The Functional Study Of PBE Gene In Arabidopsis Thaliana

Oxidative stress is one of the major threats to the plant living. The oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to clean it. All lives must maintain a reduction environment within cells which can be damaged by reactive oxygen and free radicals. Disturbances in this normal redox state can finally affect metabolism by the damage of the proteins lipids and DNA, severe oxidative stress can even cause cell death. The ubiquitin/26S proteasome pathway is considered to be an effective way of degrading unneeded or damaged proteins by proteolysis. Other than that, the ubiquitin/26S proteasome pathway also plays an important role in hormone signaling,photomorphogenesis, flower development and PCD by degrading regulatory proteins. 20S proteasome is the core particle (CP) of the 26S proteasome. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits: the outer two rings in the stack consist of sevenαsubunits each and the inner two rings each consist of sevenβsubunits. The outer two rings serve as docking domains for the regulatory particles and theαsubunits N-termini form a gate that blocks unregulated access of substrates to the interior cavity. Theβ1,β2, andβ5 subunits have protease activities which have three different substrate specificities. Sequence analysis suggests that the catalyticβsubunits diverged earlier in evolution than the predominantly structuralαsubunits and theβsubunits have high sequence identity in eukaryotic.In order to study the function of gene PBE (the gene ofβ5 subunit in Arabidopsis thaliana ), in this experiment, three vectors had been constructed: the overexpression vector of gene PBE, the GFP transient expression vector of gene PBE , and the fusion construct of PBE: : GUS . For the generation of PBE: : GUS construct,, the PBE promoter sequence (1233bp) has been cloned from Arabidopsis thaliana genomic DNA. The sequence contains TATA box and CAAT box and it has a cis-acting element involved in heat stress responsiveness HSE, a cis-acting element involved in defense and stress responsiveness TC-rich repeats and a responsive element TGA-element. The three vectors were transformed into Arabidopsis thaliana by flora dip method. After the harvest and detection of T1 mutant, we found that PBE and the GFP transient expression were mostly located in cell nucleus probably because proteasome regulated the activities of life mainly through the hydrolysis of various transcription factors which are involved in plant growth and development. We didn't detect any GUS expression in any tissue which was probably because of the low expression of the promoter. The semi-quantitative PT-PCR test indicated that the T2 mutant of overexpression line had a higher transcription level of PBE gene than the wild type does.