| Body color is one of the characteristics of fish morphological diversity,and its changing law during the growth and development of fish has attracted more and more attention.Plectropomus leopardus,with its bright red body color,is well received by the market.However,during the artificial breeding process,the body color of Plectropomus leopardus juveniles changed from red to black,which caused a lot of economic losses.Therefore,studying the molecular regulation mechanism of its "body color change" phenomenon will not only help to improve our understanding of fish body color inheritance,but also provide basic data for solving the industrial problems of Plectropomus leopardus.In this study,RNA-Seq technology was first used to sequence the transcriptome of Plectropomus leopardus juvenile,comparing the gene expression levels in the transcriptome of red and black skin tissues,screening differentially expressed genes,and mining genes and pathways related to body color.The expression levels of related genes in two skin tissues were verified by RT-q PCR.Subsequently,genes related to melanin production(ednrb,mc1 r,sox10,tyrl,tyrp1 and wnt5a)were selected,based on the sequence information of the Plectropomus leopardus genome,the amino acid ORF sequences of these genes were analyzed,and RT-q PCR was used to detect them in Expression in the skin of red and black Plectropomus leopardus juvenile.Main research experiment results:1.Using RNA-Seq technology to perform transcriptome sequencing on the skin of red and black Plectropomus leopardus juvenile,a total of 106.56 Gb data was measured.After assembly and de-redundancy,156,102 All-Unigenes were obtained,with average length,total length,N50,GC content of 1,550 bp,241,959,840 bp,3,275 bp,45.72%,respectively.Then,the All-Unigene is compared to the seven major functional databases to annotate,and finally there are 93,383(NR: 59.82%),83,083(NT: 53.22%),80,627(Swiss Prot: 51.65%),82,007(KEGG: 52.53%),73,896(COG:47.34%),72,874(Pfam: 46.68%),21,340(GO: 13.67%)All-Unigene obtained functional annotations.Using Transdecoder software to predict 78,839 CDS.Using the MISA software,59,876 SSRs were detected among 35,258 All-Unigenes,and19,887 All-Unigene encoding transcription factors were predicted.2.Analyze the expression of genes in the skin transcriptome of red and black Plectropomus leopardus juveniles,and select 25,862 differentially expressed All-Unigene,key genes of body color such as tyrp1,slc45a2,xdh,slc24a5,tyrl,hps5,pomc and pmel are annotated.Through KEGG Pathway enrichment analysis,it was found such as tyrosine metabolism,MAPK,Wnt and melanogenesis pathways are involved in the regulation of body color changes.Thirteen differentially expressed genes were screened for RT-q PCR verification.The RT-q PCR expressions of th,erk2,tyrp1,plcb1,map2k2,creb3l3,slc45a2,xdh,slc24a5,tyrl,hps5,hes1-b and trpm7 are consistent with RNA-Seq expression.It shows that the experimental results are more reliable.3.Compared with other bony fishes,Plectropomus leopardus has a higher degree of tyrl gene differentiation,while ednrb,mc1 r,sox10,tyrp1 and wnt5 a have a lower degree of differentiation.There was no significant difference in the expression of the ednrb,mc1 r,sox10 and wnt5 a genes between the red and black skins of Plectropomus leopardus.Compared with red skin,the expression of tyrl and tyrp1 gene m RNA was significantly up-regulated in black skin,indicating that tyrl and tyrp1 are involved in the regulation of Plectropomus leopardus juvenile body color from red to black. |
