| Eggshell damages lead to economic losses in the egg production industry and broken eggs are susceptible to microbial infection that might cause harm to human health potentially. How to improve the quality of eggshell is a problem for poultry breeders. Eggshell quality variation cause egg quality. The genetic variations of the uterus are associated with the eggshell mineralization which may be the key factors to the variation of the eggshell quality.. The eggshell specific matrix protein OC17catalytic cycle provides a mechanism for forming the polycrystalline mammillary caps that are deposited on the organic membrane as the first stage of eggshell formation. The genetic variation of eggshell mineralization is an effective way to improve the quality of eggshell and improve the quality of eggs. To explore the function of OC17protein on the crystallization of calcium carbonate and to found for further analysis of the function of OC17gene.We examined49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. This study focus on the eggshell significantly difference in study group, and using transcriptome sequencing and whole genome resequencing on eggshells mineralization period which of affect calcium carbonate deposition of key candidate genes and genetic variations; using the transcription splicing, rapid amplification of cDNA ends (RACE), scanning electron microscopy (SEM) technology of eggshell specific matrix proteins OC17gene cloning, expression analysis and Ser61function for calcium carbonate crystallization research. The results showed that:1. the transcriptome sequencing revealed that there were889differentially expressed genes in hen uterus which of significantly different of the eggshell strength. The gene ontology (GO) and KEGG analysis found that these differentially expressed genes mainly involved in calcium transport and extracellular organic matrix proteins biological processes and calcium signaling pathway and extracellular matrix protein receptor interaction and other biological pathways. Among them,16differentially expressed genes involved in calcium ion signaling (KO:04020) and14differentially expressed genes related to extracellular matrix (KO:04512). Whole genome-resequencing results showed that the study obtained3,671,519SNPs in protein coding genes and there are484,857and563,437in the low eggshell strength group and normal eggshell strength group, respectively. The SNPs density of each chromosome was7.19to9.27SNP/Kbp, and75.61%of the SNPs mutation was located in the intron of the genes. There are508,035indel mutations in protein coding genes, including67925and76319unique indel mutations in low eggshell strength group and normal eggshell strength group, respectively, and78%of the indel mutations in intron of the genes. The SNP (rs312834462) of CACNA1H gene was correlated with the eggshell strength.2. The OC17gene full-length sequence of cDNA was obtained by using the technique of splicing and RACE, and encoding161amino acids, including the signal peptide of19amino acids. The OC17gene was expressed specifically in the uterus, and negatively correlated with the eggshell quality.3. The calcite crystal size and morphology were affected of the OC17recombinant protein, and the deletion of the Ser in the OC17protein might delay the formation of calcite crystal<104> face. In this study, the potential of genetic mutations in genetic improvement of egg quality provides the important candidate molecular genetic marker; OC17gene expression is closely related with the eggshell quality; OC17recombinant protein found OC17matrix protein in the shell biomineralization of function for the futural study. |
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