Comparative Proteomics Analysis Of R. Mucilaginosa In Response To Heavy Metal Stress And Cloning And Sequence Analysis Of Metallothionein Gene

Microorganisms have an extremely strong adaptability to adversity environment.Under heavy metals stress, physiological and metabolic changes will happen toachieve the accumulation or detoxification in microorganisms. In this experiment, theadaptability of heavy metals of Rhodotorula mucilaginosa AN5and the change ofintracellular proteins occurred under Cu2+stress by proteomic way were analyzed. Inaddition, metallothionein gene was cloned and its sequence was analyzed bybioinformatics. Theses researches were helpful to explain the adaptation mechanismsto heavy metals in microorganism, and could supply the theoretical basis of heavymetal pollution detection and management.（1）By physiological and biochemical analysis, yeast strain AN5had the typicalculture characteristics as Rhodotorula mucilaginosa. The strain could use many carbonsources and nitrogen resources, and grew well in vitamin-free media. The optimalgrowth temperature for AN5was20℃. By26S rDNA sequence and phylogenetic treeanalysis, AN5was identified as Rhodotorula mucilaginosa.（2）Strain AN5had a great adaptability to heavy metals such as CuSO4, CuCl2,CdCl2，Pb(CH3COO)2, CrCl3, MnCl2and HgCl2. AN5could survive and grow slowlyeven in the media containing high concentration heavy metal ions of4M.（3）Protein changes of strain AN5induced by2mM CuSO4were analyzed bycomparative proteomics. After induction of2mM Cu2+the synthesis of30spotsincreased and43decreased. In addition,9spots disappear and13spots were newlysynthesized. By MALDI-TOF/TOF-MS/MS way,61proteins/peptides were identifiedfrom95spots. According to the predicted physiological functions, these proteins weredivided into six types: carbohydrate metabolism-related proteins, energymetabolism-related proteins, nucleic acid protection-related proteins, molecularchaperones, antioxidant enzyme and conserved hypothetical proteins. （4）Metallothionein gene was cloned and analyzed by PCR amplication of DNAand cDNA. The results showed that the length of metallothionein DNA was922bp,and its open reading frame was321bp, encoding106amino acids. According toprotein sequence analysis, molecular weight of metallothionein protein was estimatedabout10325.6Da, theoretical isoelectric point was8.49, with a typical hydrophobicregion in the N end position. Based on the MetallothioneinⅡ homologous model, thethird structure of metallothionein protein was built.（5）The amino acids sequence of metallothionein was similar to that ofRhodosporidium toruloides NP11by BLASTX analysis. The phylogenetic treeanalysis showed that metallothioneins were different during species.In all, the physiological characteristics, development status, adaptationmechanism of heavy metals of AN5were studied by physiological and biochemicaltechnique, the molecular and proteomics skills. Additionally, metllothionain gene wascloned and its sequence was analyzed by bioinformatics. These researches made theadaptation mechanisms of microorganism to heavy metals clear.