| Fish coloration plays an important role in survival,camouflage,courtship,reproduction and environmental adaptation,which is an important genetic and ecological factor.The body color is rich and varied,and the formation of pigment pattern is related to the synthesis and transfer of pigment,the differentiation and transport of pigment cells,and then involved in the participation and regulation of many genes.Melanophilin（Mlph）encodes a subfamily of Rab effector proteins which is an important gene for the transportation of mature melanosomes.Mlph plays fundamental roles in regulating the transportation of mature melanosomes in vertebrates,and its mutations can lead to hair or feather color fading in animals.However,the molecular function of Mlpha gene in aquatic animals need to be explored.In this study,spatial and temporal expression of Mlpha gene were investigated in Oujiang color common carp（Cyprinus carpio var.color）and zebrafish（Danio rerio）,and Mlpha gene was also knocked out by using CRISPR/Cas9technology to observe body color changes in Oujiang color common carp and zebrafish.The effect of Mlpha gene on fish melanoma body color was studied by screening the knockout mutants and the expression of related gene were examined.The relevant research results are as follows:1.Identification of SNP sites of Mlpha gene and analysis of spatiotemporal expression in Oujiang color common carpMlph gene is a member of Rab effector protein subfamily,which is an important gene for mature melanosome transport.In this chapter,two Mlpha genes of Oujiang color common carp were confirmed by gene cloning.They had 91.89%consistency in the base sequence and 78.63%similarity in amino acid sequence.They were closely clustered in the evolutionary branch of Cypiformes.The SNPs of the two Mlpha genes in Oujiang color common carp were confirmed by Sanger sequencing analysis.Mlpha1 has two associated SNPs:A/G（A69G）and G/A（G93A）,G/C（G468C）and A/G（A485G）.One SNP of Mlpha2 is G/A（G1008A）.The fluorescence quantitative expression results of six skin tones showed that the expression level of Mlpha1 in black skin was significantly higher than that in red and white skin,and the expression level of Mlpha1 in all red skin was the lowest.The expression level of Mlpha2 were opposite.In the quantitative results of Oujiang color common carp at different stages,both Mlpha1 and Mlpha2 were significantly higher at the fry stage（after incubation）than that at the embryo stage（before incubation）.Combined phenotypic observation with the result in situ hybridization,it was found that Mlpha1 and Mlpha2 probe signals were detected in the tail and gill of“Big flower”and“Pink flower”at 3 days after hatching,then migrated along the neural crest to the abdomen and the whole skin in subsequent days.While in“Full red”,Mlpha1 and Mlpha2 were both distributed in the caudal gills and abdomen at 3 days after hatching,and the probe signal of Mlpha1was significantly less than that of Mlpha2 in the whole red body of“Big flower”at the same field of vision and the same period.The above studies indicated that Mlpha is an indispensable functional gene in Oujiang color common carp,which may be involved in pigment transport.There may be functional differentiation between Mlpha1 and Mlpha2:Mlpha1 may mainly participate in melanin transport,and Mlpha2 may be involved in the transport of other pigments.2.Effect of Mlpha gene knockout on black stripe in zebrafishIn this study,spatial and temporal expression of Mlpha gene were investigated during different developmental stages in zebrafish,and Mlpha gene was also knocked out by using the CRISPR/Cas9 technology to exam body color changes in the F2homozygous mutants in zebrafish.The results indicated that Mlpha gene did not double in zebrafish and be expressed at all the collected developmental stages.The highest expression level was identified on the abdomen of zebrafish at 5 days post hatching,when obvious melanin band appeared.After Mlpha gene was knocked out,the larval individuals of F1 mosaic mutant zebrafish displayed slow expansion of melanin,and the adults showed fragmented black stripe.In the F2 homozygous mutant zebrafish,the Mlpha protein cannot be completely translated owing to terminator codon exist in the second exon,and small melanocyte appeared in adults skin.Furthermore,in situ hybridization and q RT-PCR expression analysis revealed that the expression levels of Rab27a and Myo Va which were related to Mlpha was significantly decreased in the F2 homozygous mutants（P<0.05）.Our study indicated that Mlpha gene played essential roles in regulating the transportation process of melanin,and its mutation could affect black stripe formation in zebrafish.These results provide a useful reference for understanding the genetic basis and mechanism of black spot body color formation in fish.3.Effect of Mlpha gene knockout on black spot and transport genes in Oujiang color common carpMlph gene plays fundamental roles in regulating the transportation of mature melanosomes in vertebrates,and its mutations can lead to hair or feather color fading in animals.In this chapter,gene clone was used to screen Mlpha knocked out chimeras,and fluorescence quantitative expression of related transport genes were carried out.The rescue experiments of Mlpha1 and Mlpha2 genes were carried out by using Mlpha knockout homozygotes of zebrafish.It was found that the majority of Mlpha knockout chimeras had obvious changes.Its phenotype showed three skin color regions,namely:black,gray and rad/white.The melanin in the light dark region was significantly reduced,and the melanin particles were in a state of shrinkage and dispersion.The genotype of chimeras had a large number of base sequence deletion.The expression analysis of transporter genes in different skin regions in the chimeras showed that the expression of Myo Va was significantly decreased in the Mlpha chimeras（P<0.05）,and the expression of Rab27a in black skin decreased significantly,while that in white skin increased significantly.These results suggest that the Mlpha genes（Mlpha1 and Mlpha2）were very conservative,and its mutations may lead to the loss of their binding function to Myo Va and Rab27a,thus affecting the transport of melanin,and Mlpha genes may also be involved in the transport of red pigment. |
