| Temperature was one of important factors affect cell growth, lipid content and fatty acid composition. Changing the ratio of unsaturated fatty acids in biological lipid was one of the important mechanisms for tolerance to low temperature stress. Organisms under low temperature stress, usually increased the unsaturated fatty acids or improved the proportion of branched-chain fatty acids within various types of cell membrane to enhance the membrane fluidity, thus ensuring the membrane to carry out normal physiological processes, such as material transport, energy conversion, information metabolism and metabolic regulation. Many articles reported that in a variety of organisms, the desaturatse, an important enzyme of polyunsaturated fatty acid synthesis in the classic way, was temperature-sensitive factors obviously.Schizochytrium, a marine lower fungi, with a simple, fast growth, simple fatty acid composition and easy to purify, was the ideal DHA fermentation bacteria.By shake flask fermentation, acid-heat extraction and GC-MS detection analysis of lipid showed that incubation temperature was one of the important environmental factors affect the Schizochytrium biomass, lipid content and fatty acid composition. Schizochytrium.sp FJU-512 could grow between 15℃and 37℃. At 15℃, the DHA content of Schizochytrium more than 60%, but biomass and oil yield far less than other temperatures. Schizochytrium grow better between 20℃and 30℃, with a higher lipid content and relatively stable DHA content. When temperature rose to 37℃, biomass, lipid content and DHA content were decreased. Total saturated fatty acids achieved the highest at 37℃, the lowest at 15℃, between 20℃and 30℃to maintain stable; total unsaturated fatty acid had a maximum at 15℃, minimum at 37℃, between 20℃and 30℃to maintain constant.For the first time using differential proteomics to investigate the molecular mechanism of how temperature regulate Schizochytrium, which promoted to deeply understand lipid metabolism of Schizochytrium. And through exploring the appropriate sample preparation method and two-dimensional electrophoresis conditions, a suitable system of two-dimensional electrophoresis to separate soluble proteins of Schizochytrium was successfully established. Finally put the method glass beads broken combined with TCA/acetone precipitation desalting treatment consider as the extraction method for Schizochytrium supernatant protein, using pH 3-10 (24cm) of the IPG strips for two-dimensional electrophoresis in order to meet the two-dimensional electrophoresis analysis of needs, while laying the foundation for the mass spectrometry identification.Selected three culture temperatures with significant different DHA yield, that is, low temperature stress (15℃), optimal temperature (28℃) and high temperature stress (37℃). Compared proteome map of Schizochytrium stimulated at different temperatures; selected difference points to MS analysis, and eight temperature sensitive factors were identified successfully. The impact of temperature on the fungus was very wide, these significantly changes proteins were related to glycolysis, kinase activities, electron chain transfer, amino acid metabolism, reactive oxygen species (ROS) scavenging, oxidative phosphorylation, nucleic acid and protein processing, energy metabolism and so on. Most of the identified proteins were involved in energy metabolism, particular in glycolysis, evidencing that the primary metabolism of Schizochytrium may be modulated for establishing a new dynamic equilibrium to adapt to low temperature.Expression differences of temperature-sensitive related gene, superoxide dismutase, heat shock protein and cytochrome b5 of Schizochytrium under temperature stress were detected by real-time fluorescence quantitative PCR. It verified the correctness of results of proteomic mass spectrometry in mRNA levels. In addition, Schizochytrium superoxide dismutase protein sequence was analyzed by bioinformatics.Preliminary study the effects of temperature-sensitive factors on the physiological processes,such as energy conversion, material transport, information metabolism, to explore how they associated with the biosynthesis of DHA and other polyunsaturated fatty acid, at the same time revealed Schizochytrium temperature adaptive mechanisms at the molecular level.
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