| U-box E3 ligase plays an important role in plant defence and plant growth and development.Previously,a cDNA library was constructed from leaves of Vitis amurensis ’Zuoshanyi’.In this research,a novel gene which showed high expression level in the cDNA library was cloned.The gene’s function was studied using molecular biology methods which may help reveal the mechanism of plant stress resistance and could be helpful in the molecular breeding of grapes through genetic engineeringAccording to sequence analysis,the gene encoding a protein that has the U-box domain,hence it was named PUB(Plant U-box Protein).Multiple sequence alignment demonstrated that VaPUB has a high sequence similarity with U-box E3 ligases and the defense related protein CMPG1.The qPCR result shown that PUB expressed in root,stem,shoot tip and leaves in ’Zuoshanyi’.And it was significantly regulated by cold,Plasmopara viticola,SA,ABA,but in a different regulation pattern among cultivars.Then the upstream sequence of PUB was amplified among cultivars for comparison.SNPs were found among cultivars,suggesting a different transcription regulation pattern among cultivars.VaPUB was ectopically expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana by the floral dip method.Over-expressing VaPUB in Arabidopsis resulted in an enhancement of cold and salt stress tolerance,however,the plant resistance to drought and downy mildew and root growth were no significant difference between transgenic lines and WT.The transcription factors,CBF1/DREB1B and CBF3/DREB1A,and cold inducing genes,COR15A,COR47,KIN1,RD29A,SuSy,were expressed in a higher level in VaPUB transgenic plants than in the wild type plant.In contrast,the expression of transcription factor CBF2/DREB1C and MYB15 was repressed by VaPUB.To further investigate the role of VaPUB in grapevine,VaPUB was transformed into Vitis vinifera’Thompson Seedless’.Over-expressing VaPUB in Vitis Vinifera ’Thompson Seedless’ affected the microstructure of leaves.In comparison with WT,the top epidermis layers in the transgenic leaves were thinner,the palisade cells were loosened with irregular shape,the stomatal apertures was wider,chlorophyll content decreased.The pathogen treatment assay shown that the Plasmopora viticola developed faster in VaPUB transgenic leaves than in WT leaves.The proteome assay shown that a total of 39 spots had a statistically significant fold change between WT and transgenic leaves,including pathogenesis-related protein PR 10,chitinase and many proteins involved in carbon and energy metabolism,oxidation reaction and protein metabolism.According to subcellular localization assay,VaPUB is located in nucleus.In addition,VaPUB was transformed into Microvine that has a dwarf structure and fruits in less than one year for a further study of its function in fruit development etc. |
