| Skin pattern is an important external morphological feature of fish,which plays an important role in fish survival and reproduction.Misgurnus anguillicaudatus from Poyang Lake is a small economic fish from Poyang Lake area.Our laboratory has selected and bred big blackspot,small blackspot and non-blackspot loaches.Their genetic performance of blackspot is relatively stable and the reproductive performance of three kinds of loach was significantly different.Therefore,our laboratory carried out the breeding of loaches from Poyang Lake with blackspot as the target trait in order to obtain loach varieties(strains)with excellent growth and reproductive performance.However,the formation pattern of blackspot in loach and its molecular regulatory mechanism are still unknown.In this thesis,we investigated the early development of embryo and the development characteristics of melanocyte of loach from Poyang Lake,and investigated the characteristics of mitfa(Misgurnus anguillicaudatus Microphtalmia associated transcription factor a,Mamitfa)gene and its m RNA expression characteristics;Transcriptome analysis was performed on the skin of three kinds of loaches,in order to provide follow-up experimental support for the pattern of blackspot and its regulatory mechanism of loach from Poyang Lake.The research results are as follows:(1)The Nikon SMZ1270 dissecting microscope was used to observe the developmental process of loach embryos and the types and distribution of pigment cells on the body surface and the formation of major patterns(stripes,bands and spots)in the early stage(embryonic stage to 60 days after hatching).The results showed that embryonic development of loach underwent 7 stages including fertilized egg,cleavage,blastocyst,gastrula,neurula,organogenesis,and hatching stage.In early development stage,larval melanocytes were first observed in yolk sac at 3 h post hatching.From larva stage to juvenile stage(at 21 d post hatching),larval melanocytes appeared on loach body surface.From juvenile stage(at 22 d)to adult stage,adult melanocytes appeared on loach body surface.Iridocytes were first observed in the eyes of 1-day-old larvae,but not on the body surface until at age of 12 days.Xanthophore appeared on body surface of 7-day-old juvenile.During 2-21 d post hatching,the melanocytes in loach were larval melanocytes,and their shape was converted from star to snowflake,finally to black dot(big light black dot-dark black dot-fine black dot).From the age of 22 days,different morphological adult melanocytes were formed on the body surface of three types of loaches.For big blackspot loaches,the chrysanthemum-shaped melanocytes were regularly aggregated into big black spots.For small blackspot loaches,round and dendritic melanocytes were gathered to form small black spots.For non-blackspot loaches,dendritic melanocytes were evenly distributed.Our results laid a theoretical foundation for the formation of genetically stable different spots in loaches.(2)The c DNA full length of Mamitfa gene was cloned and its molecular characterization was analyzed.The results showed that the c DNA full length of Mamitfa gene is 1482 bp,and the gene contained 1227 bp open reading frame which encoded 408 amino acids;the gene contained 89 bp 5’-UTR region and 166 bp3’-UTR region.There were three domains of MITF＿TFEB＿C＿3＿N,b HLH Zip and DUF3371 domains in Mamitfa gene.The relative molecular weight of protein was45.68 KDa,and the theoretical isoelectric point was 7.16.The secondary structure of the protein was predicted that Mamitfa contained 156 alpha helices(38.24%),13 beta turns(3.19%),and 195 random coils(47.79%),44 extended strand(10.78%),and no transmembrane structure.The distribution of α-helices was predicted for the tertiary structure of Ma Mitfa protein,which was consistent with the distribution in the secondary structure.The phylogenetic tree was constructed using the Neighbor-Joining(NJ)method with MEGA 7.0 software.The results showed that Ma Mitfa first clustered with mitfa of carp and zebrafish,which was consistent with the taxonomic status of the loach.(3)In this paper,we analyzed the m RNA expression characteristics of Mamitfa in each stage of embryonic development of three kinds of loaches.The results showed that there was m RNA expression of Mamitfa in all stages of embryonic development.In the embryos of big blackspot loach,m RNA expression level of Mamitfa was detected the highest at fertilized egg stage(P < 0.0001),and then at the multicellular stage and gastrula stage,and the lowest at the muscular effect stage.In the embryos of small blackspot loach,Mamitfa expression was detected the highest at fertilized egg stage(P < 0.0001),and then at the multicellular stage and mulberry stage(P <0.001),and the lowest at the gastrula stage;In the embryos of no-blackspot loach,the m RNA expression of Mamitfa was the highest at fertilized egg stage(P < 0.0001),and then at multicellular stage and mulberry stage,and the lowest at the fry stage.We analyzed the m RNA expression of Mamitfa in the intestine,brain,heart,liver,fins,dorsal skin,abdominal skin and muscle of three kinds of loach.In the big blackspot loach,the m RNA expression was the highest in the muscle and dorsal skin(P <0.0001),followed by the liver,intestine and abdominal skin,and the lowest in brain;In small blackspot loach,the m RNA expression was the highest in the muscle and dorsal skin(P < 0.0001),followed by abdominal skin,and the lowest in the liver;In non-blackspot loach,the m RNA expression of Mamitfa was the highest in the muscle and dorsal skin(P < 0.0001),followed by in the liver and abdominal skin,and the lowest in intestine.The same tissues were mixed of three kinds of loach and the m RNA expression of Mamitfa was detected in each tissue of the loach,and the expression of Mamitfa was the highest in the muscle tissue(P < 0.0001)and the lowest in the heart.(4)The transcriptome of dorsal skin of three kinds of loach was comparatively analyzed,and a total of 28 genes,which expressed differentially,were identified,from which the relevant genes and related signaling pathways that may regulate in the blackspot formation of loach were screened.GO analysis showed that most of the proteins related to microtubule polymerization,chemokine receptor activity and cytoskeleton related proteins were significantly enriched;KEGG analysis revealed no signaling pathways was directly linked to melanin,but there were pathways that may be indirectly linked to melanin or melanocyte migration,such as the resistance pathway of EGFR tyrosine kinase inhibitor,chemokine signaling pathway and the signaling pathway of mitogen-activated protein kinase(MAPK).The related genes which were involved in life activities and pathways are sgk1、clnd7、ndrg1、grb2、vegfa and cxcr4.Therefore,it is speculated that the blackspot may be regulated mainly by the related mechanism of microtubule activity and protein localization activity.In conclusion,the differences in the blackspot patterns of loach are closely related to the morphology,distribution and migratory movements of melanocytes,among which the Mamitfa gene is crucial in the development and differentiation of melanocytes.The regulation of blackspot formation of M.anguillicaudatus may be related to microtubule activity and protein localization activity.The results provide a theoretical basis for further study of the pattern formation and molecular regulation mechanism of blackspot in the loach from Poyang Lake. |
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