| The Japanese ornamental carp(Cyprinus carpio var.Koi)is a kind of high-grade ornamental fish popular in the world,which always be named "living stones in water" or " swimming art".It is a hybrid variant of carp,which is obtained by long-term artificial breeding and hybridization due to the change of breeding environment.A great number of researches have been carried out in fish color formation,but many of them focused on the model fishes,such as zebrafish(Danio rerio),medaka(Oryzias latipes)or some fishes with clearly genetic background.Due to the complicated genetic background of the koi carp,the researches of the koi carp is still in a basic level and the reasons for the koi carp body color formation also need further researches.mitfa and tyr gene is two important regulation genes involved in melanin synthesis,and mitfa as a transcription factor can combine with the "M-box" structure of the tyr gene to control the synthesis of melanin.xdh gene is a key biological synthetase in the process of of purine and pteridine metabolism.Purine pigment and its metabolites,pterin pigment and its metabolites are main components of the pigment in the chromathophores,xdh could affect the metabolism of purine compounds to influence the fish body color formation.This project selected the koi carp as experimental material,the cDNA sequence of xdh was cloned and analyzed by the bioinformatic softwares.Besides,the expression level of xdh also detected in kinds of tissues.Then,the process of body color formation was observed to determine the key periods and the samples of key stages were obtained to check the expression level of xdh,mitf and tyr.In addition,we also detected the the expression of xdh gene in the skin,scales and fins of the koi carp with different colores.Furthermore,the polyclonal antibody of xdh was also prepared to check the protein exrpression level.At the same time,the distribution of xdh in the skin of koi was check by immunohistochemical method.Moreover,the miRNAs of xdh was predicted by several softwares and the expression of them during the process of color formation was also determined.All the results of the project provide reference data for exploring the color formation mechanism.1.The cDNA sequence cloning and tissue expresson of xdh in koi carpThe total cDNA sequence of xdh gene was amplified by the RACE method,and the expression of xdh was checked in seven tissues of the koi carp by qRT-PCR method.The result showed that: the cDNA sequence of the koi carp was 4197 bp and the ORF was 4035 bp encoding 1344 amino acids,in which 5 ’-UTR was 98 bp and 3’-UTR was 145 bp.The xdh protein homological analysis displayed that the xdh of koi carp had the highest similarity with other Cyprinidae species,such as the common carp,Sinocyclocheilus grahami and zebrafish,etc.and the similarities with the mammals,birds,reptiles and other vertebrates were low.Phylogenetic analysis showed the same result with the protein homological analysis.Besides,the expression analysis showed that the xdh was detected in seven tissues and the highest and lowest epression level was found in kidney and scales,respectively.2.The observation of body color formation and expression analysis of pigmentation related genes during the process of color formationThis chapter described the process of body color formation of the red koi carp,and summarized the similarities and differences of different developmental stages,from which 6 specific stages were selected including 1,2,3,4,12,48 dph(day post hatching).The expression level of xdh,mitfa and tyr gene during the process were detected using qRT-PCR analysis.The results showed that xdh gene had the highest expression at 48 dph,which was significantly higher than that at 3 dph(P<0.05).The expression level of xdh gene in both stages was significantly higher than other four stages(P<0.05).The expression of xdh gene at 4dph was significantly higher than that at 1,2 and 12 dph(P<0.05).There was no significant difference among 1,2 and 12 dph.The expression of mitfa gene was highest at 1 dph,significantly higher than that of 48 dph(P<0.05),and the expression of mitfa at the two stages significantly higher than that of the other four periods(P<0.05).However,there were no significant differences among the 2,3,4 and 12 dph(P>0.05).The expression of tyr was highest at 1dph(P<0.01),and the tyr expression level of 4dph was significantly higher than that of 12 and 48 dph(P<0.05),but has no significant difference with that of 2 and 3 dph(P>0.05).There were no significant difference among 2,3,12 and 48 dph(P>0.05).The expression of xdh,mitfa and tyr gene exhibited differences during the process of body color formation,the expression of xdh firstly increased,then decreased and climed the peak at the last stage.The mitfa and tyr gene displayed the similar expressive tendency,which firstly climed the highest level,and then decreased following with the body color formation.3.Expression analysis of xdh gene in skins,scales and fins of the koi carp with different body colorsIn this chapter,the total RNA of skins,scales and fins of red koi,white koi,and red-white koi were sampled to analyze the expression of xdh gene in different body color koi carps.Results showed that: in the skin,the xdh gene expression of white carp was highest(P<0.05),the expression of xdh gene in white skin of red-white carp was lower than the white carp,but had no significant difference compared with the red skin of red-white carp and red carp.In scales,the xdh gene expression of red scales in red-white carp was the highest(P<0.05),and the white scales of red-white carp was in the second(P<0.05),the xdh expression in the scales of red carps and white carpsI was the lowest and there is no significant difference between them(P>0.05).In the fins,the xdh expression in the white fins of red-white koi carp was the highest,significantly higher than that of the red koi(P<0.05),but had no significant difference comparing with that of the white koi carp(P>0.05).Besides,there was no significant difference of the xdh between the red koi carps and white koi carps(P>0.05).4.Prokaryotic expression and preparation the xdh polyclonal antibodyIn this study,the sequence of xdh antigenic determinants,encoded 99 amino acids,was cloned into the pET-32 a(+)vector using the Hind III and Bam HI.Then,the positive recombinant pET-32 a(+)-xdh was transformed into the E.coli BL2(DE3)to expression the corresponding polypeptides.After IPTG induction,the target polypeptides were obtained.Then the purified target polypeptides were used to immunize rabbits for preparing polyclonal antibodies against xdh.At the last,the immunohistochemistry methods were used to detect the distribution of xdh in the skins of the koi carp.5.Prediction the target miRNAs of xdh and expressive analysis of the candidated miRNAsThe 3 ’-UTR sequence of xdh gene were used to predict the candidated miRNAs by RNAhybird software.Those miRNAs were selected from the miRNAs profiles during the body color formation The expression level of the miRNAs during the color formation were checked by the qRT-PCR.Combined with the expression trend of xdh gene and miRNAs at different developmental stages,the filtered miRNAs might influence body color formation by targeting the xdh gene. |
