Study On Skin Transcriptome Of Crimson Snapper (Lutjanus Erythropterus), And Their Pigment Genes Related Cloning, Expression And Evolution Analysis

Coloration is one of the most diverse phenotypic traits in vertebrates and it plays multiple adaptive functions like camouflage, warning or threatening of predators and species recognition. Skin coloration can be influenced by many factors, such as genetics, diet, environmental or healthy condition et al.. Neverthless, genetic is still the major determination, the kind of pigmentation related genes and their variant expressions are the major reason of diverse form coloration.Crimson snapper(Lutjanus erythropterus), which is distributed over the Indo-West Pacific and habitats throughout coral reef and hard-bottom, is one of the most economically important fish in South China Sea. The fish has a bright coloration which is an important factors determing its value. However, in the recent years, framed crimson snapper often appeared faded or black coloration, which was seriously affected its economic value. At the same time, based on the formed studies about the evolution of genus Lutjanus, we found that there are some relationships between the body color and their marine environment. To better understand the cells and genetic factors that influence the pigmentation formation, develop molecular resouces and explore the pigmentation related genes, in the present study, coloration formation of crimson snpper in the ontogenesis was traced firstly; then, microscopy and transmission electron microscopy techniques were utilized to observe the cellular types and distribution in differen skin coloration; RNA-seq was undertaken to study the gene patterns and expression profiles in the black and red color skin. The purpose of this study is to provide basic information about the color difference from the cellular level, and identify the genes potentially related to the color determination of crimson snapper as well as find out the genetic differences between the two different color traits. Results are as follows.1. In the ontogenesis of crimson snapper, melanophores were observed first, xanthophores appeared until 3dph. Four types of pigment cells of melanophore, erythrophore, xanthophore and iridopore were recognized in the dermis of the black and red color skins. The main difference between the two colorations was in the type and quantity of the pigment cells, the black skin was mainly caused by melanophore while the red skin was based on erythrophore and iridophore.2. Sequencing generated 51,438,110 clean reads from black fish skin and 49,531,098 clean reads from red fish skin. Then, these clean reads were assembled into unigenes, yielding 142,792 and 122,508 unigenes with mean length of 622 bp and 613 bp for black and red skin, respectively. 50,220 and 49,736 unigenes were annotated in black color skin and red color skin, respectively.After KEGG and COG annotation, no difference was found in the functional catagory between the two transcriptomes.3. After comparing the two color skin transcriptomes, 9,200 unigenes with significantly different expressions(ratio change ≥ 2 and q-value ≤ 0.05) were found, of which 5,972 were up-regulated in black skin and 3,228 were up-regulated in red skin. Through the GO and KEGG pathway analysis of the differentially transcribed genes, we excavated a number of uncharacterized candidate pigment genes as well as found the conserved genes affecting pigmentation in crimson snapper. Almost all the DTGs(different transcripted genes) enriched in Oxidative phosphorylation(75), Proteasome(28), Glycolysis/Gluconegenesis(34), Citrate cycle(24) were showed higher expression in the red skin, while, most of DTGs involved in tyrosine metabolism(18) and melanogenesis(37/45)were up-regulated in the black skin.4. Mitf genes play an important role in melanphore speciation, development and melanin synthesis. By RACE cloning, two mitf genes were found in crimson snapper. The full length of mitfa is 1,743 bp, encoding 408 amino acids and mitfb is 1,866 bp. encoding 462 amino acids. Both of them have the typical bHLH domain, which is quite conserved within vertebrates. By RT-PCR, mitfb showed a broader expression than mitfa in the ontogenesis and different tissue of adults, which implied that mitfb might inherited most function of ancestor mitf, played an important role in the melanophore speciation, development and melanin synthesis.5. Tyrosinase gene family, which encode for proteins implicated in melanin synthesis. It encompasses three menmbers, tyrosinase, tyrosinase-related protein1 and tyrosinase-related protein 2 in mammals and human. After FSGE(fish specific genome duplication), for tyr and tyrp1, duplications were found in teleosts. By RACE cloning, we found in crimson snpper tyrosinase gene family encompassed tyr, tyrp1 a, tyrp1 b and tyrp2. All the genes contained a typical domain of tyrosinase family as revealed with the SMART software. Multiple sequence alignment showed this domain was highly conserved between crimson snapper and human. Expression analysis revealed that all the genes involved in the melanin synthesis in crimson snapper, and tyrp1 b showed broader expression compared with tyrp1 a.Understanding this will advance our knowledge of skin color varieties in fish and help the genetic selection of fish species with consumer-favored colors. Futhermore, it can also provide some candidate pigmentation genes to investigate the correlation between coloration and sympatric speciation in snapper fishes.