Screening For Lipid-rich Mutants Of Chlamydomonas Reinhardtii And Analysis Of Transcription Level Of Key Genes In Lipid Metabolism

In the face of the fuel depletion and environment deterioration, bio-diesel attractsthe attention of the world as their renewable and clean characteristic. Microalga isregarded as one of the most potential source of oil bio-energy because of itsphotosynthetic characteristics as well as rapid growth rate, high lipid content and littlefarmland occupation. However, the high cost has still limited the oil industry frommicroalga. The purpose of this study is to obtain a fast growing and high lipid contentmicroalgae strains using mutagenetic breeding method, and to investigate theprobable factors and metabolic pathways of lipid accumulation in these strainsthrough transcript analysis of the genes of the key enzymes for oil metabolism. Ourresearches will make contributes to the mutagenesis of high lipid content microalgaand supply data for achieving the genetic engineered algal strains.In this study, we select Chlamydomonas reinhardtii CC-124as the experimentmaterial, and use the method of combining UV mutagenesis toghether withquizalofop-p-ethyl to the direct the screenign. We firstly determined the UVmutagenetic times (20s,25s) and quizalofop-p-ethyl concentrations (5.5,6.0μmol/L)in the pre-experiment. Totally Five fast-growing algal mutants were screened bycomparing the growth rate. After Nile red staining observation, two mutants4-2-2and9-2-7which have largest lipid bodies were picked out. By using the acid solutionmethod we assayed the total lipid content of wild type and mutants under differentnitrogen-limited culturing time (0th,4th,6thday). Higher level of neutral oil content hasbeen detected in these mutants than in wild type.Three different statistical softwares (GeNorm, NormFinder and Bestkeeper) areused to assess the stability of4candidate inner-reference genes (18S rRNA, COX4, COX12). COX12was proved to be the most suitable inner-reference gene for thisprocess and was used to investigate the quantitative expression level of the genes ofkey enzymes for oil metabolism, which is ACCase, PEPC2, LACS and DGAT undernitrogen-deficiency culture at0th,4thand6thday for the wild type strain of C.reinhardtii CC-124together with two mutants4-2-2and9-2-7. No difference wasfound in oil-accumulation mode the two mutants and they all have the bothadvantages for growth and lipid accumulation. Although mutant9-2-7had the higheroil content and, mutant4-2-2had the higher expression level of ACCase and DGAT2and maybe the better capability for persistent oil synthesis.