| The metalloenzyme superoxide dismutases(SODs, EC 1.15.1.1) are classified according to the metal ion cofactor required for their activity: the copper-zinc type(CuZn-SOD), the manganese type(Mn-SOD), the iron type(Fe-SOD) and the most recently described nickel type(Ni-SOD). Superoxide dismutases, which catalyze the dismutation of superoxide into hydrogen peroxide and oxygen, play a central role in protection against oxidative stress. Recently, it has been suggested that SODs, as virulence factors, may aid some intracellular pathogens in combating the respiratory burst and oxygen-dependent microbicidal activities of their hosts.Bacillus cereus M22, isolated from wheat, is a biocontrol agent against some crop diseases successfully with a high yield of SODs. It is hypothesized that SODs may involve in the biocontrol process and aid it in bacterial invasion. The objects of this research are to clone the SOD genes in strain M22 and to express those genes in Exoli BL21(DE3) or Pichia pastoris..Main results are as follows:1 Molecular cloning of Mn SOD genes from R cereus M22 and its expression in E. coliA 1317-bp length DNA fragment, deposited in GenBank?with accession numbers AY540749, of Mn-containing superoxide dismutase (Mn-SOD)-encoding gene from Bacillus cereus M22 were determined from genomic DNA by use of the direct PCR and inverse PCR(EPCR). Analysis of MnSODs revealed that the open reading frame consisted of 218 amino acids. Comparison this deduced amino acid sequences with previously isolated Mn-SOD amino acid sequences from other bacteria revealed that they have higher homology to, and complete conservation of, invariant residues found in Mn-SOD. The gene was inserted into the expression vector pET-22b(+) digested with two appropriate restriction enzymes with correct open reading frame and the recombinant pET-sodA had been transformed into Exoli BL21(DE3). After induced about 3 hours by lmM IPTG, a special fusion protein band appeared near the position of molecular weight 28KD with SDS-PAGE and was more than 24.3 % of the total soluble protein. The pET-sodA had been transformed into E. coli double mutant strain QC779 lacking both genes for Mn-SOD and Fe-SOD(sodA sodB). The recombinant enzyme could protect E. coli QC779 which could not live on the LB plate containing 10(amol/L paraquat from paraquat toxicity, and were judged to be Mn-SOD by native PAGE and enzyme activity on the basis of inhibiting by potassium cyanide(KCN) and hydrogen peroxide (H2O2).2 Molecular cloning of the CuZn SOD gene from R cereus M22 and its expression in R coliA 1320bp-length CuZn superoxide dismutase(CuZn-SOD) gene fragment containing the coding region which consists of 179 amino acids, 5'- and 3'-flanking sequence was cloned by PCRfrom genomic DNA of the endophytic bacterium Bacillus cereus M22. The deduced ammo acid sequence showed a high degree of homology to other bacterial CuZn-SOD. The first 16 amino acids were predominantly either hydrophobic or basic, and may serve as a signal pepti.de for this putative lipoprotein. The gene was inserted into the expression vector pET-22b(+) between EcoRI and XhoI sites with correct open reading frame, and the recombinant pET-sodC had been transformed into E.coli BL21(DE3). After induced about 3h by lmmol/L IPTG, a special protein band appeared near the position of molecular weight 24kD and was more than 21.3% of the total lysate proteins. The recombinant protein protected E. coli SOD null strain PN132, which could not grow on the LB plate containing 10umol/L Paraquat, from Paraquat toxicity. The SOD activity was up to 69.76U/mg in the lysate of PN132(pET-sodC) with IPTG induction, and was judged to be a CuZn-SOD on the basis of inhibited by potassium cyanide(KCN) and hydrogen peroxide (H2O2) by the native PAGE and enzyme activity.3. Construction of genomic library of R cereus M22 and cloning of Fe-SOD gene fragmentBacillus cereus M22, an endophytic bacterium isolated from wheat, was applied widely in biocontrol of plant diseases. Genomic DNA of Bacillus c...
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