Multi-Omics Correlation Analysis Of Lipid Metabolism Remodeling In Emiliania Huxleyi-Virus System

The interaction of Emiliania huxleyi virus(EhV)-host is important for global climate change and the carbon/sulfur biogeochemical cycle.The fight between the virus and host for infection and resistance is essentially an arms race for biochemical metabolism.As a large double-stranded DNA virus,Eh V has evolved a unique state of "virocell metabolism" to meet their high demands.However,there is still little understanding of the regulatory mechanism of this metabolic pattern.Micro RNAs(miRNAs)are important regulators in response to environmental stress.At present,little is known about the the relationship between miRNA and phytoplankton metabolic regulation.Here,our study focused on the molecular function of miRNA in Eh V remodeling host lipid metabolism network based on the Ehux-BOF92-Eh V99B1 model system.Integrated epigenomics,transcriptomics and lipidomics techniques were used to analyze the major pathways of lipid metabolism mediated by viruses,and identify the key lipid metabolism signal pathways targeted by miRNAs.In addition,Dual-luciferase reporter assay was used to verify the targeted relationship between miRNA and target genes.The main results are as follows:(1)Virus-mediated transcriptome remodeling linked with lipid metabolism: Analysis of the transcriptome data showed that differentially expressed m RNAs induced by viral infection were significantly enriched in fatty acid,steroid biosynthesis,photosynthesis,amino acids,and carbohydrate metabolism.The metabolic pathway centered on lipid metabolism showed that the expression levels of key enzyme encoding genes of ACER,UGCG and GBA involved in glycerolipid,fatty acid and sphingolipid metabolism pathways were significantly changed.For example,the gene of alkaline ceramidase(ACER)that catalyzes the hydrolysis of ceramide in sphingolipid metabolism was significantly up-regulated,while the gene of glucosylceramide synthase(UGCG)that catalyzes the glycosylation of ceramide to produce sphingolipids was significantly down-regulated.Therefore,it accelerated ceramide,an intermediate product of sphingolipid metabolism to degrade rapidly,preventing host cells from premature apoptosis.On the other hand,the gene of glucocerebrosidase(GBA)that catalyzes the hydrolysis of sphingolipids(GSLs)is significantly up-regulated.The synergistic effect of the down-regulated UGCG and up-regulated GBA inhibited the synthesis and accumulation of viral sphingolipids(v GSLs),thus it impacted on virus assembly and release.Taking together,it seemed that the virus-host had launched a fierce struggle in the sphingolipid metabolism pathway during infection.In addition,tween of genes conding ABC transporters involved in lipid transport were significantly differentially expressed during viral infection,indicating that viral infection affected the transport process of host cell lipid metabolites.(2)The key pathway of lipid metabolism targeted by miRNA: Based on our previous miRNA sequencing results,a total of 24 host miRNAs and 2 viral t RFs in response to viral infection were identified.Verified by stem-loop q RT-PCR,ehx-mi R-2,ehx-mi R-5,ehx-mi R-6,ehx-mi R-7 and t RF3-Arg TCT showed expressed differentially.Pathway analysis showed that the potential target genes of these differentially expressed miRNAs and t RF3 were significantly enriched in lipid metabolism,ABC transporters,photosynthesis,carbohydrates,and aminoacyl-t RNA biosynthetic metabolic processes;Subsequently,target genes negatively related to miRNAs/t RF expression were screened,and an integrated "miRNA-m RNA-lipid metabolite" metabolic pathway map was developed to present the information of lipid metabolites and genes involved in the pathway.The results showed that ehx-mi R-5 and ehx-mi R-6 might inhibited target gene h SPT(the host serine palmitoyltransferase)expression,resulting in a significant decrease in the abundance of ceramide(Cer and Cer G1);ehx-mi R-5 inhibeted target gene TGL(triglyceride lipase),resulting in a reduction in the content of glycerolipid(DAG and MAG)in the glycerolipid metabolism pathway;ehx-mi R-6 and ehx-mi R-7 inhibit the expression of acetyl-Co A acetyltransferase(ACAT)gene,resulting in an increase in the content of most fatty acids(FAs: C14-C18)through the fatty acid degradation pathway.In addition,ehx-mi R-6,ehx-mi R-7 and t RF3-Arg TCT significantly target the ABC transporter gene and regulated the transport of lipid metabolites between cells and cell compartments.(3)MiRNA targeting transcription factor: miRNAs/tRF could target many different transcription factors,such as transcription initiation factor TFIID subunit,transcription factor with containing HOX domain,Zn-finger(C2H2 type),b ZIP transcription factor,Myb superfamily and heat shock transcription factor,etc.The expression abundance values of these transcription factors were significantly changed during viral infection,suggesting that miRNAs/t RF induced by viral infection might regulate host lipid metabolism by targeting transcription factors.(4)Identification of tRF3-Arg TCT target gene-Oxygen-evolving enhancer protein 1 of photosystem II(PSBO-1)gene by luciferase reporter assay: With identification of sequencing,the sequences of psi CHECK-2-wt and psi CHECK-2-mut were cloned successfully.Compared with the group of psi CHECK-2-wt co-transfected with mimics NC(0.69±0.02),the relative luciferase activity in group of psi CHECK-2-wt co-transfected with t RF3-Arg TCT(0.84±0.02)showed a significant increase(p < 0.01),suggesting that PSBO-1 is a target gene of t RF3-Arg TCT.