宁乡猪不同发育阶段肌内脂肪沉积差异表达基因及其调控通路分析

doi:10.16431/j.cnki.1671-7236.2024.09.001

摘要/Abstract

摘要： 【目的】分析不同日龄宁乡猪背最长肌组织mRNA表达谱，筛选发育过程中与脂肪沉积相关的差异表达基因及其所参与的信号通路，并确定部分关键基因在肌内脂肪发育过程中的表达特征。【方法】选取30(D30)和250(D250)日龄宁乡猪各3头，采集背最长肌组织样品提取总RNA进行转录组测序，对测序数据进行比对和处理，筛选参与脂质代谢的差异表达基因进行GO功能、KEGG通路及蛋白互作分析，并随机选取6个差异表达基因进行实时荧光定量PCR验证。【结果】共筛选出30个与脂质代谢相关的差异表达基因，其中9个上调，21个下调。差异表达基因主要富集在脂肪酸氧化、长链脂肪酸代谢等生物过程，线粒体、细胞器膜等细胞组分，酰甘油O-酰基转移酶活性、氧化还原酶活性等分子功能；参与了脂肪酸降解、甘油酯代谢、PPAR等信号通路。蛋白互作分析筛选出ACSL1、ACAA2、HADHB、LIPG、GPCPD1、ALDH2等关键蛋白基因处于核心位置，通过其mRNA表达量不同参与脂肪沉积调控。ACSL1、CPT1A等6个基因的实时荧光定量PCR验证结果与转录组测序结果一致。【结论】 ACSL1、ACAA2、HADHB、LIPG、GPCPD1、ALDH2等作为核心基因参与宁乡猪肌内脂肪调控，并通过脂肪酸降解、甘油酯代谢等信号通路发挥调控作用。本研究结果为进一步研究猪肌内脂肪沉积相关基因的分子调控机制提供了理论参考。

关键词: 宁乡猪; 肌内脂肪; 脂质代谢; 转录组测序

Abstract: 【Objective】 This study was conducted to screen out differentially expressed genes (DEGs) and their regulatory signaling pathways related to lipid metabolism by analyzing mRNA expression profiles in longissimus dorsi muscle tissues of Ningxiang pigs at different ages，and determine the expression characteristics of some key genes during the development of intramuscular fat. 【Method】 Ningxiang pigs of 30 (D30, n=3) and 250 (D250, n=3) days old were selected, the longissimus dorsi muscle tissues were collected to extract total RNA for RNA-Seq, and sequencing data was compared and processed.The DEGs related to lipid metabolism were screened for GO function, KEGG pathway and protein interactions analysis.Six DEGs were randomly selected for Real-time quantitative PCR validation. 【Result】 A total of 30 DEGs related to lipid metabolism were screened by RNA-Seq, among which 9 genes were up-regulated and 21 genes were down-regulated.The DEGs were mainly enriched in biological process such as fatty acid oxidation and long-chain fatty acid metabolic process;Cellular components such as mitochondrion and organelle membrane;Molecular functions such as acylglycerol O-acyltransferase activity and oxidoreductase activity.The DEGs were involved signaling pathway such as fatty acid degradation, glycerolipid metabolism and PPAR.Key protein genes such as ACSL1, ACAA2, HADHB, LIPG, GPCPD1 and ALDH2 were screened out by protein interaction analysis, which were involved in the regulation of lipid metabolism through their mRNA expression.Real-time quantitative PCR validation results of six genes such as ACSL1 and CPT1A were consistent with RNA-Seq results. 【Conclusion】 This study screened some key genes such as ACSL1, ACAA2, HADHB, LIPG, GPCPD1 and ALDH2 participating in the regulation of intramuscular fat in Ningxiang pigs, and played a regulatory role through signaling pathways such as fatty acid degradation and glycerolipid metabolism.The resuts provided a theoretical reference for further research on the molecular regulation of genes related to intramuscular fat deposition in pigs.

Key words: Ningxiang pigs; intramuscular fat; lipid metabolism; RNA-Seq

中图分类号:

S813.3

夏铭隆, 肖银涛, 郑塞珍, 谭碧娥, 印遇龙, 陈家顺, 尹杰. 宁乡猪不同发育阶段肌内脂肪沉积差异表达基因及其调控通路分析[J]. 中国畜牧兽医, 2024, 51(9): 3703-3714.

XIA Minglong, XIAO Yintao, ZHENG Saizhen, TAN Bie, YIN Yulong, CHEN Jiashun, YIN Jie. Analysis of Differentially Expressed Genes and Regulatory Pathways in Intramuscular Fat Deposition of Ningxiang Pigs at Different Developmental Stages[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3703-3714.

