The Molecular Mechanism Of Slc24a5 And Mitfa Gene Regulate Melanophore Development In Cyprinus Carpio Var. Color

The Oujiang color common carp(Cyprinus carpio var.color)is a kind of special economical fish which has important ornamental and edible value,it is widely distributed in the Oujiang River basin of Zhejiang province in China.The Oujiang color common carp is a common variant of the common carp with a unusual and rich color.In this study,based on culture biology and molecular biology technology,learned the genetic and molecular mechanism of the melanin form and development.This research mainly focused on the development and distribution of Oujiang color common carp and analysed the important roles of slc24a5 and mitfa gene regulate melanophore development in Oujiang color common carp.The slc24a5 and mitfa gene cloning and expression analysis were carried out in Oujiang color common carp,in addition,the zebrafish model tool was used to study the function of slc24a5 gene for melanin happened.Part1.Observation on arising and distributing of melanophore on the embryo and laver stages of Oujiang color common carpIn this study,the four kinds of Oujiang color common carp embryos by artificial insemination was observed.The development stages of 24 hour post-fertilization(hpf),36 hpf,48 hpf and 1 day post-hatch(dph),2 dph,3 dph,5 dph,10 dph,15 dph was mainly observed.The development and occurrence of melanin on the embryo and laver were noted.According to the observation,learned that the melanin appeared on the eyes at 36 hpf firstly in RB,WR and WB when the culture temperature is 21? to 23 ?.At 25 ?,the melanin appeared at 24 hpf in the WW.After the embryos hatched,the melanin first arose in the body part(head)in the Oujiang color common carp with black patch at 3 dph,then the melanin appeared more at 5 dph.At 10 dph,it can be observed the melanin into fragments distribution.Meanwhile,there is no melanin observed in the Oujiang color common carp without black patch.Above research shows that: the oujiang color number of the formation of melanin is relatively common carp carp its inequality,less periods are delayed.Part2.slc24a5 gene regulation of Oujiang color common carp melanin formationSlc24a5 genes have the functions of delay and reduce the melanin deposit.Firstly,the slc24a5 gene of Oujiang color common carp is obtained by molecular cloning technology.The full-length cDNA sequence of this gene is 2056 bp,containing 157 bp 5' untranslated regions(UTR)and 738 bp 3' UTR and 1173 open reading frame(ORF),encoding 512 amino acids(aa).The amino acid sequence shared as high as 75% to 96% homology with other fish,and more than 60% homology with mammals.This shows that the gene has high conservative in fish.Quantitative RT-PCR indicated the expression levels in the tissues with melanin and melanin presence stages is higher than no melanin parts and development stages.Further study shows that slc24a5 knock-out in zebrafish via CRISPR/Cas9 technology,made the function loss of slc24a5 gene,caused a significant loss of melanin.At the same time the slc24a5 gene over-expression plasmid vector from Oujiang color common carp is constructed to rescue the slc24a5 mutant zebrafish,and it can be rescued successfully.All the results show that slc24a5 genes important regulation function of melanin formation and the gene structure function is highly conservative.Part3.mitfa gene regulation of Oujiang color common carp melanin formationThe mitfa gene is an important transcription factor that regulates the development and development of melanocytes.In this part,the mitfa cDNA sequences and DNA sequences of four species of Oujiang color common carp and common carp were obtained by molecular cloning technique.The results of cDNA sequence and encoded amino acid sequence showed that the nucleic acid variability of the four Oujiang color common carp and common carp mitfa gene sequences was low.At the same time,there was no significant difference in the expression of the gene in the skin tissues of the four species of Oujiang color common carp.The in situ hybridization results of RB and WR indicated that the gene expression was found in RB and WR all.According to the in situ hybridization results of different periods of RB,it is found that the expression level of mitfa gene increases with the increase of melanin in the skin.The results of this part indicate that the development and growth of melanin in Oujiang color common carp is not related to its amino acid sequence,and the mitfa gene plays an important regulatory role in the development and growth of melanocytes in Oujiang color common carp,also it is not the key role for the formation of black patch in Oujiang color common carp because the mitfa have function for other pigment cell.