Molecular Regulation Mechanism Of Body Color Development In Red Crucian Carp(Carassius Auratus,Red Var.)

Red crucian carp(Carassius auratus red var.)is an ornamental fish and popular with its vivid red body color.However,it has been found that the adult body color of this strain forms a gray-to-red change.This phenomenon is that melanophores increase first and then decrease,and the body color changes from gray to red.The research of this phenomenon is helpful for people to understand the molecular genetic mechanism of cell development and differentiation.Therefore,our study used the transcriptome high-throughput sequencing technology to detect gene expression information in red crucian carp skin tissue during different body color development period,We also studied gene expression and DNA methylation site information in adult red crucian carp skin tissues and adult white crucian carp skin tissues by high-throughput sequencing technologies combined with MethylRAD methylation sequencing technology.Meantime,we used CRISPR/Cas9 knockout technology and build expression plasmid vector technology to explore the specific function of the body color development related genes.Research results are as follows:1.Using histology observation techniques,we tracked observed the various body color development periods of pigment cells composition and changes in the red crucian carp skin.we found that melanophores first appeared in 48h after fertilization(48hpf),and xanthophore appeared in the head at 1d after hatching(ldph),and iridophore be found in the belly at the 7dph.Melanophores were gradually increased in the embryo and larvae red crucian carp skin,but skin melanophores began to collectively decrease about at the 45 dph to 60 dph.About at the 90dph melanophores completely faded.Meantime,increasing in xanthophore and erythrophore shown the red body color.2.In this study,the skin transcriptomes of red crucian carp during three different stages of body color development named of gray-color(GC),color-variation(CV),and red-color(RC)stages are first obtained respectively,which are 103,229.108,208 and 120,184 transcripts have been identified,respectively.Through SNP sequence analysis,we obtained 187,616,191,213 and 218,780 SNP sites,respectively.Bioinformatics analysis reveals that 2483,2967 and 4473 unigenes are differentially expressed between CV and GC,RC and CV,RC and GC,respectively.A part of the differentially expressed genes(DEGs)are involved in the signaling pathway of pigment synthesis,such as the melanogenesis genes(Mitfa,Pax3a,Foxd3,Mc1r,Asip),tyrosine metabolism genes(Tyr,Dct,Tyrpl,Silva,Tat,Hpda)and Pteridine metabolism genes(Gch,Xdh,Ptps,Tc)and so on.According to the data of transcriptome and quantitative PCR,those significant pigment development genes,especially for Mitfa,Tyr,Tyrpl,Dct,Tfe3a and Baxa,are closely related to the gray-to-red body color transformation in the red crucian carp.It is suggested that Mitfa and some genes,being related to melanin synthesis or melanophore development,are closely related to the gray-to-red body color transformation in the red crucian carp.3.By means of high throughput technology,we detected gene expression information of adult red crucian carp(RCC)and white crucian carp(WCC)skin tissue.56564 and 56612 unigenes have been obtained,respectively.There exist 7117 differentially expressed genes,including 3434 genes high expression in RCC and 3683 genes high expression in WCC.According to the data of transcriptome and quantitative PCR,we found that melanin synthesis related genes such as Tyr,Dct,Tyrpl and Mclr express at a low level in the RCC,and other pigment synthesis related genes,such as Foxd3,Hpda,Ftps,Gchl and Sox9b is highly expressed in the RCC.A KEGG pathway analysis indicated that the differentially expressed genes are mainly related to Melanogenesis,Gluconeogenesis and Tyrosine metabolism signaling pathway.4.We uesd Methyl RAD methylation sequencing technology to study the DNA methylation sites in the skin of RCC and WCC,and 17,147,966 and 16,568,891 methylation tags were obtained and comparison rate was 21.45%and 18.08%,respectively.We obtained 96,122 and 88,536 CCGG methylation sites in the skin of RCC and WCC and their average depths were 30.71 and 28.35,respectively.7,961 and 9,534 CCWGG methylation sites were obtained and their average depths were 9.18 and 9.02,respectively.Differentially methylated sites were found in genes associated with pigment development,such as Tyr,Dct,Foxd3,Mitfa,and Hpda.The KEGG enrichment analysis reveals that there are a large number of differentially methylated genes related to the MAPK signaling pathway,cAMP signaling pathway,melanogenesis and Hippo signaling pathway.These results indicate that DNA methylation plays an important role in the body color formation of crucian carp.5.Through the CRISPR/Cas9 knockout and expression vector technology,we found that the mutation in Mitfa gene can lead to melanophores loss in red zebrafish larvae.Over-expression Mitfa in the early embryo can increase the expression of melanocytes,but can't save the melanocytes fading away eventually.In conclusion,this study obtains a lot of differentially expressed genes and SNP loci information during different body color development period in red crucian carp,using high throughput RNA-Seq technology.The results analysis shows that the red crucian carp body color developing gene mitfa regulates the expression of Tyr,Dct and other genes,by melanin synthesis pathways controlling the transformation of body color in red crucian carp.Mitfa gene knockout and overexpression analysis confirm the important regulation function of Mitfa gene in the development of fish melanocytes.Using RNA-Seq and MethylRAD technology,we analyze comparatively the differentially expressed genes and DNA methylation site information in the adult red crucian carp and white crucian carp skin tissue,confirmed that the melanin synthesis key enzymes of Tyr gene and it DNA methylation level closely associated with body color formation.In this study,the large number of transcriptome data and DNA methylation sites information were obtained that provide molecular genetic basis for further revealing the regulation mechanism of fish body color development.