基于转录组测序技术筛选影响蛋鸡产暗斑蛋候选基因的研究

Identification of Key Candidate Genes Affecting Translucent Egg Production of Laying Hens Based on Transcriptome Sequencing Technology

本研究旨在利用转录组测序技术(RNA-seq)筛选鸡十二指肠中与暗斑蛋形成相关的功能基因和信号通路。选取连续产暗斑蛋率低和连续产暗斑蛋率高的济宁百日母鸡各10只分别作为对照组和暗斑组,采集十二指肠组织,测定钙代谢相关基因mRNA表达量,利用RNA-seq技术对差异表达基因(differentially expressed genes, DEGs)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,转录组获得高质量数据108.75 Gb且所有样本Q30>97%;共筛选得到471个DEGs,其中327个上调,144个下调,随机选取7个DEGs进行实时荧光定量PCR(qRT-PCR)验证,表达趋势与转录组测序结果基本一致;GO功能注释分析筛选到SLC8A3、SLC24A4、SLC9A2等DEGs主要富集到线粒体钙离子稳态、钠离子跨膜转运和钙离子结合的过程;KEGG通路富集结果显示,GSTT1、GSTO2、GSTA3、CLDN2、CLDN10等DEGs主要富集到细胞色素P450介导的外源物代谢通路、药物代谢-细胞色素P450、钙信号通路等;与对照组相比,暗斑组十二指肠钙结合蛋白(CALB1)的mRNA表达量显著下降(P<0.05)。综上,蛋鸡十二指肠中GSTT1、GSTO2、GSTA3基因的上调和SLC8A3、SLC24A4等基因的下调是影响暗斑蛋形成的关键因素,钙信号通路在暗斑形成中发挥了重要作用。本研究为暗斑蛋的机理研究提供了基础材料。

The aim of this study was to screen the functional genes and signaling pathways associated with translucent egg production in chicken duodenum by transcriptome sequencing(RNA-seq).Ten Jining Bari hens with low continuous translucent egg laying rate were selected as the control group,and ten ones with high continuous translucent egg laying rate were as the eggshell translucence group.Duodenum samples was collected,and the mRNA expression level of calcium metabolism related genes(DEGs)were measured.The differentially expressed genes(DEGs)were detected by RNA-seq,and the GO(gene ontology)functional an-notation and KEGG(kyoto encyclopedia of genes and genomes)signaling pathway enrichment were performed.The results showed that the total data obtained from the transcriptome was 108.75 Gb,and Q30 was>97%in all samples.A total of 471 DEGs were screened out,of which,327 ones were up-regulated and 144 ones were down-regulated.Seven DEGs were randomly selected for real-time quantitative fluorescence PCR(qRT-PCR)verification,and it was found that their expression trend was basically consistent with that of the transcripome sequencing results.GO functional annotation analysis showed that SLC8A3,SLC24A4 and SLC9A2 of the solute carrier family were mainly enriched into the mitochondrial calcium homeostasis,sodium ion transmembrane transport and calcium ion binding processes.KEGG pathway enrichment results showed that the DEGs such as GSTT1,GSTO2,GSTA3,CLDN2 and CLDN10 were mainly enriched into the cytochrome P450-mediated for-eign substance metabolism pathway,drug metabolity-cytochrome P450,calcium signaling pathway,etc.Com-pared with the control group,the mRNA expression of duodenal CALB1 in the eggshell translucence group was significantly decreased(P<0.05).In conclusion,the up-regulation of GSTT1,GSTO2 and GSTA3 genes and the down-regulation of SLC8A3 and SLC24A4 genes in the duodenum of laying hens were the key factors affect-ing the formation of translucent eggs,suggesting that calcium signaling pathway might play an important role in the formation of translucent eggs.The results of this study could provide basic materials for the mechanism re-search of translucent eggs.