Study On The Regulation Of Unsaturated Fatty Acid Accumulation In Microalgae

Microalgae has a good prospect as a safe,economical and environmentally friendly source of unsaturated fatty acids.Subject with index of unsaturated fatty acids for algae strains screened the advantage.The optimum nutrition and fatty acid methyl ester process parameters of the advantages algae strains were determined.And The effects of different culture conditions on the growth of dominant algae and the accumulation of unsaturated fatty acids were also studied.On this basis,the biosynthesis pathway of unsaturated fatty acids in dominant algae was constructed by transcriptome technique.The biomass,growth rate,total lipid content,total lipid yield and the fatty acid relative content of 10 microalgaes were compared.The results showed that Chlorella vulgaris CJ15 had high biomass,lipid content and yield,and the highest relative content of unsaturated fatty acids.C.vulgaris CJ15 was cultured by different nutritional methods?autotrophy,heterotrophication and mixed culture?,and the contents of various biochemical components?protein,polysaccharide,pigment,crude fiber,amino acid,lipid and fatty acid?were determined.The results showed that the nutrient pattern significantly affected the growth of C.vulgaris CJ15 and the metabolism of biochemical components.The content of protein and crude fiber of C.vulgaris CJ15 was the highest when glucose was used as carbon source for mixed culture,which were 6.70%and 6.75%.While the mixed culture with sucrose as carbon source was more beneficial for C.vulgaris CJ15 metabolites to the directed synthesis of polysaccharides.Under photosynthetic autotrophic conditions,C.vulgaris CJ15 alga cells had strong growth capacity and the highest biomass and biomass productivity(2.59 gl^-1and 1.85 gl^-1d^-1),and higher polyunsaturated fatty acid percentage,glucose,crude fiber yield and pigment content,oil yield and essential amino acid content.Taking all things into consideration,the most suitable nutrient method for C.vulgaris CJ15 culture of high-density,high-bioactive substances is photosynthetic autotrophy.The extraction pretreatment of C.vulgaris CJ15 fatty acid and the process parameters of methyl esterification were optimized.The crushing rate of C.vulgaris CJ15 cells on Grinding ultrasonic aid was the highest,up to 92.35±0.67%.The optimal process parameters of C.vulgaris CJ15 fatty acid methylation were determined by single factor experiment and response surface optimization experiment?KOH concentration:0.79mol/L,saponification temperature:65?,saponification time:38.20min,H₂SO₄volume fraction:3.32%,esterification temperature:61.3?,and esterification time:25min?.The effects of C.vulgaris CJ15 culture conditions?nitrogen concentration,phosphorus concentration,p H?were studied.The yield sequence of C.vulgaris CJ15 unsaturated fatty acids at different nitrogen concentrations was as follows:12.6 mmol/L>7.6mmol/L>22.6mmol/L>17.6mmol/L>27.6mmol/L>0.0mmol/L.The rela-tive content of unsaturated fatty acids was the highest when the phosphorus concentration was 0.06g/L and 0.08g/L,both of which were 70%,and the yield was the highest at the concentration of 0.06g/L,which was 0.28±0.03g/L.Therefore,the phosphorus source concentration suitable for the growth and unsaturated fatty acid accumulation of C.vulgaris CJ15 was 0.06g/L.p H had a great influence on the accumulation of unsaturated fatty acids in dominant alga strains.p H of 6.5 was the optimal p H for the accumulation of high biomass and high unsaturated fatty acids in C.vulgaris CJ15.Based on the change of the unsaturated fatty acids content in C.vulgaris CJ15,which was separately cultured in nitrogen deficiency?0.0mmol/L,treatment group?and normal condition?17.0mmol/L,control group?.Transcriptome sequencing and filtration were used on C.vulgaris CJ15 algal cells under the two culture conditions.The number of Clean reads of treatment group and control group were 42713412 and 42337990,accounting for98.64%and 97.77%of Raw reads,respectively.The Q20 and Q30 values of Clean Reads in the treatment group and the control group were greater than 90%and 80%,respectively,which shown that the quality of reads was high and the base information obtained by sequencing was reliable.Trinity was used to assemble the clean reads?de novo assembly??clustering and eliminate redundancy,A total of 44302 Unigene sequences were obtained for subsequent analysis.Various software was used to compare the Unigene in 7 functional databases?KEGG,GO,NR,NT,Swiss Prot,Pfam and KOG?.There were35292Unigenes were annotated,accounting for 79.66%of the total Unigene.|log2FC|?2 and FDR?False Discovery Rate??0.001 were used as the screening conditions to get the significantly differentially expressed genes?DEGs?.GO enrichment analysis and KEGG Pathway enrichment analysis were carried out for DEGs.And the biosynthesis pathway of C.vulgaris CJ15 unsaturated fatty acid was predicted.Beside,we found that the genes FASN,Fab H,FATA,FAD2 and SCD(C16:0-Co A?C16:1?n-7?were significantly up-regulated,the genes Fab D and TER were significantly down-regulated in the synthesis pathway of unsaturated fatty acids.That provides a theoretical basis for the future use of genetic engineering and other means to increase the content of C.vulgaris CJ15 unsaturated fatty acids.