ZBED6基因敲除猪肾脏转录组分析

doi:10.16431/j.cnki.1671-7236.2022.05.007

摘要/Abstract

摘要： 【目的】探究锌指BED结构域结合蛋白6(zinc finger BED domain-containing protein 6，ZBED6)基因敲除对猪肾脏组织基因转录表达的影响，并解析ZBED6基因调控猪肾脏代谢的靶基因及其通路。【方法】对8月龄ZBED6基因敲除巴马香猪(ZBED6 KO)和同月龄野生型巴马香猪(WT)的肾脏组织(n=3)进行总RNA提取，利用实时荧光定量PCR检测胰岛素样生长因子2(insulin-like growth factor 2，IGF2)和差异基因的表达量;以Illumina Hiseq高通量测序技术对ZBED6 KO和WT猪肾脏组织mRNA进行转录组测序(RNA-Seq)，用猪Sus scrofa 11.1作为参考基因组序列，通过生物信息学软件TopHat、Cufflink、Cuffmerge和Cuffdiff对测序数据进行分析，筛选ZBED6 KO和WT猪肾脏组织中的差异表达基因，对差异表达基因进行层次聚类和KEGG通路富集分析，并通过实时荧光定量PCR验证RNA-Seq结果中差异表达基因的可靠性。【结果】实时荧光定量PCR结果显示，ZBED6 KO猪肾脏IGF2基因mRNA表达量显著高于WT猪(P<0.05)。测序共获得78 G数据量，每个样本比对率在87.3%以上，测序质量良好;ZBED6 KO和WT猪肾脏组织之间共检测到25 213个基因，以调整后P<0.05和log₂|FoldChange|>2为筛选标准，得到299个差异表达基因，其中上调的基因103个，下调的基因199个。热图和主成分分析(PCA)结果显示，ZBED6 KO和WT组猪肾脏基因组内表达模式相似，组间表达模式不同;KEGG通路富集分析显示，差异基因主要参与视黄醇及疾病代谢相关通路。ZBED6基因敲除后，其中有9个差异表达基因(CYP2C42、AOX1、ENSSSCG00000036274、RDH16、CYP2A19、ENSSSCG00000022724、CYP26B1、CYP1A1、RDH5)被富集到视黄醇代谢通路中，可能参与调控猪肾脏代谢平衡。实时荧光定量PCR检测发现，7个差异表达基因(CYP2C42、AOX1、RDH16、CYP2A19、CYP26B1、CYP1A1、RDH5)的表达模式与RNA-Seq分析结果一致，证实RNA-Seq结果的可靠性。【结论】本试验利用RNA-Seq技术分析了ZBED6基因敲除对巴马香猪肾脏代谢的影响，其通过调控肾脏多个下游基因表达，从而影响其代谢相关信号通路，试验结果为阐明ZBED6基因功能提供了材料。

关键词: 猪; ZBED6基因; 肾脏; 代谢; RNA-Seq; 差异表达基因

Abstract: 【Objective】 This study was aimed to investigate the effect of zinc finger BED domain-containing protein 6 (ZBED6) gene knockout on gene transcription and expression in porcine kidney,and analyze the target genes and pathways of ZBED6 gene regulating porcine kidney metabolism.【Method】 Kidney tissues were collected from 8-month-old ZBED6 gene knockout Bama Xiang pigs (ZBED6 KO) and age-matched wildtype Bama Xiang pigs (WT) (n=3).The total RNA was extracted and Real-time quantitative PCR was used to analyze the expression of insulin-like growth factor 2 (IGF2) and other differentially expressed genes.Illumina Hiseq high-throughput sequencing technology was performed to evaluate the mRNA of kidney in ZBED6 Ko and WT pigs.Sus scrofa 11.1 was used as the reference genome sequence,the differentially expressed genes were analyzed and filtered by the softwares TopHat,Cufflink,Cuffmerge and Cuffdiff.Hierarchical clustering analysis and KEGG pathway enrichment of the differentially expressed genes were also performed.The reliability of differentially expressed genes in RNA-Seq results was verified by Real-time quantitative PCR.【Result】 Real-time quantitative PCR results showed that the expression of IGF2 gene mRNA in kidney of ZBED6 KO pigs was significantly higher than that of WT pigs (P<0.05).The sequencing results showed that 78 G data was obtained,and at least 87.3% of the reads were mapped to porcine genome,suggesting the quality of sequencing was high.RNA-Seq analysis showed that a total of 25 213 genes were detected in kidney of WT and ZBED6 KO pigs.Under the criteria of adjust-P<0.05 and log₂|FoldChange|>2,299 differentially expressed genes were obtained,including 103 up-regulated genes and 199 down-regulated genes.Heatmap and PCA results showed that the gene expression pattern was similar within groups,and was different between groups.KEGG pathway enrichment analysis displayed that the differentially expressed genes were mainly involved in retinol and other metabolic pathways,especially,9 differentially expressed genes were enriched to the retinol metabolism pathway,including CYP2C42,AOX1,ENSSSCG00000036274,RDH16,CYP2A19,ENSSSCG00000022724,CYP26B1,CYP1A1 and RDH5,7 genes (CYP2C42,AOX1,RDH16,CYP2A19,CYP26B1,CYP1A1 and RDH5) of which were further validated by Real-time quantitative RCR,confirmed the reliability of RNA-Seq results.【Conclusion】 The effect of ZBED6 gene knockout on kidney metabolism of Bama Xiang pigs was analyzed by RNA-Seq technology in this study,it affected its metabolic related signal pathway by regulating the expression of multiple downstream genes in kidney,which provided materials for clarifying the function of ZBED6 gene.

