Cloning And Analysing Of Eukaryotic Methionine-Lyase Genes

【Objective】To colone and analyse the structure of the two genes encoding methionionase from Trichomonas vaginalis.【Methods】The Trichomonas vagnalis were collected and cultivated.The total RNA was extracted from primitive protozoon Trichomonas vaginalis,cDNA fragments encoding Methionineγ-lyase genes were amplified by RT-PCR using random hexamer primer.Using the cDNA as a template and the primers designed exactly,a large number of TVMGL1 and TVMGL2 genes were amplified by PCR using the KOD plus.The activaty of the KOD plus was inactivated by the phenol method,and then a 'A'tail was added to the gene.The mixture was purified by the gel electrophoresis sepration.The fragments of methionine gene were linked with the pMD19-T cloning vector.The recombinant genes were transformed into the E.coli competent cells to get colonies with appropriate size and quantity.The white colonies were collected in accordance with the principle of blue-white selection,and incubated into LB/Amp medium overnight.The plasmid was extracted from the culture and the rest were preserve at -20℃.Using the Plasmid DNA as a template,the samples were tested by PCR and digestion.The strong positive samples were selected,the preserved curture was sent to shanghai sangon company for sequencing using DNA sequencer.【Results】The sequeces indicates that the DNA fragments are 1191bps and 1197bps in length,and with high homology with that reported previously(more than 99%).TVMGL1 is predicted to encode a protein of 396 amino acids with a molecular mass of 42.9 kDa,and TVMGL2 predicts a protein of 398 amino acids with a molecular mass of 43.1 kDa.Amino acid sequence comparision of TVMGL1 and TVMGL2 revealed that the two trichomonad proteins had 69% identity.Data bank searches revealed that the highest level of amino acid sequence identity for the two trichomonad proteins with the methionine-lyases from P.putida(44-45%).【Conclusion】Two L-Methionineγ-lyase isozyme genes were successfully cloned and identified from Trichomonas vaginalis.TVMGL1 and TVMGL2 had high homology.