The Regulation Mechanism Of Scarb1 And Scarb1-like Genes In Controlling Red Coloration In Oujiang Color Common Carp(cyprinus Carpio Var.color)

The red coloration in animals has a unique and special ornamental and economic value.At present,red color formation mechanism in animals are mainly focused on birds,silkworms and reptiles,seldom researches had been conducted in fish.Oujiang color common carp(Cyprinus carpio var.color),as a color variety of common carp(Cyprinus carpio L.),mainly includes four distinct color patterns: whole red(WR),whole white(WW),red with scattered big black spots(RB),and white with scattered big black spots(WB),which provides good materials to study the growth and color characteristics in animals.In this study,WW and WR color common carp were used as the materials to study the red color formation,and the methods mainly included phenotypic pigments analysis,transcriptome sequencing comparison,candidate genes identification,genes clone and expression analysis,targeted genes knockout,mutants phenotypic analysis,and carotenoids supplement experiments.The mainly results were as followed:1.Analysis of the red pigment contents in the skin in WR and WWThe red coloration in animals mainly consists of one or two pigments: pteridine or/and carotenoids,both of which can produce a range of colors,such as red,orange,yellow and white color.In this study,the contents of carotenoids and pteridine pigments in WR and WW skins with absorbance spectrophotometry methods.The results showed that carotenoids contents in WR skin was significant higher than in WW(P<0.05),but no difference was found about pteridine pigment contents between WR and WW.Then,high performance liquid chromatography(HPLC)was used to identify and quantify the carotenoid classes and composition in the skin,fin and eye of WR and WW.The results showed that higher contents of astaxanthin,lutein and several unknown carotenoids were detected in WR,but which could not be detected in the WW.In conclusion,these results indicated that the different pigments between WR and WW were the carotenoids,which existed in the form of esterification.2.Comparative analysis of the skin transcriptomes between WR and WWIn this study,RNA-seq was used to investigate the skin transcriptomes between WR and WW.A total of 98,906 transcripts were assembled in the transcriptomes and 77,601 were annotated,the transcripts annotated with KEGG and Go databases were 29,954 and 36,476,respectively.Between WR and WW,1,262 genes were special expressed in WR,and 189 were special expressed in WW.GO enrichment analysis indicated that the GO categories related to the “neuron nervous system development” and “cell adhesion” were strongly enriched in the genes specifically expressed in WR.GO categories involved in “response to stimulus”,“blood coagulation” and “striated muscle tissue development” were enriched in the genes specifically expressed in WW.A total of 139 different expressed genes were identified,including 36 genes upregulated in WR and 103 genes upregulated in WW.In these genes,none of them were related with melanin and pteridine pathway.The different expressed genes between WR and WW were mainly focused on the energy metabolism and muscle development.The expression of two genes related with energy metabolism and four genes related with muscle development were detected with q RT-PCR,which were consistent with the results in the transcriptomes.3.Identification of the candidate genes involved in carotenoids metabolism and pteridine pathwayThe q RT-PCR method was used to identify the candidate genes involved in carotenoids metabolism and pteridine pathway,which were based on the former transcriptomes.Two genes were involved in pteridine pathway: Gch1 and Xdh.Candidate genes involved in carotenoids metabolism were as followed: uptake and transport(CD36,Scarb1,ABCA1 and Np C1L1);binding and deposition(Apod,Apoe,Gstp1 and Stard3);and breakdown(Bcmo1 and Bco2).Gch1 and Xdh had no significant differences between WR and WW(P>0.05).The expression of CD36 and Np C1L1 had significant differences between WR and WW skins(P<0.05),which were very low.The expression of ABCA1 in WR was significantly higher than in WW(P<0.05).The expression of Scarb1 was very significantly higher in WR skin(P<0.01),which was no expressed or low expressed in WW skin.The binding and deposition genes had no differences between WR and WW skins.Bcmo1 and Bco2 were higher expressed in WR than in WW.Then,Scarb1 was found to have a higher expression in the fins,intestine,liver and blood in WR than in WW(P<0.01).4.Clone,structure and expression pattern analysis of Scarb1,Scarb1-like and Gch1Then the sequences,structure and expression pattern of Scarb1 was analyzed.Two Scarb1 were found in Oujiang color common carp,which located on chromosome 10,and named as Scarb1(Gen Bank: MK625701)and Scarb1-like(Gen Bank: MK618572),respectively.The m RNA full-length sequence of Scarb1 was 2,249 bp,and coding region was 1,494 bp,which encoded 497 amino acids.The coding region of Scarb1-like was 1,515 bp,which encoded 504 amino acids.Structure analysis showed that Scarb1 contained 12 exons and 11 introns,while Scarb1-like contained 13 exons and 12 introns.Then,the expression of Scarb1 and Scarb1-like among eight different tissues between WR and WW showed that both Scarb1 and Scarb1-like were highly expressed in WR tissues(P<0.05),and no expressed or low expressed in WW tissues.The highest expression of Scarb1 and Scarb1-like were in the skins and intestines in WR,respectively.Even the expression of Gch1 had no significant difference between WR and WW skins,which was also cloned in this experiment to set as the control group in the CRISPR/Cas9 experiment.Based on the sequence of Gch1(Gen Bank: KP05654)in Cyprinus carpio var.koi,a 756 bp sequence was cloned in Oujiang color common carp,located on chromosome 17,which encoded 251 amino acids.5.Disruption of Scarb1,Scarb1-like and Gch1 in Oujiang color common carpCRISPR/Cas9 technology was used to disrupt the target genes in WR Oujiang color common carp in this experiment,and Gch1 knockout,Scarb1,Scarb1-like and Gch1 knockout,and no-injected group were set as the control groups.Considering the high similarity of Scarb1 and Scarb1-like protein structures,two same sg