Integrated Analysis Of Transcriptome And Proteome To Explore The Genetic Factors Influencing Chicken Eggshell Brownness

The phynotype of brown eggshell color is diverse. Eggs from one population will present different shade of brownness. Previous studies suggested that brown eggshell color was multi-genes controlled, but the detail machnism was not clear up to now. In this study, we used pure line hens laying brown eggs as materials to explore the molecular mechanism of brown eggshell color formation.We compared the protoporphyrin IX content in eggshell, eggshell gland, serum, excreta and bile between hens laying dark brown eggs and light brown eggs respectively. Results showed that protoporphyrin IX content in dark brown eggshell was significantly higher than in light brown eggshell, suggesting that eggshell brownness is dependent on the content of protoporphyrin Ⅸ in the eggshell. Protoporphyrin Ⅸ content was significantly higher in eggshell gland of the dark group but there was no significant difference in the other tissues between the two groups, and the protoporphyrin IX content in the eggshell was significantly higher than in the shell gland. This result represented that the protoporphyrin Ⅸ was indeed accumulated in the shell gland gradually. However, we are not sure the site of protoporphyrin Ⅸ synthetized. Then we compared genes expression including ALAS1, CPOX, FECH, ABCB6, ABCB7, ABCG2and FLVCR by RT-PCR between the two groups. The expression of ALAS1in the shell gland and liver of the dark group were significantly higher than in the light group. The expression of CPOX, ABCB7, ABCG and FLVCR in the shell gland of the dark group were significantly higher than in the light group, but there were no significant difference in the liver between the two groups. This result suggested that protoporphyrin IX was synthesized in the shell gland.We performed transcriptome and proteome analysis in the shell gland of hens laying dark brown egg and light brown egg and discovered189differently expressed transcripts and335differently expressed proteins. Functional analysis of these differently expressed transcripts and proteins found that ovotransferrin (TF), specifically expressed in the poultry oviduct, was involved in the iron ion transport. We speculated that ovotransferrin might affect the accumulation of protoporphyrin IX by regulating heme synthesis via iron ion transport. Besides, down regulation of iron-sulfur cluster protein (Iba57) in the eggshell gland of chickens from the light brown group would result in the decrease of protoporphyrin IX by reducing the succinyl-CoA production. Heat-shock protein (HSP70) down regulation in the light group proved the speculation that the protoporphyrin Ⅸ content in the shell gland of chickens from light group was less that in the dark group. Translocator protein (TSPO) has the ability to transport protoporphyrin Ⅸ out of mitochondria and adenine nucleotide translocators (ANT2) is involved in importing the protoporphyrin Ⅸ into the mitochondria matrix. Down regulation of TSPO and up regulation of ANT2and TF in the shell gland of chickens from light group resulted in the increase of protoporphyrin Ⅸ transported into the mitochondria matrix for the formation of heme and then decreased the accumulation of protoporphyrin Ⅸ in the epithelial cells of shell gland. Therefore, the protoporphyrin Ⅸ deposited onto the eggshell would be reduced consequently and resulted in the shallow of eggshell brownness.