Study On The Molecular Mechanism Of Lipid Accumulation In Chlorella Vulgaris HN11 Under Nitrogen Limitation

Due to the shortages of fossil fuels and the production of greenhouse gas carbon dioxide on their combustion,biodiesel is receiving considerable attention.Microalgae are one of the most promising feedstock owing to their higher oil yields and fast growth.Developing transformed microalage by metabolic engineering technology is one of the important ways to reduce production cost and achieve the industrialization of microalage biodiesel.Characterizing the metabolic pathways involved in the biosynthesis of lipids is necessary to realize the full potential of these organisms as biodiesel resources.Marine green microalga Chlorella vulgaris HN11 accumulates significant amounts of triacylglycerols(TAGs)under nitrogen limited conditions.However,however,much is still unknown about the biological triggers that cause the production of triacylglycerols.Gene expressions occurring in Chlorella vulgaris HN11 subjected to nitrogen limited conditions at 0,24 and 48 hours were examined using RNA-Seq.A total of 24 Gb valid data,177 million effective sequences were obtained and thewty were assembled into 29499 genes and 34987 isogenes.The 16683 isogenes were annotated by comparison to the bio-database and 6245 were annotated with GO terms.5202 isogenes were annotated to the 292 pathways.A large number of differentially expressed genes among the tested materials were detected.479 and 155 differentially expressed isogenes were found in samples at 24 h and 48 h,respectively.Expressions of FBP,PBP,CuAO1,CuAO2,LPL and PK genes in Chlorella vulgaris HN11 subjected to nitrogen deplete and replete conditions at 0,4,8,12,24 and 48 hours were examined using absolute fluorescent quantitation.Results of the digital expression analysis and quantitative PCR analysis were consistent.Under nitrogen limited conditions,the expressions of FBP,PBP,CuAO1,CuAO2,LPL and PK genes in Chlorella vulgaris HN11 significantly higher than those under replete conditions in 1-12 hours.Under nitrogen limited conditions,the expression of CuAO2 was 200-5000 times than that under replete conditions.There was no significant difference in the expression of the gene FBP and CuAO1 that under nitrogen-depleted and nitrogen-replete conditions.The expression level of LPL gene under nitrogen was 7.7 times than control sample in 48 hours.The expression of PBP and PK at a lower level which under nitrogen limited conditionsBased on the analysis of gene expression results,we proposed that nitrogen limitation induced the up-regulated expression of genes coding endonuclease and anaphase-promoting complex,which caused the cells reached the late growth period earlier,leading to the carbon flow to lipid synthesis.Expression of genes encoding amino acid deaminase and oxidase were also up-regulated,indicating the increased deaminization,oxidation and hydrolysis of amino acids,which could provide the carbon skeleton for synthesis of lipids.Genes coding.enzymes involved in the TCA cycle such as pyruvate dehydrogenase were down-regulated,while other genes such as the aldehyde dehydrogenase gene were up-regulated.That may cause the accumulation of the intermediates for TCA such as NADH and oxaloacetate,resulting in the weakened TCA cycle and more carbon sources flowed to fatty acid synthesis.The fabF gene,aldehyde dehydrogenase gene,amino oxidase gene,aspartyl aminotransferase gene and anaphase-promoting complex gene may be the key genes for the lipid accumulation induced by nitrogen limitation.