ITRAQ-based Quantitative Proteomics Study Of Rhodotorula Mucilaginosa AN5 Under Cadmium Stress

With the rapid advancement of science and technology,the impact of human activities on the environment is increasing,and the accumulation of heavy metals in the natural environment is increasing.Living in an environment containing heavy metals,organisms undergo a series of complex reactions on its surface and inside.In this study,the morphological and biochemical changes of Antarctic yeast Rhodotorula mucilaginosa AN5 under cadmium stress were studied,aiming to dissect the heavy metal adaptation mechanism of Antarctic psychrophilic microorganisms.Differential proteomics studies were performed using iTRAQ-based quantitative proteomic analysis.(1)The growth curves of R.mucilaginosa AN5 which was cultured in medium with6 different concentration gradients separately were observed to determine the resistance of yeast to cadmium.The 4 m M cadmium induced stress was used to observe the morphological changes of R.mucilaginosa AN5 in logarithmic phase and stable phase and to detect the physiological and biochemical changes in cells.The growth condition of yeast was not significantly inhibited under the stress of 4 m M and 10 m M cadmium,basically consistent with the control.While the growth of yeast was obviously inhibited under the stress of 20 m M cadmium.The inhibitory degree increased as concentration of cadmium soar to more than 50 m M,under which the growth of yeast was severely inhibited and even retardant.The results of SEM displayed that some viscous substance secretion occurred on the cell surface morphology under cadmium stress,with the logarithmic sprouting rate decreasing.The cell enlarged under metal stress from 2μm×1.3 μm to 2.5-3 μm×1.3 μm along with an alteration of form from circular to rod-shaped in stable phase.The results of physiological and biochemical changes showed a consistent upward trend in some main substances,including the reactive oxygen species,Malondialdehyde and the membrane lipid peroxidation.Antioxidant enzyme activity and antioxidant content were also increased.Increased levels of antioxidants such as glutathione(GSH),catalase(CAT),peroxidase(POD),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and the activity of antioxidant enzymes such as glutathione reductase(GR)eliminated excessive ROS to maintain the balance of active oxygen.(2)Quantitative proteomics of R.mucilaginosa AN5 was analyzed by iTRAQ,and some differentially expressed proteins were verified by PRM.There were 196 proteins showed significant differences,of which 104 proteins increased and 92 proteins decreased under cadmium stress.The differential proteins were classified into 4categories by COG function annotation,including material metabolism,informationprocessing and storage,cellular processes and signals,and unknown function proteins.The result of GO Slim displayed that differential proteins were mostly annotated that molecular functions comprised catalytic activities and binding functions,membranes in cellular components,biological processes included signal transduction,cell cycle and transmembrane transport.The KEGG pathway displayed that differential proteins were mostly involved in the pathway was signal transduction,followed by cell growth and death.The PRM validation of the 196 proteins,the identification was consistent with the overall trend of quantitative results by iTRAQ.In conclusion,the antarctic yeast R.mucilaginosa AN5 showed strong adaptability to heavy metal cadmium in terms of morphological,physiological,biochemical and proteomic changes.With the deep understanding of the mechanism of heavy metal molecular resistance in polar yeast,it provides a new perspective for bioremediation of metal-contaminated environment.