RNAs were designed in exon 1 and exon 2,respectively,and disrupted in WR color common carp together.In WR mutants,the red color areas in whole body were disrupted and faded to white,and the white color was the same with the white color in WW,59 of 99 WR mutants had the similar phenotypes.Similar phenotype was also observed in the fins and the skins around the eyes in WR mutants.Then,HPLC analysis revealed that both in the whole red tissues and residual red tissues in WR mutants,the concentration of astaxanthin and lutein were decreased when compared with the wild WR,and astaxanthin and lutein could not be detected in the white skin in WR mutant.In Gch1 knockout groups,WR mutants had no obvious color variation compared with the wild WR.In Gch1,Scarb1 and Scarb1-like knockout group,the phenotypes of WR mutants were the same with the disruption of Scarb1 and Scarb1-like.Also,Gch1,Scarb1 and Scarb1-like were disrupted in RB color common carp.The disruption of Gch1 in RB had no obvious color variation compared with wild RB,and the disruption of Scarb1 and Scarb1-like in RB had similar color variations with WR mutants,the red color areas in whole body were disrupted and faded to white.Other colleagues also disrupted Tyr in WB color common carp(Xu et.al,unpublish),and in WB mutants with high efficient disruption,not only the black color in whole body faded to white,the black color in eyes faded to red.Compared with the deficiency of melanin,it was more convinced that the white color in color common carp is caused by the lack of carotenoids,because the deficiency of carotenoids not causes a fading of the black color in the eyes.In conclusion,the carotenoid uptake and transport genes Scarb1 and Scarb1-like have a dominant role in the red color formation in color common carp,and also,the pteridine may have no effects.6.The expression variations of candidate genes involved in carotenoids metabolism in WR mutants and the transcriptomes analysis between wild WR and WR mutantsCarotenoid coloration in animals may be influenced by the genetic and environment factors together.In this study,the expression of ten candidate genes involved in different processes in carotenoids metabolism in WR mutants(Scarb1 and Scarb1-like knockout)were compared with in wild WR.These genes were quantified in intestines,eyes,fins and skins in wild WR and WR mutants.In the intestines and eyes in WR mutants,genes variations with significant difference were focused on the uptake and transport process and breakdown process: ABCA1,CD36 and Np C1L1,Bco2 and Bcmo1 were upregulated in WR mutants(P<0.05).Besides,the binding and deposition genes Gstp1 and Stard3 were upregulated in the eyes in WR mutants(P<0.05).In the fins and skins,most of the ten genes had no significant difference variations between wild WR and WR mutants,only the breakdown gene Bco2 was downregulated in fins in WR mutants(P<0.05),but the breakdown gene Bco2 had no change between the red and white skins in WR mutants(P>0.05).The different variations of candidate genes involved in carotenoids metabolism may be related to the mechanism in different tissues,the intestines which mainly performed the uptake and transport,and breakdown of carotenoids;the carotenoids are mainly deposited in the skins and fins;and in the eyes,specific carotenoids were used to deposition and breakdown.In the mutants which had an inhibition in the uptake and transport of carotenoids,because of the compensatory effects in animals,several genes would be upregulated to compensate the uptake and transport of carotenoids,similar effects were also occurred in the binding and deposition,and breakdown process.Most of the candidate genes were upregulated in WR mutants suggested they had a similar function in carotenoids metabolism in Oujiang color common carp.No significant variations of the binding and deposition genes had been found in WR mutants,indicated the binding and deposition process was not the key to the red color formation.In conclusion,combining with the mechanisms of carotenoid metabolism in animals,the inhibition of uptake and transport of carotenoids in red color caused not enough carotenoids to deposit,which induced the formation of white color.Then,transcriptomes of the red skin in wild WR,the red and white skin in WR mutants were similar with our former transcriptomes between WR and WW,the energy metabolism genes and muscle development genes were upregulated in the WW.We also compared the expression variations of several candidate genes involved in the carotenoid metabolism in the transcriptomes,which were same with the results of q RT-PCR,no significant were found between wild WR and WR mutants.7.The effects of carotenoid supplement on the red coloration in WR mutantsThe redness and yellowness of carotenoids-based coloration could be enhanced by dietary pigments supplementations have been studied in many animals.In this study,a diet containing 75 mg/kg astaxanthin and 100 mg/kg lutein supplement was used to study whether the phenotypes caused by CRISPR/Cas9 disruption could be repaired in WR mutants.Color common carp fed with astaxanthin and lutein supplemented diets showed a stronger red color in red color scales than the control groups after 40 days feeding.The contents of astaxanthin and lutein increased significantly,and both the redness and yellowness in the dorsal and ventral scales of wild WR scales and red scales in WR mutants were enhanced with the supplemented astaxanthin and lutein.However,the contents of astaxanthin and lutein,and the redness and yellowness had no significant differences in the white tissues in WR mutants during the feeding time.In the control group,the contents of astaxanthin and lutein were decreased during the feeding time in the red tissues in wild WR and WR mutants,as well as the redness and yellowness.In conclusion,the red color in color common carp would be faded when had no enough carotenoids for a long time;the supplemented astaxanthin and lutein could enhance the contents of astaxanthin and lutein,and the red color in WR mutants,but had no effects on the white color in WR mutants,which indicated the successful disruption of Scarb1 and Scarb1-like in WR mutants,the white color in WR mutants lost the ability to absorb and transport the carotenoids.