基于转录组测序筛选维生素D调节肉鸡十二指肠钙和磷吸收的差异表达基因

Differentially Expressed Genes in Vitamin D Regulating Calcium and Phosphorus Absorption in Duodenum of Broiler Chickens Using RNA-Sequencing

本试验利用转录组测序技术(RNA-seq)筛选维生素D调节肉鸡十二指肠钙和磷吸收的功能基因和信号通路。以1~16日龄罗斯308肉鸡为试验动物,设计3种饲粮,分别在基础饲粮(不含维生素D)中添加0(对照)、5、10μg/kg 1,25-二羟基维生素D 3[1,25-(OH)2-D 3]。16日龄时屠宰肉鸡,刮取十二指肠黏膜,利用转录组测序技术,对差异表达基因进行GO(gene ontology)功能注释和KEGG(kyoto encyclopedia of genes and genomes)信号通路富集。结果表明:1)与对照组相比,添加5和10μg/kg 1,25-(OH)2-D 3显著改善肉鸡生长性能(P<0.05),增强股骨和胫骨矿化;但10μg/kg 1,25-(OH)2-D 3组肉鸡胫骨与股骨的重量、长度和灰分重量显著低于5μg/kg 1,25-(OH)2-D 3组(P<0.05)。2)分别进行0 vs.5μg/kg、0 vs.10μg/kg、5 vs.10μg/kg 1,25-(OH)2-D 3组间的比较,在各组间筛选出1029、939和642个差异表达基因。3)经GO功能注释,筛选出细胞膜维生素D受体(PDIA3)、细胞核维生素D受体(VDR)、Ⅱb型钠磷转运载体蛋白(SLC34A2)、无机磷转运载体蛋白2(SLC20A2)、钙结合蛋白D28k(CALB1)、钙离子转运ATP酶B1(ATP2B1)、钙离子转运ATP酶B2(ATP2B2)、钠钙交换蛋白(SLC8A1)、成纤维细胞生长因子1(FGF1)和成纤维细胞生长因子19(FGF19)等差异表达基因,这些基因富集到十二指肠中调节钙和磷吸收代谢的过程。4)KEGG通路富集结果显示,差异表达基因富集到丝裂原活化蛋白激酶(MAPK)信号通路、环磷酸腺苷(cAMP)信号通路或环磷酸腺苷-蛋白激酶A(cAMP-PKA)信号传导通路和环磷酸鸟苷-蛋白激酶G(cGMP-PKG)信号通路。5)选出4个差异表达基因进行实时荧光定量PCR(qRT-PCR)检测,结果显示qRT-PCR与转录组测序结果基本一致。综上,本试验筛选出维生素D调节肉鸡十二指肠钙和磷吸收的关键基因和信号通路,为改善肉鸡钙和磷利用率奠定理论基础。

This study was conducted to investigate the candidate genes and signaling pathways in vitamin D regulating calcium and phosphorus absorption in small intestine of broiler chickens using RNA-sequencing technology.Ross 308 broiler chickens at 1 to 16 days of age were used as experimental animals.Three levels[0(control group),5 and 10μg/kg]of 1,25-dihydroxycholecalciferol[1,25-(OH)2-D 3]were added to the basal diet without vitamin D.Broiler chickens were killed and duodenal mucosa was collected at 16 days of age.Gene ontology(GO)function annotation and kyoto encyclopedia of genes and genomes(KEGG)signal pathway enrichment of differentially expressed genes were carried out using RNA-sequencing technology.Results showed as follows:1)compared with the control group,the addition of 5 and 10μg/kg of 1,25-(OH)2-D 3 increased significantly growth performance(P<0.05)and improved tibia and femur mineralization.The weight,length and ash content of tibia and femur of broilers in 10μg/kg of 1,25-(OH)2-D 3 group were significantly lower than those in 5μg/kg 1,25-(OH)2-D 3 group(P<0.05).2)A total of 1029,939,and 642 differentially expressed genes were screened when comparing the three levels of 1,25-(OH)2-D 3(0 vs.5μg/kg,0 vs.10μg/kg,and 5 vs.10μg/kg),respectively.3)GO analysis identified candidate genes regulating calcium and phosphorus absorption and metabolism,including nuclear vitamin D receptor(VDR),protein disulfide-isomerase A3(PDIA3),type Ⅱb sodium phosphate transporter protein NaPi-Ⅱb(SLC34A2),inorganic phosphorus transporter carrier protein 2(SLC20A2),calbindin D28k(CALB1),ATPase plasma membrane Ca^2+ transporting 1(ATP2B1),ATPase plasma membrane Ca^2+ transporting 2(ATP2B2),solute carrier family 8 member A1 NCX1(SLC8A1),fibroblast growth factor 1(FGF1)and fibroblast growth factor 19(FGF19).4)KEGG analysis indicated pathways associated with the intestinal calcium and phosphorus absorption and metabolism of broiler chickens,including mitogen-activated protein kinase(MAPK)signaling pathway,cyclic adenosine monophosphate(cAMP)signaling pathway or cyclic adenosine monophosphate-protein kinase A(cAMP-PKA)signaling pathway,and cyclic guanosine monophosphate-protein kinase G(cGMP-PKG)signaling pathway.5)The results of qRT-PCR were in accordance with those of RNA-seq.In summary,the differentially expressed genes and signaling pathways in vitamin D regulating calcium and phosphorus absorption in small intestine of broiler chickens are screened.These data will be helpful to improve calcium and phosphorus absorption and retention in poultry diets.