compared with the leaves transformed with the empty vector control.These results suggest that PbrMYB21 might act as a transcriptional activator of PbrADC.4?In summary,a new stress-responsive gene PbrMYB21 was cloned from a Pyrus betulaefolia accession.The overexpression vector was used to transform tobacco(Nicotiana tabacum),and VIGS was performed by infiltration into the leaves of 30-day-old Pyrus betulaefolia seedling.Take together,physiological and molecular tests results demonstrated that PbrMYB21 plays a positive role in drought tolerance,which may be,at least in part,due to the modulation of polyamine synthesis by regulating the ADC expression.