Liver miRNA profiles of laying hens before and after reaching sexual maturity with a high energy feed during the maturation period
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摘要: 【目的】对育成期高能饲喂下开产与未开产蛋鸡的肝脏进行miRNA高通量测序分析，探明高能饮食状态下蛋鸡肝脏中影响其开产的miRNA，为提高产蛋性能打下基础。【方法】以代谢能水平为12.14 MJ/kg的高能饲粮饲喂育成期蛋鸡，通过Illumina NextSeq500高通量测序平台对开产与未开产蛋鸡肝脏进行small RNA测序，使用DESeq分析miRNA表达量，并以实时荧光定量PCR进行验证；采用miRanda对差异表达miRNA进行靶基因和靶位点预测，同时以超几何分布对差异表达的靶基因进行GO功能注释分析和KEGG信号通路富集分析。【结果】 6个样本（3个开产蛋鸡样本，3个未开产蛋鸡样本）能注释到的miRNA均超过300个，约占miRBase中已鉴定鸡miRNA的30.00%，且前体鉴定结果显示各样本注释到的miRNA有部分定位在同一前体上。与未开产组样本相比，开产组样本有9个miRNA表达上调、3个miRNA表达下调。其中，gga-miR-132c-5p、gga-miR-132b-5p、gga-miR-2184a-5p、gga-miR-132c-3p、gga-miR-132b-3p及gga-miR-2184a-3p属于同一miRNA基因簇，且gga-miR-1682、gga-miR-132b-3p和gga-miR-2184a-3p等3个上调miRNA在未开产蛋鸡样本中不表达。通过miRanda共预测得到129个差异表达潜在靶基因，以miR-203a预测得到的靶基因最多，为32个；其次是gga-miR-2184a-5p、gga-miR-148a-5p和gga-miR-12211-5p，分别预测到30、25和23个靶基因；而miR-132c-5p预测得到的靶基因最少，仅为9个。129个靶基因显著注释到8条GO功能条目上，生物学过程（Biological process）主要注释到脂质代谢过程、细胞脂质代谢过程、脂质生物合成过程、磷脂生物合成过程、磷脂代谢过程、甘油磷脂生物合成过程和解剖学结构发育，分子功能（Molecular funcion）仅注释到1条GO功能条目，即利钠肽受体活性，未发现涉及细胞组分（Cellular component）的GO功能条目；在注释到的8条GO功能条目中有6条与脂质代谢相关，涉及的靶基因有22个，占总潜在靶基因的17.10%。KEGG信号通路富集分析结果显示共显著富集到7条KEGG信号通路，其中脂肪酸降解、脂肪酸生物合成、酮体合成与降解及PPAR信号通路等4条信号通路与脂质代谢相关。【结论】育成期高能饲喂下开产与未开产蛋鸡中共存在12个差异表达miRNA，涉及129个差异表达潜在靶基因，且主要富集在肝脏脂质代谢相关过程和信号通路，说明miRNA是通过调控脂质代谢及其相关基因表达而影响蛋鸡开产。Abstract: 【Objective】In the current study,the liver miRNA profiles of sexually immature and sexually mature laying hens fed with a high energy feed during the maturation period were compared to clarify which liver miRNAs in liver influenced sexual maturity with high energy feed. The study laid a foundation for improving egg production.【Method】High-energy feed with 12.14 MJ/kg metabolic energy was used to feed laying hens during the maturation period. Liver miRNAs were detected by small RNA sequencing with the Illumina NextSeq500 platform in sexually mature (R) and sexually immature(N) laying hens. miRNA expression was analyzed using the bioinformatics software DESeq and validated using qRT-PCR. Target genes and target sites of differentially expressed miRNAs were predicted by miRanda and the differentially targeted genes were analyzed for GO and KEGG enrichment using a hypergeometric distribution.【Result】More than 300 miRNAs could be annotated in each of the 6 samples(3 R and 3 N),accounting for about 30.00% of the identified chicken miRNAs in miRBase. The results from precursor identification showed that some annotated miRNAs in each sample were located on the same precursor. Compared with the N group,the expression levels of 9 miRNAs in the R group were up-regulated and 3 miRNAs were down-regulated,among which gga-miR-132c-5p,gga-miR-132b-5p, gga-miR-2184a-5p,gga-miR-132c-3p,gga-miR-132b-3p and gga-miR-2184a-3p were in a miRNA cluster. In addition,3 upregulated miRNAs in the R group,including gga-miR-1682,gga-miR-132b-3p and gga-miR-2184a-3p,were not expressed in the N group. A total of 129 target genes of the differentially expressed genes were predicted by miRanda. The number of target genes predicted for gga-miR-203a was the highest(32), gga-miR-2184a-5p,gga-miR-148a-5p and ggamiR-12211-5p showed a smaller number(30,25 and 23,respectively) and miR-132c-5p the lowest(only 9). GO analysis of the 129 differentially targeted genes showed that there were 8 significantly enriched GO terms,including 7 biological processes:lipid metabolism,cellular lipid metabolism,lipid biosynthesis,phospholipid biosynthesis,phospholipid metabolism,glycerophospholipid biosynthesis and anatomical structure development. No cellular component terms were found and only 1 molecular function term was detected:natriuretic peptide receptor activity. In the 8 enriched GO items,6 were related to lipid metabolism,and 22 target genes were involved,accounting for 17.10% of the 129 genes. KEGG analysis showed that there were 7 significant enriched KEGG terms,of which fatty acid degradation,fatty acid biosynthesis,synthesis and degradation of ketone bodies and PPAR signaling pathway were related to lipid metabolism.【Conclusion】A total of 12 miRNAs are differentially expressed between sexually immature and sexually mature laying hens under a high energy feeding regime during the maturation period,involving 129 potential differentially expressed genes,which are mainly enriched in processes and pathways related to lipid metabolism in the liver. The data suggests that miRNAs affect sexual maturity of laying hens by regulating lipid metabolism and related genes.
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Keywords:
- laying hen /
- liver /
- miRNA /
- growing period /
- high energy feed /
- reaching sexual maturity
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