Effects of miRNA on Gene Expression of Sphingolipids Metabolism and Small RNA Analysis of Silencing NlSPT1 and NlSMase4 in Nilaparvata lugens
[Background]Sphingolipids are the second major type of membrane lipids and mediate various biological processes as signal transducers,including cell growth,development,reproduction and apoptosis.Sphingolipid metabolism is tightly regulated by sphingolipid metabolizing enzymes to maintain the homeostasis of metabolism in vivo.[Objective]The objectives of this study are to investigate the relative transcript levels of genes related to the sphingolipid metabolism pathway after silencing of the core components of microRNA(miRNA)biosynthesis pathway,NlAgo1,NlDicer1 and NlDrosha by RNA interference(RNAi),and analyze the differentially expressed miRNAs after silencing of serine palmitoyltransferase 1(SPT1)and sphingomyelinase 4(SMase4)gene combined with small RNA sequencing of Nilaparvata lugens,explore the role of miRNAs in the sphingolipid metabolism of N.lugens,and to provide a new molecular target for pest control.[Method]RNAi was performed with double stranded RNAs(dsRNAs)targeting NlAgo1,NlDicer1 and NlDrosha at 1 day post adult eclosion(1 PAE),respectively,and ds GFP was used as control.The ovaries at 5 PAE were dissected and β-actin was used as internal reference gene,the transcript levels of genes related to the sphingolipid metabolism pathway were detected by reverse-transcription quantitative PCR(qRT-PCR).miRNAs that may regulate the expression of NlSPT1 and NlSMase4 were predicated based on small RNA libraries combined with miRNA-target prediction software.Differentially expressed miRNAs after the silencing of NlSPT1 and NlSMase4 were identified and the target gene enrichment was analyzed by small RNA sequencing.[Result]RNAi-mediated silencing of NlAgo1,NlDicer1 or NlDrosha significantly up-regulated the expression of genes related to the sphingolipid metabolism pathway,including NlSPT1 and NlSMase4 in ovaries.Target gene prediction revealed 6 miRNAs and 13 miRNAs that could bind NlSPT1 and NlSMase4.The target genes of differentially expressed miRNAs that silencing NlSPT1 and NlSMase4 were significantly enriched in biological processes,including nuclear and protein binding,as well as metabolic pathways such as endocytosis,endoplasmic reticulum processing,MAPK signaling pathway,TOR signaling pathway,apoptosis,and lipid metabolism.[Conclusion]NlAgo1,NlDicer1,and NlDrosha-dependent miRNAs affect sphingolipid metabolism by influencing the expression of genes encoding sphingolipid metabolizing enzymes.The silencing of NlSPT1 and NlSMase4 induced changes in miRNA expression levels in N.lugens ovaries.These research results can provide a theoretical basis for pest control based on sphingolipid metabolism genes.
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