摘要
[目的]筛选鉴定鸡有机阴离子转运多肽1B3(OATP1B3)的互作蛋白,并探究其对鸡蛋绿壳颜色形成的影响,为提高绿壳蛋鸡群体选育准确性提供参考依据.[方法]以真核表达载体pEGFP-SLCO1B3和空载体pEGFP-C1分别转染绿壳型赤水乌骨母鸡蛋壳腺上皮细胞,通过免疫共沉淀(Co-IP)结合液相色谱与串联质谱联用技术(LC-MS/MS)筛选鸡OATP1B3互作蛋白,经GO功能注释分析、KEGG信号通路富集分析和蛋白相互作用网络分析鉴定出与鸡蛋绿壳性状形成相关的OATP1B3互作蛋白.同时,通过构建SLCO1B3基因沉默及过表达载体分别转染蛋壳腺上皮细胞,检测SLCO1B3基因过表达或沉默时部分OATP1B3互作蛋白基因(FXR1、CTTN、RPL12和RPS17)在蛋壳腺上皮细胞中的表达情况,并根据鸡蛋壳颜色分为白壳、浅绿和深绿3种类型,分析蛋壳颜色△a*值与FXR1、CTTN、RPL12和RPS17基因表达的相关性.[结果]共筛选鉴定出37个与鸡OATP1B3相互作用的蛋白,其中FXR1、CTTN、RPL12和RPS17与鸡蛋的绿壳性状有密切关系.SLCO1B3基因过表达能极显著降低蛋壳腺上皮细胞内FXR1基因的表达(P<0.01,下同),显著上调RPL12基因表达(P<0.05,下同),极显著上调RPS17基因表达;当SLCO1B3基因被沉默后,蛋壳腺上皮细胞内FXR1和CTTN基因的表达极显著上调,RPL12基因的表达显著下降.白壳类型赤水乌骨母鸡下丘脑中的FXR1基因相对表达量极显著高于绿壳类型赤水乌骨母鸡,蛋壳腺和肝脏中的RPL12基因及肝脏中的RPS17基因相对表达量则显著高于绿壳类型赤水乌骨母鸡;此外,FXR1基因在下丘脑中的相对表达量与蛋壳颜色△a*值极显著正相关,RPL12基因在蛋壳腺和肝脏中的相对表达量与蛋壳颜色△a*值极显著正相关.[结论]筛选鉴定出的37个鸡OATP1B3互作蛋白主要定位于细胞质或细胞核,具有蛋白结合能力及结构分子活性,主要参与细胞过程、生物调节和能量代谢.在鸡壳腺上皮细胞内SLCO1B3基因与FXR1、CTTN、RPL12和RPS17基因间存在调控关系,其中,FXR1和RPL12基因在鸡蛋绿壳性状的形成过程中发挥着重要作用.
Abstract
[Objective]The purpose of the study was to screen and identify chicken organic anion transporting poly-peptide 1B3(OATP1B3)interacting proteins,to explore their effects on the formation of green shell color in eggs,and to provide a reference basis for improving the accuracy of selection of green-shelled laying hen populations.[Method]Eu-karyotic expression vector pEGFP-SLCO1B3 and empty vector pEGFP-C1 were used to transfect eggshell gland epithelial cells of green-shelled Chishui black bone hens,respectively.The chicken OATP1B3 interacting proteins were screened by co-immunoprecipitation(Co-IP)coupled with liquid chromatography and tandem mass spectrometry(LC-MS/MS).By GO functional annotation analysis,KEGG signaling pathway enrichment analysis and protein interaction network analy-sis,OATP1B3 interaction proteins related to the formation of green shell trait in eggs were identified.At the same time,by constructing SLCO1B3 gene silencing and overexpression vectors to transfect eggshell gland epithelial cells respec-tively,the expression of some OATP1B3 interaction protein genes(FXR1,CTTN,RPL12,and RPS17)in the eggshell gland epithelial cells was detected when SLCO1B3 gene was overexpressed or silenced.And they were categorized into three types of eggshells according to the eggshell color,namely,white shell,light green,and dark green.The correlation between eggshell color △a* value and the expression of FXR1,CTTN,RPL12 and RPS17 genes was analyzed.[Result]A total of 37 proteins interacting with chicken OATP1B3 were identified,among which FXR1,CTTN,RPL12 and RPS17 were closely related to the green shell trait of eggs.The overexpression of SLCO1B3 gene could extremely significantly re-duce the expression of FXR1 gene in the epithelial cells of the eggshell glands(P<0.01,the same below),significantly up-regulate the expression of RPL12 gene(P<0.05,the same below).0.05,the same below),and extremely signifi-cantly up-regulate RPS17 gene expression.When SLCO1B3 gene was silenced,the expression of FXR1 and CTTN genes in eggshell gland epithelial cells was extremely significantly up-regulated,and the expression of RPL12 gene was signifi-cantly decreased.The relative expression of FXR1 gene in the hypothalamus of white-shelled type Chishui black bone hens was extremely significantly higher than that of green-shelled type Chishui black bone hens,while the relative expres-sion of RPL12 gene in the eggshell glands and liver as well as that of RPS17 gene in the liver was significantly higher than that of the green-shelled type Chishui black hens.Moreover,the relative expression of FXR1 gene in hypothalamus ex-tremely significantly positively correlated with the value of eggshell color △a*,and the relative expression of RPL12 gene in eggshell gland and liver was extremely significantly positively correlated with eggshell color △a* value.[Conclu-sion]The 37 chicken OATP1B3 interacting proteins identified by screening are mainly localized in the cytoplasm or nucleus,with protein-binding ability and structural molecular activity,and are mainly involved in cellular processes,bio-logical regulation and energy metabolism.There is a regulatory relationship between SLCO1B3 gene and FXR1,CTTN,RPL12 and RPS17 genes in the epithelial cells of chicken shell gland,in which FXR1 and RPL12 genes play an important role in the formation of green shell trait in eggs.
基金项目
贵州省科技计划项目(黔科合支撑[2022]重点34)
贵州省教育厅自然科学研究项目(QKHZC[2022]Zhongdian 34)
国家科技期刊平台
NSTL
万方数据