| Microcystins are a group of cyclic heptapetides which has strong toxicity. Microbial degradation is considered as the main removal pathway for MCs in natural water bodies. At present, more than 60 strains of MCs degrading bacteria have been isolated, and most of them have the same MCs degradation mechanism. Previous studies showed that at least 3 hydrolytic enzymes are involved in the degradation process, which are encoded by mlrA, mlrB and mlrC genes respectively. These genes gathered together with mlrD gene which encodes transport protein to form a 5.8 Kb-long gene cluster. Although mlrA gene had been detected in many MCs degrading bacteria, only 4 complete mlrA gene sequences were obtained. Moreover, the detection of other three genes mlrB?mlrC and mlrD have not been performed for most of MCs degrading bacteria, and hence few full sequence has been obtained. Due to lack of information of complete sequences, the origin and evolution process of the MCs genes is not yet clear. A MCs degrading bacterium Sphingopyxis sp. X20 has been isolated from the sediments of DianChi Lake in previous studies. In this thesis, we obtained the complete sequence of mlr gene cluster through gene cloning technology. The bioinformatic analyses of mlrA, mlrB, mlrC and mlrD as well as the enzymes encoded by these genes were conducted. In addition, the key gene mlrA was verified through heterogeneous gene expression. The main research contents and results are reported as follows:(1) The complete sequence of mlrA gene of strain X20 was obtained by PCR amplification. The full length of mlrA gene is 1011 bp. It encodes 336 amino acids, which include 163 hydrophobic amino acids and 58 polar amino acids. MlrA was predicted to contain 6 across membrane regions and a signal peptide with the cleavage site between the alanine and leucine amino acids at positions 26 and 27. The sequence of mlrA of strain X20 was 99% similar to that of a previously reported MCs-degrader Sphingopyxis sp. C-1 and these two isolates clustered together in the phylogenetic tree constructed using mlrA gene sequences of X20 and other MC-degrading bacteria. The heterogeneous expression product of mlrA can degrade MCLR to linear MCLR, suggesting that the obtained sequence of mlrA is correct.(2) mlrD gene sequence was obtained by PCR amplification method. The full length of mlrD gene is 1272 bp, encoding putative protein with a calculated molecular weight of 44.849 kDa. MlrD has 423 amino acids with 221 hydrophobic amino acids and 80 polar amino acids. There are 10 across membrane regions, but no signal peptide was observed in the MlrD of X20. The sequence of mlrD of strain X20 was 99% similar to that of a previously reported microcystin-degrader Sphingopyxis sp. C-1.(3) The complete sequence of mlrC gene was obtained by PCR walking technology. The full length of mlrC gene is 1587 bp. The protein encoded by mlrC gene has 528 amino acids, which contain 193 hydrophobic amino acids and 113 polar amino acids. No across membrane region or signal peptide were observed in MlrC. Phylogenetic analysis suggested that mlrC gene of X20 is highly homologous to that of strain C-1.(4) Because the mlrB sequence of X20 can not be obtained by PCR walking technology, mlrB gene was cloned by constructing a genomic library of X20. The full-length of mlr B sequence is 1581 bp. The putative translated protein of mlrB contains 193 hydrophobic amino acids and 113 polar amino acid. The predicted molecular mass of MlrB is 57.6 kDa. No across membrane region or signal peptide was detected in MlrB. The results of phylogenetic analysis show that mlrB gene sequence is highly homologous to that of strain C-1. |
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