| Avian eggshell pigment can strengthen the eggshell and filter harmful solar radiation,and the eggshell coloration is the result of crypsis or mimetism to avoid predation and plays an important roles for avian.In addition,the blue eggshell color has been proposed as a sexually selected signal of the laying female's genetic quality to its mate in order to induce a higher allocation of paternal care.Biliverdin content in eggshell is the key role for the formation of blue egg in birds,but the source of eggshell pigment has not been resolved.Moreover,the molecular mechanism of the blue egg formation in poultry may be different due to the different genetic backgrounds.The objective of this study was to research the molecular mechanism of the blue egg formation in ducks,we chose the blue-shelled ducks and white-shelled ducks to be the experimental subjects.At first,to reveal the synthesis characteristics of biliverdin in ducks,we detected the biliverdin contentof eggshell,serum,liver and shell gland,and then localization of HO-1 of shell gland in duck was performed.Then,the analyzing and enrichment differentially expressed genes and miRNA related to the formation of the blue egg traits in shell gland of blue-and white-shelled ducks by using high-throughput transcriptome sequencing,in order to explore the molecular mechanism of duck blue-shell egg formation and lay a theoretical foundation for the breeding of blue-shelled ducks.The results show that:The biliverdin content in eggshell of blue-shelled ducks was significantly higher than that in eggshell of white-shelled ducks(P<0.01).There were no significant difference in biliverdin concentration in serum,shell gland,liver and excreta between the two groups.In addition,HO-1 protein was high expression in shell gland epithelium cells of blue-and white-shelled ducks.The 6 samples were obtained 34.64Gb Clean Data,andeach library produced more than 5.29G clean bases with a Q30 percentage over 95.41%.The biological repetition samples had higher correlation in the different two groups,and the correlation was more than 0.8276.Compared with the shell gland of white duck,there were 457 up-regulate genes and 311 down-regulate genes.The DEGs were predictednine groups(1 to 9)using hierarchical clustering methods,were defined according to the expression profiles.KEGG database analysis showed that the biliverdin synthesis were enriched in the transcriptome,including ten enzymes required for biliverdin synthesis,and only ALAS1 was significantly higher expression in blue-shelled ducks.The protein network mapping was obtained between these enzymes required for biliverdin synthesis and differentially expressed genes,and the result showed that there were ten differentially expressed genes,which may involve in the regulation of biliverdin synthesis,and most of them were in contact with ALAS1.Subsequently,hierarchical clustering methods wasperformed based on RPKM values.We found that PER2,NFE2L1 and ALAS1 were clustered at a group.The cluster analysis of transporter gene and biliverdin biosynthetic gene,we found that there were similar expression pattern between transporter gene and biliverdin biosynthetic gene,for example TAL and HMOX-1,SLC13A5 and ALAS1.The results of miRNA showed that all 269 miRNAs were screened in the shell gland of blue shelled ducks and white shelledducks,including known 245 miRNAs and new 24 miRNAs.Compared with white shelled ducks,the shell gland of blue shelled ducks had 169 differently expressed miRNAs,including 85 up-regulated and 84 down-regulated,and total 4,872 target genes were obtained,but the miRNAs were not significant difference.The correlation coefficient were higher between all of the shell gland samples in blue and white shelled ducks.The Top Gene Ontology(TopGO)analysis of target genes showed that molecules function including heme binding(count=23,P=0.008)were existed,and most of them were accompanied by function of electron and Fe2+ transport.Biliverdin synthetic gene that heme oxygenase 1(HMOX1)and a large number of organic matter transport and metabolism genes were predicted by gga-miR-133b,gga-miR-193b-3p and gga-miR-449a.In conclusion,biliverdin in eggshell is a primary pigment for blue egg-shell in ducks,which is most likely synthesized and secreted from epithelium cells of shell gland to eggshell in ducks.RNA-Seq analysis of the shell gland in blue-and white-shelled egg ducks suggested that ALAS1 may be a key role in formtation of blue egg in ducks,and NF-E2L1 and PER2 may be the essential elements in the formation of blue egg through regulating the expression of ALAS1 in duck shell gland.In addition,some of transport gene such as TAL and SLC13A5 ma)nvolve in the formation of the blue egg in duck,through regulating the synthesis of intermediate product in thebiliverdin biosynthetic pathway.These results suggested that the formation of the blue-shelled egg trait may be related to the biliverdin synthesis of shell gland in ducks. |
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