The Differential Expression Of MRNA And MiRNA In Cold Sensitive And Cold Tolerant Litopnaeusvannamei Under Low Temperature

The Litopenaeus vannamei(L.vannamei)is one of the most widely cultured shrimp species in the world.The specie is a warm water species.The suitable water temperature is 18-35℃ for survival,feeding and growth of L.vannamei.When the water temperature is lower than 18℃,the survival,feeding and growth rate of shirmp will decreases.Therefore,the breeding benefit will be limited by extreme weather,season and region.It is urgent to study the cold resistance mechanism of L.vannamei and improve its cold tolerance.In this study,we performed transcriptomic sequencing of mRNA and miRNA on L.vannamei(cold-tolerant and cold-sensitive)under control temperature(28°C),cold-stress(16℃)and recovery to 28℃.The purpose of this experiment is to obtain differentially expressed genes(DEGs),differentially expressedmiRNAs,differentially expressed miRNA-target and regulatory pathways under low temperature in L.vannamei through transcriptome sequencing of mRNA and miRNA.The main results of the present study are as follows :1.Low temperature transcriptome of L.vannamei84.5 gigabases(Gb)of sequences were totally generated from 12 L.vannamei hepatopancreas libraries.De-novo assembly generated a total of143,029 unigenes,of which 34.08% were annotated in the Nr database.We analyzed the DEGs between nine comparison groups and detected a total of21,026 DEGs.Enrichment analyses of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways analysis revealed that pathways only significantly enriched by DEGs between different temperatures in GH2 but not in GH1 included lysosome,sphingolipid metabolism and nitrogen metabolism,which may are involved in cold-tolerance.According to our transcriptome data and genes reported as cold-tolerant related,we obtained 21 candidate genes that may be associated with adapting to cold-stress in L.vannamei,including C-Type lectin,Acid ceramidase,zinc proteinase,Synapse-associated protein of 47 k Da,DEAD/DEAH box helicase,caspase-3,etc..Furthermore,12 most significantly DEGs under cold-stress in L.vannamei from transcriptome analysis were selected for quantitative real-time PCR(qPCR)validation.The result of most mRNA of confirmed by qPCR is similar to mRNA sequencing.2.MiRNA sequencing analysis of L.vannamei under low temperatureBy using Solexa sequencing,57,48 and 58 known mature miRNAs were obtained from smallRNA libraries at control temperature,low temperature and recovery to 28℃,respectively.25 miRNAs were significantly differential expressed in M16 vs M28,and nine miRNAs were significantly different expressed in two comparison groups(M16 vs M28;MR vs M28).Furthermore,the result of three miRNAs of confirmed by qPCR were similar to miRNA sequencing.3.MiRNA target gene prediction and verificationBy predicting target gene for differentially expressed miRNA,17,641,11,570 and 17,252 miRNA-targets were obtained from the differentially expressed miRNAs of M16 vs M28,MR vs M28 and MR vs M16,respectively.Moreover,we also observed 4 genes that significantly differential expressed both in cold regulated target genes of miRNAs and transcriptome,including the nuclear export mediator factor Nemf-lik,synapse-associated protein,seleno proteins and DEAD-boxRNA helicase Variant 1.Then,it was verified by qPCR that it expressed in the hepatopancreas of L.vannamei under low temperature at different time.The KEGG enrichment analysis of the target gene revealed that the most significant pathway was fatty acid degradation in three comparison groups.