| Chlorella vulgaris can accumulate lipid more than 40% of dry weight(DW) when grow in BG-11 medium for 18 days, so it is a potential strain of oleaginous microalgae. In this research, the aglae cells were cultivated in BG-11 medium which contain different NaNO3 concentrations(18.0, 9.0, 4.5 and 3.6 mmol·L-1), we measured the biomass and main biochemical components of C. vulgaris in different time courses, combining the changes of photosynthetic physiological parameters and pigment-protein complexes, to explore the carbon flux and response of photosynthetic apparatus during algae cells accumulating lipid in nitrogen limitation. The results showed that:(1) The differences of biomass of C. vulgaris among four nitrogen concentrations were not remarkable, but there was a considerable increace of the lipid accumulation in 4.5 and 3.6 mmol·L-1 groups(p<0.05), the maximum lipid content and productivity were obtained in 4.5 mmol·L-1 group(48.32% of DW and 0.0931 g·L-1·d-1). Algae cells mainly contained C16:0, C16:3, C18:1 and C18:2, the content of saturated fatty acids increased during the cultivation, indicated a higher saturation degree of fatty acids. The content of carbohydrate and soluble protein in these four groups all decreased for accumulating lipid, however, in 4.5 and 3.6 mmol·L-1groups, the soluble protein degraded rapidly for carbohydrate synthesis within the first three days, which suggested that different nitrogen concentrations induced different metabolic patterns in C. vulgaris, the transiently accumulated carbohydrate converted to lipid with the extension of cultivation under nitrogen limitation. Results of RT-PCR showed that, AGPase gene in 18.0 mmol·L-1group exhibited high expression in 0-96 h, the expression of SP gene was low but tended to enhance, in 3.6 mmol·L-1group, the expression of AGPase gene was lower than that in higher nitrogen group but there was a high transcript level of SP gene in 24-96 h, which resulted in the transient accumulation of carbohydrate.(2) In four different initial nitrogen concentrations, the Chl a, Chl b and Car content of C. vulgaris all increased rapidly then decreased slowly to keep constant,and they showed positive correlation with initial nitrogen concentrations. However, Car/Chl ratios rose fluctuatingly and Car/Chl ratios in 4.5 and 3.6 mmol·L-1 groups were markedly higher than which in higher nitrogen groups(p<0.05),suggested nitrogen limitation induced C. vulgaris tend to accumulate carotenoids. The chlorophyll fluorescence parameters of PSⅡshowed that: Fv/Fm, Yield and ETR all briefly raised then showed decreasing trend. Oxygen evolution rates rised to the peak in the first day then reduced quickly, it closed to the detection limit at the fourth day, respiration rates also raised to the maximum during 1-2 days then decreased to the minimum at the fifth day and keep stable, which showed that the PSⅡ activity reduced constantly but maintain the basic metabolic activity. The ratios of F683/F718 decreased first then increased continuously were related to LHCⅡ reversible dissociated from PSⅡ core complex which protected it from damaged by high light.(3) Six pigment-protein complexes bands were isolated through disc SDS-PAGE electrophoresis. According to 77 K fluorescence emission spectrum, band 1-3 had two fluorescence peaks at 670 nm and 709 nm, the fluorescence peaks of band 4, 5 and 6 were at 677 nm, 678 nm and 674 nm respectively.The results of secondary electrophoresis showed that band 1-3 contained peptides of 82 kDa, 83 kDa and 66 kDa, the peptides in band 4 were 47 kDa, 43 kDa, 37 kDa, 32 kDa and25-30 kDa, band 5 contained 25-32 kDa peptides. Combining with the results of mass spectrometry,we named these complexes as CPⅠa2, CPⅠa1, CPⅠ, CPa, LHCP and FP. The colors of CPⅠ and CPa became shallow gradually with the processing of culture, especially CPa, which suggested that PSⅡ core complex damaged more severely in the two photosystems. |
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