沐川乌骨黑鸡肌内脂肪沉积相关microRNAs的筛查与鉴定

doi:10.16431/j.cnki.1671-7236.2021.08.004

摘要/Abstract

摘要： 试验旨在挖掘高肌内脂肪（IMF）含量和低IMF含量鸡胸肌中差异表达的microRNA（miRNA）（DEM），以期从miRNA角度探究鸡IMF沉积的分子调控机制。以120只沐川乌骨黑鸡为研究对象，采用高通量测序分别完成3个IMF含量极高（H组）和极低（L组）鸡胸肌组织的miRNA测序，测序结果经生物信息学分析，鉴定H和L组中差异表达的miRNAs，随机选择6个miRNAs进行实时荧光定量PCR验证。IMF含量测定结果表明，每只鸡平均100 g胸肌中IMF含量为4.08 g，H和L组IMF含量存在极显著差异（P<0.01）；6个测序文库结果表明，每个测序文库获得Clean reads均超过10 000 000条，Clean reads占Raw reads的百分比>93%，21~23 nt的small RNA（sRNA）数量占总sRNA的百分比>50%，其中22 nt长度的sRNA所占比例最高，平均GC碱基含量为48.95%，获得的测序数据可靠；6个测序文库中sRNA注释为rRNA、tRNA、snRNA、snoRNA以及重复序列的比例较低，分别为2.74%、1.85%、6.21%、1.58%、2.82%和2.81%；6个测序文库共鉴定已知miRNAs成熟体578个，miRNAs前体500个，鉴定差异表达的miRNAs 23个，其中H组中上调表达的miRNAs有16个，下调表达的miRNAs有7个；miRNA靶基因预测分析结果表明，23个差异表达miRNAs共预测靶基因628个；GO分析结果表明，共鉴定了30个显著富集GO条目，包括6个细胞成分、13个生物学过程和11个分子功能；KEGG富集分析表明，差异表达miRNA的靶基因显著富集于胰岛素信号通路、半乳糖代谢、脂肪细胞因子信号通路、鞘糖脂生物合成、糖酵解及淀粉和蔗糖代谢；实时荧光定量PCR结果表明，gga-miR-124a-3p、gga-let-7a-5p在H组中表达量极显著或显著高于L组（P<0.01；P<0.05）；gga-miR-19a-3p在H组中表达量极显著低于L组（P<0.01），gga-miR-6553-3p和gga-miR-128-3p在H组中的表达量显著低于L组（P<0.05），其表达趋势与测序结果一致。以上结果表明，差异表达的miRNA gga-miR-124a-3p、gga-let-7a-5p、gga-miR-6553-3p、gga-miR-128-3p和gga-miR-19a-3p参与脂肪生成，是鸡IMF含量调控重要的候选miRNAs，为阐释miRNA调控鸡IMF沉积分子机制提供理论基础。

关键词: 鸡; 肌内脂肪; miRNA; 高通量测序

Abstract: This study was aimed to identify differentially expressed microRNA(miRNA) (DEM)in the breast muscle between high intramuscular fat (IMF) and low IMF content chickens, and explore the miRNA regulatory mechanisms involved in IMF deposition in chicken.120 Muchuan Black-bone chickens were employed in this study.High-throughput RNA sequencing (RNA-Seq) was performed to identify miRNA in the breast muscle between three high IMF (H group) and low IMF (L group) content chickens.The sequencing data was analyzed to identify DEMs.Six miRNAs were randomly selected for quantitative analysis using Real-time quantitative PCR.The results of IMF contents determination showed that the average IMF level of 120 chickens was 4.08 g per 100 g muscle, and there was extremely significant difference in IMF content between H and L groups (P<0.01).Results of 6 RNA-Seq libraries showed that more than 10 000 000 Clean reads were obtained for each library, and the Clean reads ratio was greater than 93%, more than 50% of small RNA (sRNA) ranged from 21 to 23 nt in length, and the 22 nt sRNA was the most abundant.The average GC content was 48.95%.These results indicated that the sequencing data were reliably produced in our study.The proportion of sRNA annotated as rRNA, tRNA, snRNA, snoRNA and repetitive sequence in the 6 RNA-Seq libraries was 2.74%, 1.85%, 6.21%, 1.58%, 2.82% and 2.81%, respectively.A total of 578 known miRNAs and 500 pre-miRNAs were identified from the 6 chicken sequence libraries, with 23 DEMs were identified between two groups, of which 16 were upregulated and 7 were downregulated.The prediction analysis of miRNA target genes showed that 23 miRNAs which were differentially expressed between two chicken groups targeted a total of 628 genes.GO analysis results showed that a total of 30 significantly enriched GO terms were identified, including 6 cellular components, 13 biological process and 11 molecular function.KEGG pathway analysis showed that the target genes were mainly involved with insulin signaling pathway, galactose metabolism, adipocytokine signaling pathway, glycosphingolipid biosynthesis, glycolysis and gtarch and sucrose metabolism.Real-time quantitative PCR results indicated that the expression of gga-miR-124a-3p and gga-let-7a-5p in H group was significantly higher than that in L group (P<0.01;P<0.05), the expression of gga-miR-19a-3p in H group was significantly lower than that in L group (P<0.01), the expression of gga-miR-6553-3p and gga-miR-128-3p in H group were significantly lower than that in L group (P<0.05), and 5 DEMs expression pattern were consistent with sequencing data.The results showed that gga-miR-124a-3p, gga-let-7a-5p, gga-miR-6553-3p, gga-miR-128-3p and gga-miR-19a-3p were associated with adipogenesis in animal, which might be important for IMF deposition in chicken and would provide a foundation for clarifying the molecular regulatory mechanisms of miRNA involved in IMF deposition in chicken.