参考文献

[1] WANG Z,LI Y,WU L,et al.Rosiglitazone-induced PPARγ activation promotes intramuscular adipocyte adipogenesis of pig[J].Animal Biotechnology,2023,34(8):3708-3717.
[2] SUN Y,CHEN X,QIN J,et al.Comparative analysis of long noncoding RNAs expressed during intramuscular adipocytes adipogenesis in fat-type and lean-type pigs[J].Journal of Agricultural and Food Chemistry,2018,66(45):12122-12130.
[3] ZHANG Z,LIAO Q,SUN Y,et al.Lipidomic and transcriptomic analysis of the longissimus muscle of Luchuan and Duroc pigs[J].Frontiers in Nutrition,2021,8:667622.
[4] 田紫金，李碧侠，吴萍.猪只肌内脂肪沉积调控机制研究进展[J].猪业科学，2023，40(11):114-117. TIAN Z J，LI B X，WU P.Research progress on the regulatory mechanism of intramuscular fat deposition in pigs[J].Swine Industry Science，2023，40(11)：114-117.(in Chinese)
[5] 杨帅，史明月，李文霞，等.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 S，SHI M Y，LI W X，et al.Screening and expression analysis of genes related to porcine intramuscular fat deposition[J].China Animal Husbandry & Veterinary Medicine，2022，49(5)：1651-1661.(in Chinese)
[6] 聂靖茹，马黎，严达伟，等.大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析[J].中国畜牧兽医，2022，49(8)：2855-2868. NIE J R，MA L，YAN D W，et al.Analysis of differentially expressed genes and regulation pathways of intramuscular fat deposition in Large Diqing Tibetan pigs at different growth stages[J].China Animal Husbandry ＆ Veterinary Medicine，2022，49(8)：2855-2868.(in Chinese)
[7] 李鹏飞，杜海燕.猪肉脂肪含量和质量影响因素的研究进展[J].畜禽业，2006，23：14-16. LI P F，DU H Y.The research advance of factors affecting pork fat content and quality[J].Livestock and Poultry Industry，2006，23：14-16.(in Chinese)
[8] 任慧波，杜丽飞，朱吉，等.宁乡猪种质特性调查与研究[J].养猪，2011，6：57-61. REN H B，DU L F，ZHU J，et al.Investigation and study on germplasm characteristics of Ningxiang pigs[J].Swine Production，2011，6：57-61.(in Chinese)
[9] 王芳，张跃博，蒋谦，等.宁乡猪皮下脂肪与肌内脂肪组织转录组差异分析[J].遗传，2023，45(12)：1147-1157. WANG F，ZHANG Y B，JIANG Q，et al.Analysis of transcriptome differences between subcutaneous anointramuscular adipose tissue of Ningxiang pigs[J].Hereditas (Beijing)，2023,45(12)：1147-1157.(in Chinese)
[10] MALGWI I H,HALAS V,GRUNVALD P,et al.Genes related to fat metabolism in pigs and intramuscular fat content of pork:A focus on nutrigenetics and nutrigenomics[J].Animals,2022,12(2):150.
[11] 刘乔.基于转录组分析不同生长阶段民猪的特异性表达基因与时序特征[D].哈尔滨：东北农业大学，2023. LIU Q.Analysis of specific expressed genes and temporal characteristics of Min pigs at different growth stages based on transcriptomics[D].Harbin：Northeast Agricultural University，2023.(in Chinese)
[12] 李小金.基于RNA-Seq技术对皖南花猪和大约克猪背最长肌差异基因的筛选[D].合肥：安徽农业大学，2015. LI X J.Screening of differentially expressed genes in the longissimus dorsi muscles of the Wannanhua pig and the Yorkshire pig based on RNA-Seq technology[D].Hefei：Anhui Agricultural University，2015.(in Chinese)
[13] TANG Y，ZHANG W，WANG Y，et al.Expression variation of CPT1A induces lipid reconstruction in goat intramuscular precursor adipocytes[J].International Journal of Molecular Sciences，2023，24(17)：13415.
[14] LI Z Q，LI J J，LIN Z Z，et al.Knockdown of CPT1A induce chicken adipocyte differentiation to form lipid droplets[J].Brazilian Journal of Poultry Science，2022，24(4)：1-8.
[15] PELLIEUX C，AASUM E，LARSEN T S，et al.Overexpression of angiotensinogen in the myocardium induces downregulation of the fatty acid oxidation pathway[J].Journal of Molecular and Cellular Cardiology，2006，41(3)：459-466.
