The Physiological Response And Carbon Fixation Mechanism Of Phaeatylum Tricornutum Under Different CO₂ Concentrations

The marine diatom Phaeodactylum tricornutum is a photo-autotrophic eukaryotic microalga with a central carbon metabolic network which is composed of the Calvin cycle, tricarboxylic acid cycle, pentose phosphate pathway and glycolysis pathway. The central carbon metabolic network provides carbon skeleton and reducant for the synthesis of fatty acids. P. tricornutum has been used as a potential feedstock for biodiesel production and an ideal species in microalgal industry due to its high photosynthetic efficiency, vigorous growth, and plentiful content of unsaturated fatty acids（EPA and DHA） and fucoxanthin. The genome of P. tricornutum has been completely sequenced which made it being a model diatom species and a wonderful species for photosynthesis study.In this thesis, we performed a systematic study of physiological and biochemical mechanisms of P. tricornutum in response to different CO₂ concentration. The main results are as follows:（1） We detected the cellular neutral lipid content and relative fluorescence intensity of normal and nitrogen deficiency cultivated P. tricornutum which was stained with N ile red and BODIPY 505/515. The results showed that both staining methods can rapidly and sensitively determinate the fluorescence intensity of P. tricornutum intracellular neutral lipids, and we also found that changes of intracellular neutral lipid’s fluorenscence intensity after cultured for 3, 5, 7, 9, 11 days were well matched with the changes of intracellular neutral lipid content which were detected by TLC/FID. Moreover, there is a linear relationship between the relative fluorescence intensity and neutral lipid content of P. tricornutum.（2） We performed a comparative analysis of central carbon metabolic network related key enzymes’ activity and relative mRN A expression level in P. tricornutum under three different CO₂ concentrations. Besides, the mechanism of differences in growth and lipid content in P. tricornutum under different CO₂ concentrations was explored. The results showed that P. tricornutum with high-CO₂ cultivation had not only rapid growth but also increased lipid content. Enzyme activity and mRNA expression of three Calvin cycle-related enzymes（Rubisco, PGK and PRK） were also increased under high-CO₂ concentration, which suggested the enhancement of Calvin cycle activity. This may account for the observed rapid growth rate. In addition, high activity and high level of mRN A transcript of G6 PDH and 6PGDH, which produce NADPH through oxidative pentose phosphate pathway, were observed in high CO₂ cultured cells. These results indicated that the activity of oxidative pentose phosphate pathway was enhanced and might play an important role in rapid growth and lipid synthesis under high- CO₂ concentration.（3） We determinated the enzyme activity and mRNA expression of C₄ pathway related key enzymes of P. tricornutum which was cultured under normal and low-CO₂ concentration. The enzyme activity and real time-PCR results showed that the activity of C₄ pathway related enzymes, such as NAD-ME, PEPCK, PEPC and NAD-MDH, significantly increased under low-CO₂ cultivation. Meanwhile, the relative mRNA expression level of genes localized in mitochondria, such as pepc2, mdh and pyc, had also upregulated under low-CO₂ cultivation. These results showed that partial reactions involved in NAD-ME type of C₄ pathway and all reactions involved in PEPCK type of C₄ pathway participated in carbon fixation process in P. tricornutum under low-CO₂ concentration, suggesting the coexistence of NAD-ME type and PEPCK type C₄ photosynthetic pathways in P. tricornutum.This study focuses on the physiological and biochemistrical changes in P. tricornutum under different CO₂ concentrations. We found that enhanced activity of oxidative pentose phosphate pathway may be related to the rapid growth and lipid synthesis in P. tricornutum under high-CO₂ cultivation. Under low-CO₂ cultivation, there might be a coexistence of NAD-ME type and PEPCK type C₄ photosynthetic pathways in P. tricornutum.