Screening Of Temperature Sensitive Cell Division Variants Of C.reinhardtii And Neutral Lipid Accumulation

Microalgae is a kind of plant with fast growth speed,simple generation cycle and strong environmental adaptability.It is suitable for open and large-scale cultivation of microalgae,which can directly convert solar energy into chemical energy and accumulate oil for potential industrial applications.Chlamydomoans reinhardtii is a unicellular eukaryotic green algae that can photosynthesize to accumulate neutral lipids for biodiesel production.Chlamydomonas sp.is an important model strain for studying green microalgae.For industrial application,algal strains with high specificity and stable traits can be obtained through mutation breeding,selective breeding,cell fusion and genetic engineering.In this study,UV-induced mutagenesis was used to screen Chlamydomonas reinhardtii mutant strains with high biomass and oil production.Firstly,the temperature-sensitive(ts)mutant of Chlamydomonas reinhardtii were screened by the sensitivity of mutant strain to the restrictive temperature.Secondly,the cell division cycle mutants(cdc)were further screened by identifying the giant cells as a terminal phenotype.Finally,these ts cdc mutant of Chlamydomonas reinhardtii were analyzed by the content of neutral lipids,triacylglycerol(TAG),fatty acid composition and DNA content.The results are as the following:1.7500 C.reinhardtii single colonies were UV mutagenesized with the average lethality of65%.15 temperature-sensitive(ts)mutants were identified at the restrictive temperature,in which condition the thermal stability of the mutated gene has changed.2.The ts mutants of C.reinhardtii were further screened by microscope.Three ts mutants of C.reinhardtii were further identified as cell division cycle(ts cdc)mutants.These ts cdc mutants grow normally at 22°C.When the temperature increased to restrictive temperature of 34°C,the cells continue to grow but do not divide,eventually identified as the terminal phenotype of giant cells.3.Neutral lipids fluorescence in C.reinhardtii ts cdc mutant cells was monitored by fluorescence microscopy.It was found that these ts cdc mutants of C.reinhardtii do not accumulate neutral lipids at 22°C.Only when these mutants were transferred to restrictive temperature,the volume of the cell begins to increase and produces neutral lipid fluorescence.4.TAG accumulation in ts cdc mutant of C.reinhardtii were validated using thin layer analysis(TLC).The result showed that the wild type grown in the absence of nitrogen condition and ts cdc mutants grown in the complete condition could accumulate TAG.In the above two conditions,both C.reinhardtii wild-type cells and ts cdc mutant cells were unable to undergo normal cell division and accumulate TAG.5.The neutral lipids were seperated from the polar fats in C.reinhardtii ts cdc mEtants and analyze their fatty acid compositional by GC-MS.The results showed that C.reinhardtii ts cdc variants showed an increase in saturated fatty acids and a decrease in polyunsaturated fatty acids at the restrictive temperature,suggesting that the fatty acid components shifted mainly from polar lipids to the neutral lipids.6.The nuclear DNA of C.reinhardtii ts cdc mutants was analyzed using a fluorescence microscope.It was found that the ts cdc mutant of C.reinhardtii showed only one distinct nuclear DNA fluorescence and without a flagellar under the restrictive temperature conditions.According to the cell cycle of C.reinhardtii,the cell cycle of these ts cdc mutants is arrested after DNA synthesis but before the cell division.In summary,this study random mutagenesized C.reinhardtii to screen and isolate the temperature-sensitive cell division cycle mutant(ts cdc)strains.These ts cdc mutant of C.reinhardtii increase their cellular volume without further cell division at the restrictive temperature and accumulate neutral lipids or TAG under the nutrient complete conditions.These mutants could contribute to an enhanced lipid production per cell or programmed lipid accumulation,i.e.to use temperature as a signal to induce neutral lipid formation.This will be an important next step to evaluate the feasibility of isolating mutants with properties that,without compromising productivity,allow an efficient transition from biomass production to lipid accumulation in a microalgal based biodiesel production process.