基于转录组测序筛选影响从江香猪产仔数的候选基因

Selection of candidate genes affecting litter size of Congjiang Xiang pig by transcriptome sequencing

【目的】筛选出影响从江香猪产仔性状的基因调控网络,揭示其繁殖分子机理,为后期开展从江香猪繁殖性能研究及良种选育提供理论依据。【方法】以高产仔家系(平均产仔数≥12头,遗传稳定)和低产仔家系(平均产仔数8~10头,遗传稳定)从江香猪为研究对象,选择初情期不同产仔家系从江香猪卵巢组织各3个,使用Illumina HiSeq^(TM)2000测序仪进行转录组测序(RNA-Seq),并对筛选获得的差异表达基因进行GO功能富集分析及KEGG信号通路富集分析,以寻找与从江香猪产仔数相关的基因调控网络。【结果】从高产仔家系和低产仔家系从江香猪卵巢组织中测序获得的纯净序列(Clean reads)均超过5000万条,且有96.00%以上的Clean reads能比对上猪参考基因组。以|log_2FC|≥1.0且P<0.01为标准,筛选获得高产仔家系和低产仔家系从江香猪卵巢组织差异表达基因212个,其中上调表达基因138个、下调表达基因74个。采用实时荧光定量PCR对随机选择的10个差异表达基因进行定量分析,发现OSAP、MSMB、FGFBP1、RLN、HAS1和PLBD1基因在高产仔家系从江香猪卵巢中的相对表达量显著高于低产仔家系从江香猪(P<0.05,下同),而EDG7、PTX3、BSP1和MRO基因在低产仔家系从江香猪卵巢中的相对表达量显著高于高产仔家系从江香猪,与RNA-Seq测序结果一致。212个差异表达基因共富集在48个GO功能条目上,包含分子功能(Molecular function)、生物学过程(Biological process)和细胞组分(Cellular component);经KEGG信号通路分析发现共有70个差异表达基因注释到特定的代谢信号通路上,其中显著性富集的KEGG信号通路有类固醇生物合成通路(Steroid biosynthesis)、钙信号通路(Calcium signaling pathway)、卵巢类固醇生成通路(Ovarian steroidogenesis)、心肌细胞肾上腺信号通路(Adrenergic signaling in cardiomyocytes)和肥厚型心肌病通路(Hypertrophic cardiomyopathy,HCM)。在卵巢类固醇生成通路上发现SCARB1、STAR、COX2、CYP11A、CYP17A、17βHSD和CYP19A等7个基因与从江香猪的繁殖性能存在密切联系。【结论】从江香猪卵巢类固醇生成通路上的STAR、CYP11A、CYP17A、17βHSD和CYP19A基因与其发情排卵密切相关,于发情期上调表达能促进卵泡发育成熟及类固醇激素合成,通过增加排卵数量而促使从江香猪表现高产,故可作为从江香猪繁殖性状的候选基因。

【Objective】In order to provide theoretical basis for the breeding performance studies and breeding,gene regulatory networks and the molecular mechanism of reproduction traits on Congjiang Xiang pig were screened.【Method】Three ovary tissues were selected from the high litter size family(number of average litter size≥12,stable inheri-tance)and the low litter size family(number of average litter size was 8-10,stable inheritance)at puberty,and transcriptome sequenced(RNA-Seq)by Illumina HiSeq^(TM)2000,and GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes(DEGs)to find the gene regulation networks related to litter size of Congjiang Xiang pigs.【Result】More than 50 million Clean reads were obtained from sequencing results of ovaries from high litter size family and low litter size family,and more than 96.00%of the Clean reads could be mapped with the reference genome of pig.With|log2FC|≥1.0 and P<0.01 as the conditions,212 DEGs in ovary between high yield family and low yield family were found,and among which 138 were up-regulated genes,74 down-regulated genes.Ten randomly selected DEGs was verified by real-time fluorescence quantitative PCR,the expression levels of OSAP,MSMB,FGFBP1,RLN,HAS1 and PLBD1 genes in the ovary of high litter size family were significantly higher than those in the low litter size family(P<0.05,the same below).However,the expression levels of EDG7,PTX3,BSP1 and MRO genes were in the ovary of low litter size family were significantly higher than those in the high litter size family.The results showed that the sequencing results were consistent with the results of qRT-PCR.A total of 212 DEGs were enriched in 48 GO functional items,including molecular function,biological process and cellular component.Through KEGG pathway analysis,a total of 70 DEGs were annotated to specific metabolic pathways.Significant enrichment pathways included steroid biosynthesis,calcium signaling pathway,ovarian steroidogenesis,adrenergic signaling in cardiomyocytes and hypertrophic cardiomyopath(HCM).On the ovarian steroidogenesis pathway,seven genes including SCARB1,STAR,COX2,CYP11 A,CYP17 A,17 P17 A and CYP19 A1 genes were found to be closely related to reproductive performance of Congjiang Xiang pigs.【Conclusion】STAR,CYP11 A,CYP17,17βHSD and CYP19 A1 genes in the ovarian steroidogenesis pathway are closely related to estrus and ovulation in pigs.These genes which are up-regulated during estrus can promote follicle development and maturation,steroid hormone expression,enhance high reproduction of Congjiang Xiang pigs by increasing the number of ovulation.Thus these genes can be used as candidate genes for breeding traits in Congjiang Xiang pigs.