A Preliminary Study On The Regulation Of PUFA Synthesis By Physicochemical Factors In Phaeodactylum Tricornutum

The growth and life activities of microalgae are affected by various physical and chemical factors in the culture environment.By artificially adjusting these factors,the growth of microalgae can be effectively promoted,and the synthesis and accumulation of unsaturated fatty acids can be enhanced.This is of great significance and prospect for using microalgae as a source of unsaturated fatty acids and realizing their application in commercial production.In this study,the total lipid（TFAC）and fatty acid content of Phaeodactylum tricornutum were investigated under nitrogen starvation,low temperature stress,10^-6mol L^-1Fe²⁺,2 mmol L^-1dithiothreitol（DTT）and 2.5 mg L^-1methyl jasmonate（MJ）.The purpose of this study is to determine the mechanism of polyunsaturated fatty acid（PUFA）synthesis in P.tricornutum at the molecular level by using modern omics techniques.The results showed that:（1）the highest TFAC yield was detected in the nitrogen starvation group until the end of stress;the highest docosahexaenoic acid（DHA）,eicosapentaenoic acid（EPA）and PUFA yields were detected in the low temperature stress group.The contents of DHA,EPA and PUFA were also increased in DTT and MJ treatment groups.The accumulation of EPA and PUFA was not detected in Fe²⁺treatment,but the factors had a positive effect on the accumulation of DHA and TFAC.（2）Real-time quantitative PCR results showed that the PTD4 gene was highly expressed under nitrogen starvation.The expression of PTD5a gene was inhibited under nitrogen starvation and low temperature stress,and MJ could effectively promote the expression of this gene.Three groups of treatments can promote the expression of PTD5b gene,and the effect of MJ is the most significant.PTD6 gene was up-regulated under MJ treatment,and nitrogen starvation severely limited its expression.（3）The results of lipidomics analysis of the algae under low temperature stress,DTT and MJ treatment showed that glycerophospholipids and glycerolipids were closely related to the synthesis mechanism of PUFA under stress response.DTT could increase the DHA content in phosphatidylethanolamine（PE）and thioisorhamnosyldiacylglycerol（SQDG）and the EPA content in phosphatidyl-glycerol（PG）,phosphatidylcholine（PC）,phosphatidylserine（PS）and monogalactosyldiacylgl-ycerol（MGDG）,and inhibit the accumulation of DHA in PC.MJ treatment only reduced the DHA content in PA;low temperature stress promoted the accumulation of DHA in PC and DGDG and EPA in PE,and reduced the content of DHA in PA and MGDG.It is speculated that these three physical and chemical factors are more eukaryotic pathways that affect the PUFA synthesis of the algae.（4）A total of 661differentially expressed proteins were identified by TMT quantitative proteomics analysis.According to the functional analysis of differentially expressed proteins,the transcription factors involved in the PUFA metabolic process of the algae under low temperature stress,DTT and MJ treatment were preliminarily screened.These transcription factors belong to AP2/EREBP,C2H2 zinc finger,RING zinc finger and heat shock transcription factor（HSF）transcription factor family;the interaction between these selected transcription factors and the cis-acting elements of PTD5a and PTD5b genes was predicted,and multiple possible transcription factor binding sites were predicted.Among them,the most likely transcription factor binding site in the2000 kb upstream of the PTD5a gene was DREB1E（ATGTCGGC）and ZBTB32（TGTACAGTAT）,and the transcription factor binding sites in the 2000 bp upstream of the PTD5b gene were MOT2（ATATA）and SKN7（GGCCAT）,but its specific expression regulation needs further experimental verification.In summary,this paper concludes that:（1）Low temperature,DTT and MJ are beneficial to improve the synthesis and accumulation of PUFA in P.tricornutum;（2）PTD5b gene plays a more critical role in the upstream of PUFA synthesis.（3）Low temperature,DTT and MJ could increase the accumulation of DHA and EPA.