Full Length CDNA Isolation Of CaPUB59 And Its Preliminary Functional Identification

Plants form a special defense mechanism to prevent all kinds of the biological and biological stresses in a long-tern natural selection process.Previous researches found that ubiquity-26s proteosome pathway(UPP)participate in plants growth and adversity stress process.PUB protein decided the specificity of substrate in the ubiquitin-proteasome as a subfamily of the ubiquitin-protein ligase E3 family.According to the report,PUB protein was widespread in many plants.It participate in various of abotic and biotic stresses,signal transduction pathways and associated with flowering plants self-incompatibility and so on.In this study,the full length cDNA of an E3 candidate gene(CaPUB59)was screened from pepper leaves normalizatoin cDNA library.Analyzing its structure,expression,and the influence of CaPUB59 overexpression to tobacco phenotype via analyzing its T1 generation of tobacco lines.The main research results were as follows:1.Screening pepper cDNA library by using the specific primer which was designed by pepper EST of E3 protein homologous.An positive clone(CaPUB59)of an E3 protein analogues was obteined.Its full length was 2053bp,and has a open reading frame encoding 521 amino acids.The deduced protein molecular weight was 56.71 kDa.There is a conservative U-box structure in N-terminal and 7 WD40 repeats in C-terminal.It is similar to the sixth group of arabidopsis AtPUB.2.Building the vector of subcellular localization which was fusing wih GFP protein.The fusion protein plasmid of GFP-CaPUB59 was expressed in Onions skin cells via gene gun.CaPUB59 protein was observed in the nucleus.3.Real time PCR results shows that CaPUB59 expressed in roots,stems,leaves,flowers and fruits of mature seedlings,especially high expressed in fruits.CaPUB59 expression was reduced under cold treatments and inhibited under hot treatments.There was no obvious difference between experimental group and control group under mechanical damage,CdCl2,mannnitol treatments and ralstonia solanacearum infection.CaPUB59 had no significantly expression change under exogenous ABA,SA and ethylene processing.The experimental group CaPUB59 expression little lower than control group within 24 hours under Auxin treatment,and higher with:in 24 hours under MeJA treatment.4.13 CaPUB59 overexpression plants and their T1 progenies were obtained through tobacco genetic transformation.Studying the impact of CaPUB59 overexpression in tobacco resistance to adversity by utilizing T1 genetation transgenic plants.Found that CaPUB59 overexpression can significantly reduce the resistance to high temperature,osmotic stress,drought treatment and Ralstonia solanacearum infection.But there was no obvious impact of resistance to cold and salt stress.In summary,through the expression profiles analysis and overexpression plants stress analysis,CaPUB59 maybe participate in various adversity stress as a negative adjustment factor.