Fatty Acid Synthesis And Transcriptome Analysis On Temperature Stress In Schizochytrium

Docosahexaenoic acid(DHA)is a kind of unsaturated fat that is extremely important for human health,which contributes to the risk reduction of cardiovascular disease,brain development,enhancement of children's memory,and blood lipids decreasing.schizochytrium is the most promising marine fungus with simple fatty acid species and high DHA content.However,there is still unclear synthesis mechanism on DHA of Schizochytrium limacinum,as well as its yield needs to be improved.The aim of this study was to explore the key transcription factors in the DHA synthesis pathway in schizochytrium by transcriptomics combined with Weighted Gene Co-Expression Network(WGCNA)analysis,so as to provide some theoretical support for elucidating the mechanism of DHA synthesis.The main research contents are as follows:(1)The experimental strains were sterilized,and the purified strains were obtained by spread plate and picking single colonies.The final experimental strains were identified as schizochytrium by molecular biological means.The optimal scheme of culture medium was as follows: glucose 7 g/L,yeast powder 1 g/L,peptone 1 g/L.The growth curve showed that schizochytrium grew fastest at 25?,and both high and low temperature stresses inhibited schizochytrium growth.The total fatty acid content was the lowest at30°C,when DHA accounts for only 6% of the total fatty acids.The content of total fatty acids was the highest at 15?,when DHA accounts for only 64% of total fatty acids.(2)Transcriptome data at different culture temperatures(15?,20?,25?,and 30?)were obtained using Illumina Hiseq sequencing technology.The results of KEGG pathway analysis showed that fatty acid synthase(Fas)gene and fatty acid desaturase gene(Fad E)were significantly up-regulated at 30? than at 20?.The key transcription factor MYB(MYB3R1,schi20061090)for the synthesis of DHA by Schizochytrium limacinum,and related genes such as MAPK,CYB5D1,and CYP450 were finally figured out by the weighted gene co-expression network analysis(WGCNA)technology.The q PCR validation of MYB,FAD,CYP450,FAS,and PFA1 was conducted by q PCR technology,which revealed that compared with that at 30?,MYB,CYP450,and PFA1 of in schizochytrium at 20? were up-regulated by 83%,46%,and 343%,respectively,and the FAD and FAS were down-regulated by 36% and 51%,respectively.(4)Total of 42 MYB family members,6 FAD members and 14 CYP450 family members were identified in the schizochytrium genome.Of the 42 MYBs,24 were1R-MYBs,13 were 2R-MYBs,and 5 were 3R-MYBs,and no 4R-MYBs were found.A total of 8 motifs were found in the 42 MYB proteins,each of which had a Myb?DNA-binding domain,in addition to 26 members with a Myb?DNA-bind?6domain and 2 members with a Dna J domain.A total of 3 motifs were found in FAD,and all of them had FA?desaturase conserved domains,of which the Lipid?DES domain and Cyt-b5 domain were also found in 4 members.Except for schi20041340,which contained both the p450 domain and the Cyt-b5 structural domain,other CYP450 s only contained the Cyt-b5 domain,and 10 motifs were found in CYP450.Collinearity analysis showed that both MYB and CYP450 of schizochytrium underwent gene family expansion by means of segmental duplication,tandem duplication,and dispersed duplication,while no segmental duplication and tandem duplication events were found in FAD.The Ka/Ks value of duplicated gene pairs of MYB and CYP450 was less than 1,indicating that the MYB and CYP450 gene families of schizochytrium were under purifying selection during expansion.In this study,the main conclusions were as follows:(1)Low temperature stress is beneficial to the accumulation of total fatty acids and DHA in Schizochytrium,while the opposite is true under high temperature stress.(2)When the content of total fatty acids is higher,the proportion of DHA in total fatty acids is greater,and the proportion of saturated fat is smaller.(3)There are both FAS pathway and PKS pathway for fatty acids synthesis in schizochytrium,and the efficiency of fatty acids synthesis by PKS pathway was much higher than that by FAS pathway.(4)The expression of related genes in PKS pathway are dominant under low temperature stress,when fatty acids synthesis efficiency is relatively high and the main product DHA.The expression of related genes in FAS pathway is dominant under high temperature stress,when the synthesis efficiency of fatty acids is low,and the products are mainly saturated fatty acids.(5)The transcription factor MYB plays a key regulatory role in DHA synthesis in schizochytrium.