猪肌内脂肪沉积相关基因的筛选及其表达特性分析

doi:10.16431/j.cnki.1671-7236.2022.05.006

摘要/Abstract

摘要： 【目的】通过分析马身猪和大白猪背最长肌转录组测序数据，筛选肌内脂肪沉积相关基因，并对其表达特性进行分析。【方法】采集180日龄马身猪和大白猪背最长肌，采用RNA-Seq技术对其进行转录组测序，筛选差异表达基因并进行GO功能和KEGG通路富集分析;采用GeneCards在线查询基因功能，进一步筛选与肌内脂肪沉积相关的差异基因;采用实时荧光定量PCR技术检测差异基因在2个品种猪不同组织以及肌内脂肪细胞成脂分化过程中的表达变化。【结果】获得2个品种猪背最长肌差异表达基因共280个，其中128个表达显著上调，152个表达显著下调。GO功能富集分析注释到46个条目，其中生物过程24条，分子功能8条，细胞组分14条。KEGG通路分析显著富集到PPAR信号通路、MAPK信号通路和脂肪细胞因子信号等脂肪沉积相关通路。GeneCards功能查询获得TRAF2、DUSP1、ACOT4、NR4A1、SLC27A6、PLIN5共6个与脂肪沉积相关的基因。实时荧光定量PCR分析表明，马身猪和大白猪背最长肌中NR4A1、DUSP1、PLIN5基因表达量差异显著或极显著(P<0.05;P<0.01)，TRAF2、ACOT4和SLC27A6基因表达量差异不显著(P>0.05)，但表达变化趋势与测序结果一致;马身猪腹部皮下脂肪组织中NR4A1和PLIN5基因表达量极显著高于背部皮下脂肪组织(P<0.01)，而大白猪背部皮下脂肪组织中NR4A1和DUSP1基因表达量极显著高于腹部皮下脂肪组织(P<0.01)，PLIN5基因表达量显著高于腹部皮下脂肪组织(P<0.05)。在背部皮下脂肪组织中，NR4A1、DUSP1和PLIN5基因在大白猪中的表达量极显著高于马身猪(P<0.01);在腹部皮下脂肪组织中，NR4A1和DUSP1基因在大白猪中的表达量显著高于马身猪(P<0.05)，PLIN5基因表达量在2个品种间无显著差异(P>0.05)。体外培养的猪肌内脂肪细胞分析表明，随着成脂天数的增加，NR4A1基因表达量呈下降趋势，DUSP1和PLIN5基因表达量均呈上升趋势。【结论】本研究获得了NR4A1、DUSP1、PLIN5 3个与猪肌内脂肪沉积相关的候选基因，可为后续进一步探讨猪肌内脂肪沉积调控机制提供一定的参考。

关键词: 马身猪; 大白猪; RNA-Seq; 差异表达基因; 肌内脂肪

Abstract: 【Objective】 The aim of this study was to screen the genes related to intramuscular fat deposition and analyze the expression characteristics based on studying the transcriptome sequencing data of longissimus dorsi muscle in Mashen and Large White pigs.【Method】 The longissimus dorsi muscle samples of Mashen pigs and Large White pigs at the age of 180 days old were collected and sequenced by RNA-Seq technology to screen differentially expressed genes.Founctional enrichment analysis of the differentially expressed genes was conducted by GO function and KEGG pathway.GeneCards was used to query gene function online and further screen differentially expressed genes related to intramuscular fat deposition.Real-time quantitative PCR was used to analyze the expression characteristics of the differentially expressed genes in different tissues of the two varieties and intramuscular adipocyte differentiation process at the different stages.【Result】 There were 280 differentially expressed genes in longissimus dorsi muscle between Mashen and Large White pigs,of which 128 were significantly up-regulated and 152 were significantly down-regulated.GO function enrichment analysis identified 46 items,including 24 in biological processe,8 in molecular function,and 14 in cellular component.Meanwhile,KEGG pathway analysis recognized that the differentially expressed genes were involved in PPAR,MAPK and adipokine signaling pathways.Six genes,TRAF2,DUSP1,ACOT4,NR4A1,SLC27A6 and PLIN5,which related to lipid deposition,were obtained by GeneCards function search.Real-time quantitative PCR results showed that the expression of NR4A1,DUSP1 and PLIN5 genes in longissimus dorsi muscle between Mashen and Large White pigs were significantly or extremely significantly different (P<0.05 or P<0.01),while the expression of TRAF2,ACOT4 and SLC27A6 genes were not significantly different (P>0.05),but the expression trend was consistent with the sequencing results.The expression of NR4A1 and PLIN5 genes in abdominal subcutaneous adipose tissue of Mashen pigs were extremely significantly higher than those in dorsal subcutaneous adipose tissue (P<0.01),whiles the expression levels of NR4A1 and DUSP1 genes in dorsal subcutaneous adipose tissue of Large White pigs were extremely significantly higher than those in abdominal subcutaneous adipose tissue (P<0.01),and the expression of PLIN5 gene of Large White pigs was significantly higher than that in abdominal subcutaneous adipose tissue (P<0.05).In the dorsal subcutaneous adipose tissues,the expression of NR4A1,DUSP1 and PLIN5 genes in Large White pigs was extremely significantly higher than those in Mashen pigs (P<0.01).In the abdominal subcutaneous adipose tissues,the expression of NR4A1 and DUSP1 genes in Large White pigs were significantly higher than those in Mashen pigs (P<0.05),and the expression of PLIN5 gene was not significantly different (P>0.05).During the porcine intramuscular adipogenic differentiation,the expression of NR4A1 gene showed a down-regulated trend,and the expression of DUSP1 and PLIN5 genes showed an up-regulated trend.【Conclusion】 In this study,three candidate genes (NR4A1,DUSP1 and PLIN5) related to porcine intramuscular fat deposition were obtained,which could provide a reference for further exploring the regulatory mechanism of porcine intramuscular fat deposition.

Key words: Mashen pigs; Large White pigs; RNA-Seq; differentially expressed genes; intramuscular fat

中图分类号:

Q786

杨帅, 史明月, 李文霞, 员佳乐, 孙迪, 路畅, 杨阳, 蔡春波, 高鹏飞, 郭晓红, 李步高, 曹果清. 猪肌内脂肪沉积相关基因的筛选及其表达特性分析[J]. 中国畜牧兽医, 2022, 49(5): 1651-1661.

YANG Shuai, SHI Mingyue, LI Wenxia, YUN Jiale, SUN Di, LU Chang, YANG Yang, CAI Chunbo, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Screening and Expression Analysis of Genes Related to Porcine Intramuscular Fat Deposition[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1651-1661.

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