| There are a wide variety of colors and coloration patterns in fish,and fish is an excellent model for studying the molecular processes of skin color pigmentation.There are three body color polymorphisms which can be stably inherited in Misgurnus anguillicaudatus from Poyang Lake area,namely,large-blackspot,small-blackspot and non-blackspot loaches.In order to investigate the association between tyrosinase genes and the formation of three different spots in Misgurnus anguillicaudatus,we studied the function of tyr gene in Misgurnus anguillicaudatus(designated as Matyr)on blackspot and color formation.Firstly,the full-length c DNA of Matyr was cloned with RACE-PCR method,then the spatio-temporal expression of Matyr gene in three kinds of loaches was analysised.At last,under the condition of black and white culture background,the body color and blackspots on the body surface of loach were detected and the expression of Matyr were analyzed.The results could provide the support for studying the mechanism of the blackspot formation of loaches.The results was as follows:(1)The Matyr c DNA sequence is 2020 bp in length,containing an open reading frame(ORF)of 1617 bp,a 5’ UTR region of 121 bp and a 3’ UTR region of 282 bp,encoding a hydrophilic protein containing 538 amino acids,with a relative molecular mass of 61.04 k Da and a theoretical isoelectric point of p I=5.85.There were a signal peptide and a transmembrane region at each end of Ma Tyr,and an epidermal growth factor-like domain(EGF like)and a tyrosinase domain(Pfam tyrosinase)containing two copper ion binding sites in the middle.In addition,there was a dileucine signal behind the transmembrane region.Secondary structure prediction showed that Ma Tyr contained 307(57.06%)random coils(Cc),160 α-helices(Hh),60 extended chains(Ee)and 11 β-folded(Tt).The predicted tertiary structure showed that the Ma Tyr tertiary structure model contained two copper ions inside,and the distribution of α-helices was basically the same as that in the secondary structure.The results of multiple sequence alignment and phylogenetic analysis showed that the Tyr sequences of Misgurnus anguillicaudatus(designated as Ma Tyr)had high homology with Cyprinidae,and were clustered firstly with Triplophysa tibetana and Leptobotia taeniops in evolution,and then clustered with other cyprinid fish,which was consistent with the taxonomic status of Misgurnus anguillicaudatus.(2)The spatio-temporal expression of Matyr genes in three kinds of Misgurnus anguillicaudatus was localized by quantitative real-time PCR(q RT-PCR)and wholemount in situ hybridization(WISH).The q RT-PCR results showed that the Matyr gene was expressed at all stages of embryonic development in the loach,with an overall pattern of expression from low to high.Relatively obvious Matyr expression could be detected at the beginning of the muscle effect stage,in which the expression of Matyr gene in the large-spotted loach was highly significantly elevated at that period.In adult loach,the Matyr gene is highly expressed mainly in muscle and dorsal skin,with very low expression in internal organs such as the heart and hepatopancreas.The WISH results showed that Matyr hybridization signals with similar location could be found in all developmental stages of three kinds of loaches from fertilized eggs to 6 day post fertilisation(DPF).From ocular melanin period,many dot-like hybridization signals can be observed in the head of the larvae,which gradually extend to the dorsal,and the signal distribution in the larvaes of the large-blackspot loach was more than that in the non-blackspot loach.(3)The black/white background adaptation experiment was conducted for 60 days to investigate the m RNA expression of Matyr gene in response to the change in body color of loach after 30 days of black/white background adaptation,after 30 days of black/white background domestication and then switching the background color for another 30 days,and after 60 days of continuous domestication in black/white background.The results showed that the apparent body color of Misgurnus anguillicaudatus showed light color on white background and dark color in black background,and the body color reversibly changed with the change of tank background color.With the extension of domestication time,the body color of loach further converged to the background color,the black spots on the body surface of WW group became lighter in color,while the abdomen skin of BB group also showed obvious dark tones.The m RNA expression level of loach’s Matyr gene in the black background group was generally higher than that in the white background group,and was mainly expressed in the brain,dorsal skin,muscle and abdominal skin of loach,with very low expression levels in the visceral organs.In addition,the expression levels of Matyr in the skin of loaches that had undergone white background domestication were all extremely low or even non-expressed.In the case of transfer from white to black background and continuous domestication in black background for 60 days,Matyr gene showed relatively high level of expression in loach’s brain,while both W and WW groups showed higher Matyr expression level than the control group.In addition,after30 days of background domestication,the expression levels of Matyr in both dorsal and abdominal skin of group B were significantly increased compared to the control group(P<0.01),while the expression levels in abdominal skin of group W were significantlydecreased compared to the control group(P<0.01),and the expression of this gene was not detected in the dorsal skin of group W.After changing the background color,the expression of Matyr in the skin of WB group was significantly higher(P<0.01)compared to W group,while the expression of BW group was significantly lower(P<0.01)compared to B group,and the expression level of Matyr in the abdominal skin of BW group was extremely significantly lower than that in the control group(P<0.01).After 60 days of domestication under the same background,the relative expression of Matyr in the skin of the BB group was significantly lower(P<0.01)compared to the B group,while Matyr was in a state of no expression in the WW group.In summary,the change in body color of the loach is reversible and can produce convergent changes with the black/white background color.The Matyr genes may be involved in the formation of different black spots on the skin and the darkening of body color in Misgurnus anguillicaudatus by controlling the amount of melanin production,and play a role in organ development. |
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