| Botia superciliaris,an endemic cobitid fish in China,is traditionally appreciated by Chinese consumers because it is tender and delicious.In recent years,as a new type of ornamental fish,it is becoming more and more popular because of its beautiful black and yellow skin stripes.Then no studies have been reported on the genetic basis of the skin striations of the Botia superciliaris.In this paper,the wild Botia superciliaris was used as the research material,RNA-seq and small RNA-seq technique was used for the first time to study the transcriptome of the black and yellow skin samples of the Botia superciliaris.We illustrated the key genes and important miRNAs involved in the skin pigmentation process of the Botia superciliaris and constructed miRNA-mRNA regulatory network for the different pigmentation between black and yellow skins.These results will help to elucidate the molecular mechanism of skin pigmentation for the Botia superciliaris and be helpful to molecular assisted breeding of skin color of ornamental fish.The main results of this study are as follows:(1)In this study,RNA-seq sequencing technology was used to carry out mRNA sequencing of the black and yellow skin of the Botia superciliaris,a total of 49.08 million(M)(49.08 ± 0.85 M,n = 3)and 51.72 M(51.72 ± 0.32 M,n = 3)Raw reads were generated,then full-length transcripts were de novo assembled using Trinity based on Clean data,and resulted in 159,327 Unigenes that were used as reference sequences.Subsequently,all Unigenes were compared against protein databases for annotation,and 20,178 high-confident Unigenes were obtained.(2)The RPKM values of 20,178 highly confident unigenes were analyzed with edgeR software to reveal the expression differences.A total of 3169 genes exhibited significantly different expression patterns(Fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05),including 1891 up-regulated genes(59.67%)and 1278 down-regulated genes(40.33%)in black vs yellow skin.(3)Functional enrichment analysis was performed on 3169 differentially expressed genes,and the result showed that these genes were enriched in 50 GO terms and 10 KEGG pathways(q≤ 0.05),which included melanogenesis,and contained 21 up-regulated genes and 5 down-regulated genes in black vs yellow skin.(4)Using small RNA-sequencing,a total of 355 miRNAs were identified in the black and yellow skin tissues of the Botia superciliaris,including 9 conserved and 38 novel miRNAs.Furthermore,87 differentially expressed miRNAs were identified between black and yellow skins(Fold-change ≥ 2 or ≤ 0.5 and q ≤ 0.05),including 50 up-regulated and 37 down-regulated miRNAs in black vs yellow skin.Through target gene prediction,the miR-217-5p was predicted to be related to pigment formation by targeting Zgc gene.(5)The miR-217-5p inhibited and overexpressed Botia superciliaris were constructed through in vivo injection.The skin melanin content detection,the qRT-PCR quantification of miR-217-5p and its target gene Zgc,and the dual-luciferase reporter assay revealed that miR-217-5p can indeed inhibit pheomelanin formation by directly targeting Zgc.In conclusion,high-throughput sequencing was used for the first time to study the mRNA and miRNA transcriptome of the black and yellow skin samples of the Botia superciliaris,and important genes and miRNAs related to the formation of skin stripe pigment were identified,and the miRNA-mRNA regulatory networks for different skin pigmentation were constructed in this study.miR-217-5p can reduce the pheomelanin formation by target Zgc. |
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