Cloning And Expression Analysis Of Color Related Genes TYR,DCT In Cynoglossus Semilaevis

Melanin widely exists in animal tissues.The reason why the animal body surface has different colors and abundant pigment patterns is mainly caused by the difference in the distribution and development of melanin and related cells.Different pigment patterns have important functions such as biological camouflage,courtship and demonstration,especially in fish.As one of the main breeding species of flatfish in our country,Cynoglossus semilaevis often suffers from two body color anomalies as melanism and albinism,which has caused great economic losses to the breeding industry.As the research object,Cynoglossus semilaevis cloned two individual color related gene TYR,DCT’s c DNA sequence,and analyzes their gene sequence,this paper discusses the TYR gene and DCT expression in eyes,skin,liver,preliminary reveals its role in the melanin formation and growth process,to delve into Cynoglossus semilaevis’ s body color regulatory mechanism laid a foundation.The main research results are as follows:1.Cloning and tissue expression of TYR gene in Cynoglossus semilaevisThe coding region sequence of TYR gene in Cynoglossus semilaevis was obtained by RT-PCR,and the expression characteristics of the gene were analyzed by fluorescence q-PCR.The full-length CDS sequence of TYR gene in Cynoglossus semilaevis was 1620 bp,encoding 539 amino acids,and the predicted molecular weight of TYR protein encoded in the Cynoglossus semilaevis was 61.008 k D.Amino acid homology analysis showed that TYR of Cynoglossus semilaevis had a homology of up to 80%-85% with other fishes,and more than 50% homology with reptiles,birds and mammals,proving that this gene has a high evolutionary conservation in fishes.The results of fluorescence quantitative PCR showed that TYR gene was highly expressed in eyes,and was also highly expressed in black skin and liver,which was significantly higher than other tissues.The expression of the gene encoding TYR in black skin may be related to the control of melanin synthesis.2.Cloning and tissue expression characteristics of DCT gene in Cynoglossus semilaevisDCT gene is an important member of tyrosinase family,and its expression can affect the activity of tyrosinase and the efficiency of melanin synthesis.In this part,the coding region sequence of DCT gene in Cynoglossus semilaevis were obtained by gene cloning.The full-length CDS sequence of DCT gene in Cynoglossus semilaevis was 1551 bp,encoding 516 amino acids,and the predicted molecular weight of theencoded DCT protein was 57.948 k D.Amino acid homology analysis showed that the homology of DCT in Cynoglossus semilaevis was 77.33%-88.05% with other fishes,and more than 50% homology with reptiles,birds and mammals,which proved that the gene also had a high evolutionary conservation in fishes.RT-PCR results showed that the DCT gene was highly expressed in the eyes,was also highly expressed in the liver and black skin,and was hardly expressed in other tissues,which was similar to the expression pattern of TYR.Thus,DCT plays an important synergistic role with TYR in the synthesis of melanin in Cynoglossus semilaevis.3.Expression and regulation of TYR and DCT genes in Cynoglossus semilaevis at different developmental stagesIn this part,fluorescence quantification technique was used to show the temporal and spatial expression differences of larvae and tissues in early stage of development.The expression of TYR and DCT genes in the larval tissues of Cynoglossus semilaevis from 1d to 20 d after membrane emergence showed an overall increasing trend,indicating that these two genes play a crucial role in the metamorphism and development of Cynoglossus semilaevis.From 3 months to 12 months after membrane emergence,the expression of TYR and DCT in black skin was significantly higher than that in white skin.However,the expression of TYR in the normal skin(black)on the ocular side of the Cynoglossus semilaevis showed a significantly increasing trend(p<0.05),there was no significant difference in the expression of melanotic skin,albinic skin and normal skin(white)on the non-ocular side.The expression of DCT gene in normal skin(black)on the ophthalmic side,melanic skin on the ophthalmic side,and normal(white)skin on the ophthalmic side increased significantly(p<0.05).Statistical analysis showed that although TYR and DCT of Cynoglossus semilaevis could regulate the synthesis of melanin,they might have different roles in the formation of melanin and the growth and development of Cynoglossus semilaevis,which still needs to be proved by follow-up experiments.