Abstract:Microphthalmia-associated transcription factor (MITF) is a transcription factor with a typical basic helix-loop-helix-leucine zipper structure. It is a key transcription factor in the melanin synthesis pathway in animals by activating downstream genes to initiate melanin synthesis. In order to explore the regulatory action of mitf during body color variation in Amphilophus citrinellus, the full-length cDNA of the mitf gene was cloned using the rapid amplification of cDNA ends technique, and the difference in gene expression in different developmental stages of embryos, different stages of body melatonin, and various tissues in A. citrinellus were analyzed through qRT-PCR. Two mutant sequences of mitf were obtained. The full-length of mitf1 cDNA was 1816 bp, including a 158 bp 5′UTR, a 428 bp 3′UTR, and a 1230 bp ORF coding a polypeptide of 409 amino acids. The full-length of mitf2 cDNA was 1638 bp, including a 160 bp 5′ UTR, a 428 bp 3′UTR, and a 1050 bp ORF coding a polypeptide of 349 amino acids. The analysis of the homology and phylogenetic tree results showed that mitf1 and mitf2 clustered into a small branch in the phylogenetic tree, which had the highest homology with Cichlidae fish and low homology with mammals. The results of the qRT-PCR showed that mitf1 and mitf2 were expressed to varying degrees in various tissues of adult fish, among which the expression in the eyes was the highest and was significantly higher than in other tissues (P<0.05), and the expression in muscle, brain, and kidney tissue was also higher. The mitf1 and mitf2 genes were expressed in all stages of embryonic development, and the expression level was highest in the cleavage stage, which was significantly higher than the other embryonic stages. With the body color transition from black to orange, the expression of mitf1 and mitf2 genes decreased gradually in the fish skin, scales, and tail fins. It showed that the expression of mitf was positively correlated with changes in body color from black to yellow in A. citrinellus, which may be related to the dynamic change in the differentiation and distribution ratio of pigment cells in the body color development stage of fish. Through understanding the molecular basis of fish body color development and variation, this study accumulates data for fish pigment cell development and the artificial improvement of fish body color.
