Genome-Wide Analysis Of U-box E3 Ubiquitin Ligases Gene Family In Chinese White Pear (Pyrus Bretschneideri) And Identification Of PbPUB18 Drought-Resistance

The cultivated area and yield of pear（Pyrus）in China are the third largest fruit in China compared with apples and citrus.Pear is widely distributed in China,however,pear is subject to geographical and climatic conditions and other environmental factors due to extensive cultivation.The production is often harmed by drought,waterlogging,cold,saline-alkali soil and other abiotic stress.Drought stress seriously affected the yield and quality of pear.In order to adapt to the adverse environmental conditions,plants have evolved complex and effective mechanisms to defend adverse environmental conditions.The identification of genes related to abiotic stress in pear and the cultivation of new varieties with strong drought resistance and good characters have became the important work for the development of pear industry.We identified the PUB gene related to abiotic stress.Ubiquitin/26S proteasome system（UPS）is an extremely large and complex proteolytic mechanism,which widely exists in eukaryotic cells.A large number of studies have shown that UPS is involved in the regulation of a variety of processes,plant growth and development,signal transduction,immune response,embryonic development,biostress and abiotic stress,and its pathway is highly conservative.In this pathway,ubiquitin ligases bind directly or indirectly to substrate proteins and play an important role in ubiquitin.However,there are few studies about ubiquitin ligases in pears.In this study,we carried out bioinformatics analysis of U-box E3 ubiquitin ligase gene family in pear Furthermore,a new stress response gene PbPUB18,was cloned from pear（Pyrus betulaefolia）.The expression level of PbPUB18 under different stress treatments and subcellular location were analyzed.Heterologous over-expression of PbPUB18 in Arabidopsis thaliana and virus-induced gene silencing（VIGS）PbPUB18 gene in pear were performed to study its function under drought stress.The main results are as follows:1.In the present study,we identified 62 Pbr PUBs in the genome of Chinese white pear.Among them,50 Pbr PUB genes were mapped on 17 chromosomes and 12 other Pbr PUB genes were located on scaffold contigs.Based on the phylogenetic relationship,62 PUB genes were clustered into five groups.The conserved motif and gene structure analysis provided further evidence to support the classification result of phylogenetic tree.Cis-acting element analysis indicated that PUB genes might participate in diverse biological processes,especially in the response to abiotic stresses.Based on RNA-data from“Dangshansuli”at seven tissues,we found that PUB genes exhibited diverse of expression level in seven tissues,and q RT-PCR experiment further support the reliable of RNA-seq data.To identify candidate genes associated with resistance,we conducted q RT-PCR experiment to explore the expression level of pear seed plant under four abiotic stresses,including:ABA,dehydration,salt and cold treatment.2.This study showed that the expression level of PbPUB18 was greatly induced under,dehydration,salt,cold and ABA treatment.Overexpression of PbPUB18 in Arabidopsis enhanced tolerance to drought stresses.Compared with wild type,transgenetic Arabidopsis had higher survival rate,lower conductivity content,lower malondialdehyde content,higher Fv/Fm lower accumulation of H₂O₂ and O₂^-after drought treatment.In contrast,down-regulation of PbPUB18 in Pyrus betulaefolia by virus-induced gene silencing（VIGS）resulted in elevated drought sensitivity.After drought treatment,compared with wild type,silencing lines had higher conductivity content,higher MDA content,higher Fv/Fm,higher H₂O₂ and O₂^-accumulation.Take together,physiological and molecular tests results demonstrated that PbPUB18 plays a positive role in drought tolerance.