Study On The Interaction Mechanism Between Tetr Protein And Its Cis-elements In Comamonas Testosterone

Steroid may cause adverse health effects in humans and wild life,such as reproductive disorders,malformations,development abnormalities,cancers.It may be the biggest challenge for human reproductive health in the 21 st century.Comamonas testosteroni is a kind of microorganism that can use a variety of steroid compounds as the carbon and energy source.3,17?-Hydroxysteroid dehydrogenase(3,17?-HSD)from Comamonas testosteroni is a testosterone inducible protein and a key enzyme in steroid degradation.The TetR which located downstream of 3,17?-HSD,might be a repressor for the expression of 3,17?-HSD in Comamonas testosteroni.The study attempted to construct a knockout mutant strain of TetR gene,and analyzed the regulatory relationship between TetR gene and 3,17?-HSD.The TetR gene expression vector was constructed and the site of TetR active protein binding to the regulatory sequence of 3,17?-HSD was decided.Understanding the induction complete molecular regulatory mechanism of the 3,17?-HSD gene and TetR which helps to promote potential application value in the treatment of polluted environment with androgenic hormones.The experiment was mainly divided into two parts.The first part: 331 bp TetR mutation vector pK18-TetR was constructed.Genetic recombination method was used to construct TetR knockout mutant.The growth trend of mutant and wild bacteria was analyzed by spectrophotometry.The expression and genetic stability of 3,17?-HSD in wild-type and mutant strains induced by testosterone were analyzed.Defined the regulatory relationship between TetR gene and 3,17?-HSD.The second part:The 660 bp TetR gene was cloned into the expression vector of pET-15 b,and the TetR active protein extraction conditions were optimized.The sites in the upstream of the 3,17?-HSD gene which TetR protein combined were analyzed by electrophoretic mobility shift assay(EMSA)and gold nanoparticle(AuNPs)light absorption,and try to release the TetR protein repressor effect with testosterone.Results: 1)The TetR gene was knocked out and the mutant strain was successfully constructed.Moreover,the growth trend of mutant and wild bacteria is not much different,and mutant bacteria have good biological stability.The expression of 3,17?-HSD was enhanced in the mutant under testosterone-induced condition,and the reverse genetics proved that the TetR protein is a inhibitor of 3,17?-HSD,the mutant has good genetic stability.2)The pET-15b-TetR expression vector was successfully constructed.The TetR active protein was extracted,which can specifically bind to the 3,17?-HSD palindromic sequence,and binding ability is palindromic sequence 2 >palindromic sequence 1.However,a better binding force may have a synergistic effect.The combination of testosterone and TetR protein can release the binding of TetR protein and palindromic sequence,in other words,to eliminate the repressive effect of TetR protein on the expression of 3,17?-HSD.As a repressor of 3,17?-HSD,TetR can bind to two repeats in the 3,17?-HSD regulatory sequence to reduce its expression.Testosterone can abolish the repressive effect of TetR on 3,17?-HSD.Regulation mechanism consistent with lactose operon model theory,TetR mutant has good genetic stability and has the potential to be applied to the environmental management of steroid hormone pollution.