Identification Of Reference And Biomarker Proteins,and Characterization Of Expression Patterns For Proteins Involved In Triacylglycerol Biosynthesis In Chlamydomonas Reinhardtii

The utilization of traditional fossil fuels brought a lot of problems including environmental pollution,energy crisis,and ecological destruction.One of the effective solutions to alleviate the pressure from energy and environment is developing efficient,clean and renewable bio-energy.Microalga were regarded as the most promising third generation bio-energy resource because of high lipid content,high energy-efficiency,high CO₂ fixation rate,simple equipments for large-scale cultivation,and abundance of wild algal strain resources.It is also concluded that no competition with human beings for food production and no competition with agriculture for land use.Based on genome sequence and established mutant libraries,Chlamydomonas reinhardtii became the model organism for molecular biology research and was applied to study molecular mechanism of lipid biosynthesis,stress responses,photosynthesis,respiration,flagella motility,carbon dioxide fixation and exogenous proteins expression.As the application of“omics”,researchers are now able to accomplish high-throughput screening for important genes involved in essential biological process.However,it is hard to focus on target genes or pathways based on“omics”data.In this study,two target proteomics strategies,one is antibodies-based and the other one is Mass MRM-based,were applied to quantitatively analyze the proteins involved in lipid accumulation,additionally,the reference and biomarkers proteins suitable for abiotic stresses were identified.The main results are listed as below:1.C.reinhardtii cells grown at log phase were treated at normal,dark,cold,heat,salinity and glucose conditions,the phenotype,cell density and total protein content of 6treatments at 5 time points?0 d,1 d,2 d,4 d and 6 d?were compared.It is found that heat stress affected most to the growth of C.reinhardtii,both cell density and total protein content decreased dramatically.Relative less influence to cell density and total protein content were observed from cold,dark,salinity and glucose treatments.2.Western blot analysis was carried out for 20 candidate proteins,the expression patterns under different treatments and time points were obtained.Pearson's correlation coefficient?PCC?was calculated between the abundance of candidate proteins and cell density or total protein content to identify reference proteins.At the end,OEE2 and BCR1were recommended as reference proteins for normal condition,RK1 and ATPs-A were recommended as references for dark treatment,RMT1 was recommended as references for cold treatment,ATPs-A was recommended as references for heat treatment,HistoneH3,PD1 and ATPs-6 were recommended as references for salinity treatment,RMT1,ATPs-6and GAP23 were recommended as references for glucose treatment.3.Average of relative fold change?ARF?was calculated for different time points between treatment and control to identify biomarkers.PRPL18,RBCS2,BCR1 and Cre10.g420200 were choose as broad-spectrum biomarkers,ATPs-6 was identified as biomarker for cold treatment,GAP2,Cre17.g734900 and HSP70A were identified as biomarker for heat stress and FAB2 was biomarker for glucose treatment.4.The C.reinhardtii cells grown at log phase were cultivated under normal?control?and nitrogen-depletion condition.The phenotypic differences under two treatments at 5times points?0 d,1 d,2 d,4 d and 6 d?were compared.The culture under nitrogen-depletion condition showed yellow colour,reduced cell dividing rate and total protein content,while lipid droplets and lipid content were increased.5.Western blot analysis was carried out for 29 candidate proteins selected among 113reported for protein samples collected in normal?control?and nitrogen-depletion treated C.reinhardtii cells.Twelve of them showed induced abundance along with nitrogen-depletion,support that they may play positive roles in the lipid biosynthesis pathway.6.Mass-based multiple reaction monitoring?MRM?was applied for proteomics analysis of C.reinhardtii samples using 113 reported proteins involved in lipid biosynthesis as target.Under nitrogen deprivation condition,30 target proteins were identified by MRM assay.Among them,8 proteins were found overlap with Western blot analyzed protein illustrated above,and four of them showed consistent induced expression pattern.7.Based on reported transcriptomic and proteomic data derived from mass spectrometry,twenty-two proteins related to lipid synthesis were selected as candidate.Antibodies were generated and Western blot analysis was carried out for control and nitrogen-depletion treated C.reinhardtii cells.It was found that 13 proteins showed induction expression pattern at nitrogen-depletion condition.8.In total,72 antibodies against C.reinhardtii proteins were generated,and Western blot analysis were carried out for sample collected at different time point under multiple treatments.The data for protein expression patterns will provide unique impact on the understanding of gene functions,the antibodies also will be important resources for protein-related investigation and mechanisms study for biological processes.