Key words: chicken; intramuscular fat; miRNA; high throughput sequencing

中图分类号:

S831.99

喻世刚, 王钢, 廖娟, 谢伟. 沐川乌骨黑鸡肌内脂肪沉积相关microRNAs的筛查与鉴定[J]. 中国畜牧兽医, 2021, 48(8): 2713-2726.

YU Shigang, WANG Gang, LIAO Juan, XIE Wei. Screening and Identification of the microRNAs Associated with Intramuscular Fat Deposition in Muchuan Black-bone Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2713-2726.

参考文献

[1] 巨晓军,束婧婷,章明,等.不同品种、饲养周期肉鸡肉品质和风味的比较分析[J].动物营养学报,2018,30(6):2421-2430. JU X J,SHU J T,ZHANG M,et al.Comparison analysis of meat quality and flavor of different breeds and feeding periods of broilers[J].Chinese Journal of Animal Nutrition,2018,30(6):2421-2430.(in Chinese)
[2] LONERGAN S M,DEEB N,FEDLER C A,et al.Breast meat quality and composition in unique chicken populations[J].Poultry Science,2003,82(12):1990-1994.
[3] 吕亚宁,贺琛昕,兰旅涛.猪肌内脂肪与肉品质的关系及其影响因素的研究进展[J].中国畜牧兽医,2020,47(2):554-563. LYU Y N,HE C X,LAN L T.Research advances on the relationship between intramuscular fat and meat quality and influence factor of intramuscular fat in pigs[J].China Animal Husbandry & Veterinary Medicine,2020,47(2):554-563.(in Chinese)
[4] FORTIN A,ROBERTSON W M,TONG A K W.The eating quality of Canadian pork and its relationship with intramuscular fat[J].Meat Science,2005,69(2):297-305.
[5] ZHANG M,LI D H,LI F,et al.Integrated analysis of miRNA and genes associated with meat quality reveals that gga-miR-140-5p affects intramuscular fat deposition in chickens[J].Cellular Physiology and Biochemistry,2018,46(6):2421-2433.
[6] 李俊英,李慧锋,姜润深,等.优质鸡肌内脂肪含量与屠体及肉质性状间的关系[J].中国畜牧杂志,2004,12:16-19. LI J Y,LI H F,JIANG R S,et al.Correlation analysis between intramuscular fat and body composition and meat traits in quality chickens[J].Chinese Journal of Animal Science,2004,12:16-19.(in Chinese)
[7] 舒婷,沙尔山别克·阿不地力大,姜维,等.拜城油鸡肌内脂肪沉积规律及其与肉品质的关联性分析[J].中国家禽,2018,40(6):5-9. SHU T,SHAERSHANBIEKE A,JIANG W,et al.Deposition rule of intramuscular fat of Baicheng You chicken and its correlation with meat quality[J].China Poultry,2018,40(6):5-9.(in Chinese)
[8] CHARTRIN P,MÉTEAU K,JUIN H,et al.Effects of intramuscular fat levels on sensory characteristics of duck breast meat[J].Poultry Science,2006,85(5):914-922.
[9] LEVEILLE G A.In vitro hepatic lipogenesis in the hen and chick[J].Comparative Biochemistry and Physiology,1969,28(1):431-435.
[10] QIU F F,XIE L,MA J E,et al.Lower expression of SLC27A1 enhances intramuscular fat deposition in chicken via down-regulated fatty acid oxidation mediated by CPT1A[J].Frontiers in Physiology,2017,8:449.
[11] RANRAN L,YANFA S,GUIPING Z,et al.Genome-wide association study identifies loci and candidate genes for body composition and meat quality traits in Beijing-You chickens[J].PLoS One,2013,8(4):e61172.
[12] NASSAR M K,GORAGA Z S,BROCKMANN G A.Quantitative trait loci segregating in crosses between New Hampshire and White Leghorn chicken lines:Ⅲ.Fat deposition and intramuscular fat content[J].Animal Genetics,2013,44(1):62-68.
[13] YE M H,CHEN J L,ZHAO G P,et al.Associations of A-FABP and H-FABP markers with the content of intramuscular fat in Beijing-You chicken[J].Animal Biotechnology,2010,21(1):14-24.
[14] CUI H X,ZHENG M Q,LIU R R,et al.Liver dominant expression of fatty acid synthase (FAS) gene in two chicken breeds during intramuscular-fat development[J].Molecular Biology Reports,2012,39(4):3479-3484.
