Long Non-Coding RNA Identification And Function Analysis In The Marine Microalga Emiliania Huxleyi After Virus Infection

The coccolithophore Emiliania huxleyi,a cosmopolitan eukaryotic unicellular alga,is the most abundant coccolithophore in the marine system,forming large oceanic blooms that cover thousands of square kilometers.It is a key species for current studies on global biogeochemical cycles and climate modeling,because of the ability of high-yielding of CaCO3 and dimethyl sulfide(DMSP).E.huxleyi’s massive annual blooms in the marine system are routinely infected and terminated by E.huxleyi virus(EhV),a specific nucleocytoplasmic large double-stranded DNA virus.The lysis of E.huxleyi affects the release of calcite skeleton and the climate-bioactive gas dimethyl sulfide(DMS),which can affect cloud formation.Therefore,the interaction between the E.huxleyi and EhV is a major factor determining the fate of carbon and sulfur in the ocean,playing an important roles in carbon and sulfur cycle,climate change,the reshape of community structure and geological evolution.With the gradually recognition of the genome structural characteristics,horizontal gene transfer phenomenon and the rich genetic hereditary diversity of virus and it hosts,Eh-EhV has become a key host-pathogen model system.However,the complex interaction mechanism between them is not very clear,especially lack sufficient experimental evidence to systematically elucidate the virus-host interaction mechanism at the molecular level.Long non-coding RNA(lncRNA)is an important non-coding transcript larger than 200 nt in length and have extremely low protein coding ability or without protein coding ability,but it plays important roles in eukaryotic gene regulation at transcription post-transcription and epigenetic level.lncRNA is commonly used as a functional regulatory element to participate in the regulation of gene expression in eukaryotic biological processes,such as cell differentiation and individual developmental.It becomes the current hot topics in the study of molecular biology and genetics.Current research of lncRNA has been focused on animals and plants such as humans,mice,model organisms and economic species.However,the investigation of marine phytoplankton lncRNA that respond to virus infection has not been performed.In this study,the lncRNA transcriptomes involved in virus infected E.huxleyi BOF 92 by EhV-99 B1 were investigated by using Illumina HiSeq 4000 high-throughput sequencing technology combined with bioinformatics and molecular biology experiments.Three cDNA libraries,generated 6 and 45 h after viral infection(Exp)were compared with three libraries from the corresponding times uninfected cultures(Con).We constructed viral infection-associated mRNA and lncRNA transcriptome expression profiles,screened and validated differentially expressed mRNAs and lncRNAs in viral infections.Through the analysis of two different transcriptomes,our results revealed the differential changes of related transcripts under viral infection and their metabolic regulation pathways.The binding sites of lncRNA and miRNA were predicted by miRanda and RNA 22,and their possible interaction pattern were studied by using the dual-luciferase reporter system.The main results are as follows:(1)A total of 38554 mRNAs with an average length of 1117 bp were identified.Quantitative expression analysis showed that 15374 and 7334 differentially expressed mRNAs were obtained after virus infection 6 h and 45 h,respectively,including 5422 differentially expressed genes at two time points.10 differentially expressed mRNAs were selected randomly and verified by qRT-PCR.The results showed that transcriptome analysis is reliable.(2)A total of 24193 lncRNAs with an average length of 900 nt were identified,including 12449 long intergenic non-coding RNAs(lincRNAs)and 11744 anti-sence lncRNA.We obtained 4797 and 876 diff-erentially expressed lncRNA after virus infection early and late stages,respectively.The 520 lncRNA were the common differentially expressed genes in the two time point.The qRT-PCR results showed that more than 73%of 20 differentially expressed lncRNA were consistent with the sequencing results,indicating that lncRNA transcriptome analysis is reliable.(3)GO and KEGG analysis showed that the differential expression genes were mainly involved in metabolic pathway.The biosynthesis of secondary metabolites,biosynthesis of antibiotics,carbon metabolism,biosynthesis of amino acids,fatty acid metabolism,porphyrin and chlorophyll metabolism,biosynthesis of steroid skeletons,biosynthesis of steroids and glycolysis or gluconeogenesis are remarkable enrichment results of mRNAs.The Intersection of co-located and co-expressed target genes of lncRNA was significantly enriched to antibiotic biosynthesis,glycolysis or gluconeogenesis,secondary metabolic biosynthesis and fatty acid metabolism.The co-enrichment metabolic pathways of differentially expressed mRNAs and lncRNA target genes indicated that lncRNA might be involved in the metabolic regulation of secondary metabolism,carbon metabolism and lipid metabolism in the host after virus infection.(4)The binding sites prediction between 20 lncRNAs and 5 miRNAs indicated that lncRNA can bind to multiple miRNAs and miRNA can also bind multiple lncRNA as well.Multiple binding sites might exist between the same lncRNA and the same miRNA.We finally selected LNC-011228 and Ehe-miR6 for validated the possible interaction pattern using the psiCHECK2 dual-luciferase reporter system.Wild-type and mutant-type dual-luciferase recombinant vectors containing LNC-011228 and Ehe-miR6 binding sites were constructed,and the recombinant vector was co-transfected into 293T cells with miRNA.The results showed that Ehe-miR6 significantly inhibited the relative luciferase activity of the psiCKECK2-wt recombinant vector,indicating that there may be a targeting relationship between LNC-011228 and Ehe-miR6.