| The green-shell trait is an important economic trait in green-shelled hens,but the degree of green-shelled egg coloration can vary greatly due to genetic differences among individuals.In order to analyze the molecular mechanisms underlying the differences in greenshell egg coloration,this study aimed to analyze the molecular mechanisms of greenshell traits in Chishui wooshell chickens by transcriptomic(RNASeq)and proteomic tandem mass spectrometry tagging(TMT)techniques,and to provide a theoretical basis for the study of greenshell egg shell coloration.In this experiment,the pure strain of 280-day-old Chishui wolf-bone chickens was used as the research object.The eggshell color of each chicken was measured continuously for 14 days using an eggshell color tester,and the eggshell color was divided into two groups:dark green eggs(SL)and light green eggs(QL).Three chickens from each group were slaughtered,egg shell glands were collected,and differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)from different groups were screened by RNA-Seq and proteomics TMT techniques,and GO(Gene Ontology,GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)enrichment analyses were performed on these significantly differentially expressed genes and proteins,in order to explore the candidate genes,proteins and signaling pathways regulating the color of green eggshells.By analyzing the possible mechanisms by which these genes and proteins affect greenshell egg color,we laid the foundation for the next screening of molecular markers for greenshell egg color traits.The main findings are as follows:1.RNA-Seq analysis of eggshell gland tissues of Chishui black bone chickenIn this study,sequencing analysis of the dark green and light green groups of eggshell gland transcriptome screened 93 DEGs,59 of which were significantly upregulated and 34 significantly down-regulated in the dark green group.The DEGs were mainly significantly enriched in sodium transport,negative ion binding,sarcoplasmic reticulum membrane,mineral uptake,and linoleic acid metabolism by the combined analysis of GO and KEGG.In conjunction with the deposition of biliverdin onto the eggshell surface,TF,SCNN1 family genes,CYP450 family genes,SLC family genes,FAM family genes,and signaling pathways of aldosterone regulating sodium reabsorption,α-linolenic acid metabolism,and arachidonic acid metabolism were found to be potentially important genes and pathways affecting eggshell color.The q RT-PCR technique was used to detect the m RNA expression of six of the screened genes in eggshell gland tissue to verify the reliability of the data results.2.Histoproteomic analysis of eggshell gland tissue of Chishui black bone chickenThe eggshell gland tissues of hens laying dark green and light green groups of Chishui woebone chickens were analyzed by proteomics TMT technique.A total of5958 quantifiable proteins were identified,and 102 DEPs were significantly differentially expressed in the dark green and light green groups,of which 46 were significantly up-regulated and 56 were significantly down-regulated in the dark green group.Subcellular localization revealed that most of these DEPs are mostly localized in the nucleus,cytoplasm,cell membrane and mitochondria and are mainly involved in biological processes such as hormone response,transmembrane transport activity,transmembrane enzyme excitation,transport activity,cell stimulation response,activation,growth factors,epithelial cell regulation,coagulation regulation,and chemical reactions.Significantly differential expression of HSD17B7,TXNRD2,and KNG1 proteins with SLC family proteins was found by functional analysis to help catalyze or inhibit heme synthesis and affect the amount of biliverdin deposited onto the eggshell surface,while the function of carbohydrate transmembrane transport activity and the signaling pathway of steroid hormone biosynthesis may be key signaling functions and pathways affecting biliverdin synthesis or transport in green eggs.3.Combined RNA-Seq and proteome analysisSequencing of the eggshell gland transcriptome revealed that SLC34A2,and SLC20A1 genes were significantly upregulated in the dark green group;studies of the eggshell gland proteome revealed that SLC35A5,SLC22A15 b,SLC4A4 and SLC2A1 proteins were significantly upregulated in the dark green group,and subcellular localization revealed that the SLC family of significant DEPs were all localized to the cell membrane.In this study,it is hypothesized that the upregulation of some of the SLC family of transporter genes and proteins,which are effective mediators between cell adhesion,have different effects on the transport of inorganic ions such as calcium through the upregulation of some genes and proteins,affecting the eggshell calcification process and leading to differences in the amount of biliverdin deposition.By correlation analysis of transcriptomic and proteomic KEGG signaling pathways,steroid hormone biosynthesis was enriched in both histologies,and combined with the analysis of green eggshell formation mechanism,steroid hormone biosynthesis has a regulatory role in the process of green eggshell color formation.In summary,the color differences of green-shell eggs of Chishui woks are regulated by multiple genes,among which SLC family genes and proteins play an important role in the regulation.In summary,the color differences of green-shell eggs of Chishui woks are regulated by multiple genes,among which SLC family genes and proteins play an important role in the regulation.This study provides a theoretical basis for revealing the regulatory mechanism of egg shell formation in Chishui woosebone chickens with different degrees of green color. |
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