[15] ZHANG R,LIN Y,ZHI L,et al.Expression profiles and associations of adiponectin and adiponectin receptors with intramuscular fat in Tibetan chicken[J].British Poultry Science,2017,58(2):151-157.
[16] LI Q,ZUO L L,LIN Y Q,et al.Cloning and expression of SFRP5 in Tibetan chicken and its relationship with IMF deposition[J].Animal Biotechnology,2016,27(4):231-237.
[17] AMBROS V.The functions of animal microRNAs[J].Nature,2004,431(7006):350-355.
[18] LI S,NGUYEN T D,NGUYEN T L,et al.Mismatched and wobble base pairs govern primary microRNA processing by human microprocessor[J].Nature Communications,2020,11(1):1926.
[19] SIENGDEE P,TRAKOOLJUL N,MURANI E,et al.Transcriptional profiling and miRNA-dependent regulatory network analysis of longissimus dorsi muscle during prenatal and adult stages in two distinct pig breeds[J].Animal Genetics,2013,44(4):398-407.
[20] HUANG H Y,LIU R R,ZHAO G P,et al.Integrated analysis of microRNA and mRNA expression profiles in abdominal adipose tissues in chickens[J].Scientific Reports,2015,5:16132.
[21] SUN Y,WANG S,LIU H,et al.Profiling and characterization of miRNAs associated with intramuscular fat content in Yorkshire pigs[J].Animal Biotechnology,2020,31(3):256-263.
[22] 付守艺,陈亚迪,王杰,等.鸡miR-124a-3p的组织表达谱及其与ACAA2基因的互作关系[J].农业生物技术学报,2018,26(6):949-958. FU S Y,CHEN Y D,WANG J,et al.Tissue expression profile of miR-124a-3p and its interaction relationship with ACAA2 gene in chicken (Gallus gallus)[J].Journal of Agricultural Biotechnology,2018,26(6):949-958.(in Chinese)
[23] ROMANO G,NIGITA G,CALORE F,et al.miR-124a regulates extracellular vesicle release by targeting GTPase rabs in lung cancer[J].Frontiers in Oncology,2020,10:1454.
[24] DAS S K,STADELMEYER E,SCHAUER S,et al.microRNA-124a regulates lipolysis via adipose triglyceride lipase and comparative gene identification 58[J].International Journal of Molecular Sciences,2015,16(4):8555-8568.
[25] 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)
[26] OHKURA T,YOSHIMURA T,FUJISAWA M,et al.Spred2 regulates high fat diet-induced adipose tissue inflammation,and metabolic abnormalities in mice[J].Frontiers in Immunology,2019,10:17.
[27] BONEY C M,GRUPPUSO P A,FARIS R A,et al.The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes[J].Molecular Endocrinology,2000,14(6):805-813.
[28] SUBRAMANIAM S,OZDENER M H,ABDOULAZIZE S,et al.ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans[J].FASEB Journal,2016,30(10):3489-3500.
[29] YAN J,GAN L,CHEN D,et al.Adiponectin impairs chicken preadipocytes differentiation through p38 MAPK/ATF-2 and TOR/p70 S6 kinase pathways[J].PLoS One,2013,8(10):e77716.
[30] 仇杨,刘帅,宁小敏,等.过表达miR-128a抑制大鼠前体脂肪细胞分化[J].农业生物技术学报,2013,21(4):388-395. QIU Y,LIU S,NING X M,et al.Inhibition of the pre-adipocyte differentiation in rat(Rattus norvegicus) caused by over-expression of miR-128a[J].Journal of Agricultural Biotechnology,2013,21(4):388-395.(in Chinese)
[31] CHEN C,DENG Y,HU X,et al.miR-128-3p regulates 3T3-L1 adipogenesis and lipolysis by targeting PPARG and Sertad2[J].Journal of Physiology and Biochemistry,2018,74(3):381-393.
[32] CHEN L,ZHANG T,ZHANG S,et al.Identification of long non-coding RNA-associated competing endogenous RNA network in the differentiation of chicken preadipocytes[J].Genes,2019,10(10):795.
