| With the approaching possible depletion of fossil fuels in the near future,the development of renewable energy would be an important part of the world.Microalgae biofules as a new type of renewable energy has been widely accepted.In this study,Euglena gracilis has been referred to be one of the greatest biodiesel material which can accumulate abundant lipids in some certain centain conditions.In addition,it is also a good source of food and nutritional supplements because of its rich active ingredients.This research has combined the phototrophic character of Euglena gracilis(wild type,WT)with its heterotrophic culture condition,to screen those mutants which could grow fast and accumulate more bio-active molecules.We treated the WT with a gradient of concentrations of nitro-guanidine(NTG)or different time of ultraviolet(UV)light treatments,and then cultivated them under normal light conditions in the organic medium.It is desirable to obtain the mutated strains which can grow faster and with higher accumulation of activity ingredients such as fatty acids and paramylon.In addition,in order to understand the mechanism of photosynthesis,we cultivated WT cells under the condition of heterotrophic and then transformed them into phototrophic condition,to analyze differential expression about chloroplast transcriptome under two cultivation conditions and study the correlation between the chloroplast transcription and the culture conditions of the WT.The results are as following:(1)In this study,total 34 mutants were successfully obtained with the above two mutagenic treatments.Among them,the growth rate of the mutants obtained by NTG mutagenesis is relatively higher than that of the mutants obtained by UV treatment.At the NTG concentration of 0.8mg / m L,we obtained the mutants with higher growth rate and biomass accumulation compared with the WT.The growth rate of mutans obtained by UV mutagenesis was less than that of the WT.(2)After comparing the growth curves and the maximum cell numbers of themutants,6 mutants were selected for the active ingredient content,using the WT as the control.The sampling time was on 4th day(logarithmic phase)and 9th day(cell acme phase).(3)We measured the fatty acid content and composition on the 4th and 9th days in the experiment of fatty acid determination.We found that total fatty acids(TFAs)on the 9th day were higher than those on the 4th day.Among them,the TFA content of the mutant A4 was the highest,reaching to 171.34 ?g/mg dw.The TFA content of the other mutants were 122.57-140.07 ?g/mg dw while the WT was the lowest,with a content of 97.91 ?g/mg dw.Interestingly,in both wild and mutants,the content of saturated fatty acids(SFA)was the highest,and the polyunsaturated fatty acids(PUFA)was the secondly,the monounsaturated fatty acids(MUFA)were the lowest.The contents of SFA and PUFA in A4 were higher than those in other mutants,the MUFA content was similar to that of the other strains,and the main PUFA of A4 is C18:2?6,C20:4(AA)and C22:6?3(DHA).(4)In the paramylon(Pm)content experiment we founded that most of the mutants and WT Euglena on the 4th day had higher contents of Pm than those on the9 th day.On the 4th day,mutant A4 contained the highest Pm content and in mutants of A11 and WT,accounting for 60.64%,56.57% and 55.02% of their dry weight respectively.(5)Transcriptome.We focused on the chloroplast transcriptome this time.A total of 75 transcripts were obtained,and include 48 protein encoding genes.There were 13 genes involved in photosynthesis that were mainly genes of photosynthetic system I(PSI)and photosynthetic system II(PSII)genes.However,only 2 genes showed significantly differential expression(Fold change?2,p?0.05)after the cultivation switch.which were ribosome-related protein gene rpl5 and photosynthesis-related protein gene psb H,the former was up-regulated and the latter was down-regulated respectively.Thus,the chloroplast transcriptome indicated that in some case,the regulatory probably is majorly at the post-transcriptional level.In general,we obtained 6 Euglena mutants by exploring the appropriate condition of NTG and UV mutagenesis treatment and comparing their highest cellconcentrations.Afterword we measured the content of fatty acids and Pm.Analyzing fatty acids by GC-MS to explore the accumulation of fatty acid in different periods preliminarily provides a possible way for the exploration and large-scale industrialization for the microalgae lipds.In addition,we also analyzed the differential gene expression under the conditions of light and dark,which provided the basis for exploring the association mechanism of chloroplast transcriptome in Euglena gracilis. |
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