[16] LI A，LI Y Y，WUQIE Q B，et al.Effect of ACADL on the differentiation of goat subcutaneous adipocyte[J].Animal Bioscience，2023，36(6)：829.
[17] 杨朝云.黄牛HADHB基因转录调控及其对脂肪细胞增殖和分化的影响[D].银川：宁夏大学，2022. YANG Z Y.Study on the transcriptional regulation of HADHB gene and its effect on the proliferation and differentiation of adipocytes in cattle[D].Yinchuan：Ningxia University，2022.(in Chinese)
[18] COLEMAN R A，LEWIN T M，MUOIO D M.Physiological and nutritional regulation of enzymes of triacylglycerol synthesis[J].Annual Review of Nutrition，2000，20(1)：77-103.
[19] CUI M，WANG Y，SUN B，et al.miR-205 modulates abnormal lipid metabolism of hepatoma cells via targeting acyl-CoA synthetase long-chain family member 1(ACSL1) mRNA[J].Biochemical & Biophysical Research Communications，2014，444(2)：270-275.
[20] 田宏山.ACSL1基因对牛脂肪细胞中不饱和脂肪酸合成的影响[D].兰州：甘肃农业大学，2020. TIAN H S.Effect of ACSL1 gene on unsaturated fatty acid synthesis in bovine adipocytes[D].Lanzhou：Gansu Agricultural University，2020.(in Chinese)
[21] MUKHERJEE R，YUN J W.Long chain acyl CoA synthetase 1 and gelsolin are oppositely regulated in adipogenesis and lipogenesis[J].Biochemical and Biophysical Research Communications，2012，420(3)：588-593.
[22] LI L O，MASHEK D G，AN J，et al.Overexpression of rat long chain Acyl-CoA synthetase 1 alters fatty acid metabolism in rat primary hepatocytes[J].Journal of Biological Chemistry，2006，281(48)：37246-37255.
[23] LI T，LI X，MENG H，et al.ACSL1 affects triglyceride levels through the PPARγ pathway[J].International Journal of Medical Sciences，2020，17(6)：720-727.
[24] 单保森.ACSL1和CAT基因对二花脸猪皮下前体脂肪细胞成脂分化的影响机制研究[D].南京：南京农业大学，2019. SHAN B S.Mechanism of ACSL1 and CAT genes on adipogenic differentiation of sub-predecessal fat cells in Erhualian pigs[D].Nanjing：Nanjing Agricultural University，2019.(in Chinese)
[25] MILTIADOU D，HAGER-THEODORIDES A L，SYMEOU S，et al.Variants in the 3'-untranslated region of the ovine acetyl-coenzyme A acyltransferase 2 gene are associated with dairy traits and exhibit differential allelic expression[J].Journal of Dairy Science，2017，100(8)：6285-6297.
[26] 李文娟.鸡肉品质相关脂肪代谢功能基因的筛选及营养调控研究[D].北京：中国农业科学院，2008. LI W J.Filtration of functional genes related to lipid metabolism of meat quality and nutritional regulation in chickens[D].Beijing：Chinese Academy of Agricultural Sciences，2008.(in Chinese)
[27] 颜忠康.乙酰辅酶A酰基转移酶2介导脂质代谢作用机理的初步探究[D].合肥：安徽大学，2021. YAN Z K.A preliminary study on the mechanism of lipid metabolism mediated by acetyl-coenzyme A acyltransferase 2[D].Hefei：Anhui University，2021.(in Chinese)
[28] 纪艳芹.ACAA2基因对绵羊前体脂肪细胞分化的影响及其相关育种材料创制[D].扬州：扬州大学，2017. JI Y Q.The effect of ACAA2 gene on sheep preadipocyte differentiation and the creation of related breeding materials[D].Yangzhou：Yangzhou University，2017.(in Chinese)
[29] HONG C，DENG R，WANG P，et al.LIPG:An inflammation and cancer modulator[J].Cancer Gene Therapy，2021，28(1-2)：27-32.
[30] JUSTINE E Y，HAN S Y，WOLFSON B，et al.The role of endothelial lipase in lipid metabolism，inflammation，and cancer[J].Histology and Histopathology，2018，33(1):1-10.
[31] 李武峰，孙瑜彤，关家伟，等.驴肌内脂肪沉积关键调控因子研究[J].畜牧兽医学报，2021，52(2)：364-375. LI W F，SUN Y T，GUAN J W，et al.Key regulatory factors of intramuscular fat deposition in donkey[J].Acta Veterinaria et Zootechnica Sinica，2021，52(2)：364-375.(in Chinese)
[32] YU Y H，LIAO P R，GUO C J，et al.PKC-ALDH2 pathway plays a novel role in adipocyte differentiation[J].Public Library of Science，2016，11(8)：e0161993.