[33] HARDING R L,VELLEMAN S G.microRNA regulation of myogenic satellite cell proliferation and differentiation[J].Molecular and Cellular Biochemistry,2016,412(1-2):181-195.
[34] SHEN X,LIU Z,CAO X,et al.Circular RNA profiling identified an abundant circular RNA circTMTC1 that inhibits chicken skeletal muscle satellite cell differentiation by sponging miR-128-3p[J].International Journal of Biological Sciences,2019,15(10):2265-2281.
[35] ZHANG M,LI F,SUN J W,et al.lncRNA IMFNCR promotes intramuscular adipocyte differentiation by sponging miR-128-3p and miR-27b-3p[J].Frontiers in Genetics,2019,10:42.
[36] FENG S,ZHU X,FAN B,et al.miR-19a-3p targets PMEPA1 and induces prostate cancer cell proliferation,migration and invasion[J].Molecular Medicine Reports,2016,13(5):4030-4038.
[37] LEE S,LEE H,BAE H,et al.Epigenetic silencing of miR-19a-3p by cold atmospheric plasma contributes to proliferation inhibition of the MCF-7 breast cancer cell[J].Scientific Reports,2016,6:30005.
[38] KANGAS R,MORSIANI C,PIZZA G,et al.Menopause and adipose tissue:miR-19a-3p is sensitive to hormonal replacement[J].Oncotarget,2018,9(2):2279-2294.
[39] XIANG H,ZHONG Z X,PENG Y D,et al.The emerging role of Zfp217 in adipogenesis[J].International Journal of Molecular Sciences,2017,18(7):1367.
[40] WANG W,DU Z Q,CHENG B,et al.Expression profiling of preadipocyte microRNAs by deep sequencing on chicken lines divergently selected for abdominal fatness[J].PLoS One,2015,10(2):e0117843.
[41] WONG T S,MAN O Y,TSANG C M,et al.microRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression[J].Journal of Cancer Research and Clinical Oncology,2011,137(3):415-422.
[42] GIROUD M,KARBIENER M,PISANI D F,et al.Let-7i-5p represses brite adipocyte function in mice and humans[J].Scientific Reports,2016,6:28613.
[43] SUN T,FU M,BOOKOUT A L,et al.microRNA let-7 regulates 3T3-L1 adipogenesis[J].Molecular Endocrinology,2009,23(6):925-931.
[44] LI G,LI Y,LI X,et al.microRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing[J].Journal of Cellular Biochemistry,2011,112(5):1318-1328.
[45] ZHANG F,LI D,WU Q,et al.Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows[J].Journal of Animal Science and Biotechnology,2019,10:38.
[46] WONG R H,SUL H S.Insulin signaling in fatty acid and fat synthesis:A transcriptional perspective[J].Current Opinion in Pharmacology,2010,10(6):684-691.
[47] QIU F F,NIE Q H,LUO C L,et al.Association of single nucleotide polymorphisms of the insulin gene with chicken early growth and fat deposition[J].Poultry Science,2006,85(6):980-985.
[48] DUPONT J,MÉTAYER-COUSTARD S,JI B,et al.Characterization of major elements of insulin signaling cascade in chicken adipose tissue:Apparent insulin refractoriness[J].General and Comparative Endocrinology,2012,176(1):86-93.
[49] DUPONT J,CHEN J,DEROUET M,et al.Metabolic differences between genetically lean and fat chickens are partly attributed to the alteration of insulin signaling in liver[J].The Journal of Nutrition,1999,129(11):1937-1944.
[50] FUKUHARA A,MATSUDA M,NISHIZAWA M,et al.Visfatin:A protein secreted by visceral fat that mimics the effects of insulin[J].Science,2005,307(5708):426-430.
[51] YE Y,LIN S,MU H,et al.Analysis of differentially expressed genes and signaling pathways related to intramuscular fat deposition in skeletal muscle of sex-linked dwarf chickens[J].BioMed Research International,2014,2014:724274.