基于TMT定量蛋白质组学技术筛选调控鸡蛋绿壳性状的候选蛋白

Screening of Candidate Proteins Regulating Egg Green-shell Traits Based on TMT Quantitative Proteomics Technology

【目的】蛋壳颜色作为绿壳蛋的重要经济性状,不仅影响着消费者的购买偏好,还直接关系到绿壳蛋的市场定价,且绿壳蛋颜色的深浅受多个基因的协同调控。试验旨在挖掘调控绿壳蛋蛋壳颜色深浅的候选蛋白及其关键信号通路,为优化绿壳蛋鸡的选种选育和提升绿壳蛋的经济价值提供材料。【方法】以纯系高产期的赤水乌骨鸡为研究对象,采用串联质谱标签(TMT)定量蛋白质组学技术分析产浅绿蛋(QL)和产深绿蛋(SL)的赤水乌骨鸡蛋壳腺组织中的差异表达蛋白,对其进行GO功能和KEGG通路富集分析,筛选调控绿壳蛋颜色深浅的候选蛋白并进行Western blotting验证。【结果】共鉴定到5958个可定量蛋白,包含差异表达蛋白102个,其中46个上调,56个下调。GO功能分析发现,差异表达蛋白在生物过程中主要富集于联受体信号通路、维生素A应答、对雌激素的反应等条目;分子功能主要富集于丝氨酸水解酶活性、碳水化合物跨膜转运蛋白活性、视黄醇结合等条目;细胞组分主要富集于蛋黄、胶原三聚体、血液微粒等条目。KEGG通路分析中显著富集到6条通路,其中类固醇激素生物合成通路可能通过影响蛋壳钙化过程来改变蛋壳颜色的形成。Western blotting结果显示,SL组赤水乌骨鸡蛋壳腺中HSD17B7和TXNRD2蛋白表达水平极显著高于QL组(P<0.01),表达趋势与蛋白组学研究结果一致。【结论】差异表达蛋白中SLC22A15b、SLC35A、SLC2A1、SLC4A4、HSD17B7、TXNRD2的显著上调和KNG1的显著下调可能与蛋壳颜色的形成有关,研究结果可为阐明绿壳蛋色素沉着的分子机制提供参考。

【Objective】As an important economic trait of green-shell eggs,eggshell color not only affects consumers’purchase preference,but also directly affects the market pricing of green-shell eggs,and the color of green-shell eggs is regulated by multiple genes.The purpose of this study was to explore the candidate proteins and key signaling pathways that regulated the color of green-shell eggs,and provide materials for optimizing the selection and breeding of green-shell chickens and improving the economic value of green-shell eggs.【Method】Chishui Black-bone chickens in the high-yield period of the pure line were used as the research objects,the differentially expressed proteins in the eggshell gland tissues of Chishui Black-bone chickens producing light green eggs(QL)and dark green eggs(SL)were analyzed by tandem mass tags(TMT)quantitative proteomics technology.GO function and KEGG pathway enrichment analysis were performed to screen candidate proteins that regulated the color depth of green shell eggs and verify them by Western blotting.【Result】A total of 5958 quantifiable proteins were identified,including 102 differentially expressed proteins,of which 46 were up-regulated and 56 were down-regulated.GO functional analysis showed that differentially expressed proteins were mainly enriched in the receptor signaling pathway,vitamin A response and response to estrogen in biological process.Molecular function was primarily enriched in the items of serine hydrolase activity,carbohydrate transmembrane transporter activity,retinol-binding,etc.Cellular component was mainly enriched in items such as egg yolk,collagen trimer and blood particles.Six pathways were significantly enriched in KEGG pathway analysis,among which steroid hormone biosynthesis pathway might change the formation of eggshell color by affecting the eggshell calcification process.Western blotting results showed that the expression of HSD17B7 and TXNRD2 proteins in eggshell glands of Chishui Black-bone chickens in SL group were extremely significantly higher than that in QL group(P<0.01),and the expression trend was consistent with the proteomics results.【Conclusion】The significant up-regulation of SLC22A15b,SLC35A,SLC2A1,SLC4A4,HSD17B7 and TXNRD2 proteins,and the significant down-regulation of KNG1 protein in differentially expressed proteins might be relate to the formation of eggshell color.The results could provide reference for elucidating the molecular mechanism of pigmentation in green-shell eggs.