The Complete Mitochondrial Genome Of Mastacembelus Armatus And Development Of Immune Genes Based On Transcriptome

Mastacembelus armatus,belonging to Mastacembelidae,Synbranchiformes,Osteichthyes,is mainly distributed in tropical and subtropical rivers and lakes.Its fresh muscle contains high content of crude protein and abundant amino acids.M.armatus is very popular with consumers as a high grade table fish.Recently,the wild population has declined gradually due to overfishing and various ecological imbalance.The aquaculture of this spiny eel has flourished in Guangdong,Fujian and Guangxi provinces in recent years.At present,studies on this spiny eel mainly focused on its biology,physiology and genetics.This study successfully sequenced complete mitochondrial genomes of M.armatus and Mastacembelus erythrotaenia using Sanger sequencing technology and further analyzed their sequence composition and phylogenetic information.Besides,this study also carried out transcriptome sequencing on the spleen of M.armatus before and after infection by Aeromonas veronii based on Illumina sequencing technology,and further obtained a lot of functional genes and molecular markers.We also got a lot of information on different expression genes through comparing gene expression levels before and after infection.Finally,many TLR genes were also screened out in the results of trancriptome assembly.Some main conclusions were summarized as follows:1.The complete mitochondrial genomes of M.erythrotaenia and M.armatus(HN)were 16493 bp and 16487 bp in length,respectively.The complete mtDNAs of the two spiny eels included 13 protein-coding genes,22 transfer RNA genes,two ribosomal RNA genes and a control region.Except for COI(GTG)gene,12 protein-coding genes of the two spiny eels all started with typical ATG codons.The phylogenetic trees strongly supported that M.armatus from Hainan(HN)had a closest relationship with other species in Mastacembelus.The phylogenetic analysis also showed that Sinobdella sinensis clustered with Macrognathus aculeatus and their sequence composition was also very similar with only a little difference,which might mean they represented the same species.The phylogenetic tree based on 11 protein-coding sequences from some species of Mastacembelidae and Synbranchidae showed that Mastacembelidae clustered with Synbranchidae and formed a clade independent from other species,which supported the view that Mastacembelidae belongs to Synbranchiformes.2.This study obtained 110,328 unigenes with 44.42% GC content through assembly and annotation for the transcriptome sequencing of Zig-zag eel spleen.Out of 110,328 unigenes,27,098 were successfully annotated by public protein databases including NR,Uniprot,KEGG and KOG.The unigenes that annotated by GO were distributed into 47 functional categories.In addition,15,219 unigenes were included in 25 categories in KOG database and 16,523 unigenes were distributed in 387 pathways of six classes.3.The results of different expression analysis showed that a total of 1278 significantly differentially expressed unigenes with 767 unigenes upregulated and 511 unigenes downregulated.These significantly differentially expressed unigenes were assigned into 43 GO categories through GO enrichment analysis and 6 categories were significantly enriched including extracellular region part,immune system process,response to stimulus,multi organism process,locomotion and molecular function regulator.The KEGG analysis showed that a total of 16 pathways were significantly enriched and all of them participated in immune regulation,mainly including Cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,HTLV-I infection,TNF signaling pathway and so on.4.Based on the results of transcriptome analysis,this study successfully filtrated 11 TLR cDNA sequences including TLR2,TLR3,TLR5 M,TLR5S,TLR7,TLR8,TLR9,TLR14,TLR21,TLR22 and TLR23.Except for TLR21 and TLR23,they all contained complete coding region.The results of SMART domain prediction showed that these TLRs from the spleen transcriptome of Zig-zag eel all contained many LRR domains,one transmembrane domain and TIR domain.The analysis of sequence similarity and phylogeny showed that these TLRs of Zig-zag eel were homologous to other teleosts.The results of tissue expression detection showed that the highest expression of most TLRs occurred in important immune organs like spleen and gill.After infection,the responses of TLRs were different.The expression of most TLRs downregulated,while TLR5 S upregulated dozens of times.The positive selection analysis based on site model of PAML showed that TLR2,TLR5 M,TLR5S,TLR7,TLR8,TLR9,TLR14 and TLR22 all had undergone positive selection pressure.