Functional Identification Of Hydrophilin Leak Conferred Abiotic Stress Tolerance To Deinococcus Radiodurans

Deinococcus radiodurans R1which exists in various extremeenvironments is extre mely resistant not only to ionizing and ultravioletradiation but also to desiccation, oxidants, high salinity, lowtemperature stress resistance. The genome of D. radiodurans R1includes DR₁172（leaK）, a homologous gene with LEA of plant,encoded a protein in size30.9kDa containing298amino acids. Theprotein which is rich in hydrophilic amino acids is a kind of stablehydrophilic protein and belongs to a group3LEA proteins. In this study,the function of LeaK in response to stress resistance was characterizedthrough gene mutation, transcription analysis, Biolog test, heterologousexpression, achieved the following progress:The leaK deletion mutation strain （ΔleaK） was constructed by thefusion PCR. The mutant strain and wild type were dealt with NaCl,sorbitol, H₂O₂and desiccation individually. The results indicated tha tthe expression of leaK enhanced bacterial salt tolerance, droughtresistance, anti-oxidation, but could not increase the UV radiationresistance of the cell. However, the various abiotic stress experimentswere carried out for the transgenic E.coil and we found that LeaK cannot enhance the resistance of cells to high salt, hyperos molality, dryingand UV radiation stress.In order to research whether the deletion of leaK gene impa ct othergenes of gene cluster, the RT-PCR assays were carried out in thi ssection. The results displayed that leaK gene is cotranscribed withDR₁168, DR₁169, DR₁170and DR₁171. Furthermore, Quantitativereal-ti me PCR analysis demo nstrated that the fo ur genes expression ofleaK clusters al most had not changed in the mutant and wild type. Thedata suggested that leaK gene mutation had no effect on the expressionof other genes.The QRT-PCR results showed that, with the extension ofincubation time, the relative expression of DR₁172had trended down in growth different periods of D. radiodurans and maintained low levelexpression in the stationary phase, speculating that Lea K p rotein couldbe related to the growth of bacteria. Further more, the relativeexpression quantity of main genes related to oxidative stressupregulated significantly in the mutant ΔleaK under50mM H₂O₂treatment. The results indicated that LeaK may have the ability tore move oxygen free radicals. And they also could act as molecularchaperone to protect the activity of enzymes. Biolog assays revealed achange in the carbon source utilization of the mutant ΔleaK. Compa redto wild type DR, the utilization of five carbon resources was reducedefficiently and four increased significantly. Biolog resultsde monstrated that the deletion of leaK gene influenced the metabolismcapability of D. radiodurans R1.In this study, we constructed the reco mbinant plas midspET28a-leaK and transfor med into Escherichia coli BL21（DE3） toachieve high-efficiently expression of LeaK pr otein. The LeaK proteinwas researched preliminarily to lay the foundation for carrying outLeaK protein antibody preparation and exploring LeaK protein functionin vitro.