Key words: pigs; ZBED6 gene; kidney; metabolism; RNA-Seq; differentially expressed genes

中图分类号:

田文杰, 王丹丹, 王圣楠, 马月辉, 蒋琳, 宋子仪. ZBED6基因敲除猪肾脏转录组分析[J]. 中国畜牧兽医, 2022, 49(5): 1662-1670.

TIAN Wenjie, WANG Dandan, WANG Shengnan, MA Yuehui, JIANG Lin, SONG Ziyi. Transcriptome Analysis of Porcine Kidney with ZBED6 Gene Deletion[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1662-1670.

参考文献

[1] MARKLJUNG E,JIANG L,JAFFE J D,et al.ZBED6,a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth[J].PLoS Biology,2009,7(12):e1000256.
[2] JEON J T,CARLBORG O,TORNSTEN A,et al.A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus[J].Nature Genetics,1999,21(2):157-158.
[3] WANG D,PAN D,XIE B,et al.Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets[J].PLoS Genetics,2021,17(10):e1009862.
[4] HUANG Y Z,ZHAN Z Y,SUN Y J,et al.Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle[J].Genome,2013,56(6):327-334.
[5] HUANG Y Z,HE H,ZHAN Z Y,et al.Relationship of polymorphisms within ZBED6 gene and growth traits in beef cattle[J].Gene,2013,526(2):107-111.
[6] 马燕,贺三刚,李文蓉.绵羊ZBED6基因多态性检测及群体遗传结构分析[J].石河子大学学报(自然科学版),2016,34(4):431-435. MA Y,HE S G,LI W R.Study on the polymorphisms and population genetic structure analysis of ZBED6 gene in two different sheep breeds[J].Journal of Shihezi University(Natural Science),2016,34(4):431-435.(in Chinese)
[7] YOUNIS S,SCHONKE M,MASSART J,et al.The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(9):E2048-E2057.
[8] HUANG Y Z,ZHANG L Z,LAI X S,et al.Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts[J].Scientific Reports,2014,4:4570.
[9] JIANG L,WALLERMAN O,YOUNIS S,et al.ZBED6 modulates the transcription of myogenic genes in mouse myoblast cells[J].PLoS One,2014,9(4):e94187.
[10] LIU X,QIU S,MEI L,et al.A high-resolution melting analysis with an unlabeled probe for CRISPR/Cas9-induced ZBED6 knockout pigs detection[J].Journal of Aoac International,2021,104(3):541-545.
[11] WANG X,JIANG L,WALLERMAN O,et al.Transcription factor ZBED6 affects gene expression,proliferation,and cell death in pancreatic beta cells[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(40):15997-16002.
[12] WANG X,YOUNIS S,CEN J,et al.ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation[J].Diabetologia,2021,64(10):2292-2305.
[13] WANG X,JIANG L,WALLERMAN O,et al.ZBED6 negatively regulates insulin production,neuronal differentiation,and cell aggregation in MIN6 cells[J].FASEB Journal,2019,33(1):88-100.
[14] AKHTAR A M,YOUNIS S,WALLERMAN O,et al.Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(25):7743-7748.
[15] OLSEN T,BLOMHOFF R.Retinol,retinoic acid,and retinol-binding protein 4 are differentially associated with cardiovascular disease,type 2 diabetes,and obesity:An overview of human studies[J].Advances in Nutrition,2020,11(3):644-666.
[16] MODY N.Alterations in vitamin A/retinoic acid homeostasis in diet-induced obesity and insulin resistance[J].Proceedings of the Nutrition Society,2017,76(4):597-602.
[17] RAILA J,BUCHHOLZ I,AUPPERLE H,et al.The distribution of vitamin A and retinol-binding protein in the blood plasma,urine,liver and kidneys of carnivores[J].Veterinary Research,2000,31(6):541-551.
[18] GERLACH T H,ZILE M H.Metabolism and secretion of retinol transport complex in acute renal failure[J].Journal of Lipid Research,1991,32(3):515-520.
[19] GAVRILOV V,HARMAN-BOEHM I,AMICHAY D,et al.Kidney function and retinol status in type 2 diabetes mellitus patients[J].Acta Diabetologica,2012,49(2):137-143.
[20] DUESTER G,FARRES J,FELDER M R,et al.Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family[J].Biochemical Pharmacology,1999,58(3):389-395.
[21] PERSSON B,KALLBERG Y,BRAY J E,et al.The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative[J].Chemico-Biological Interactions,2009,178(1-3):94-98.
[22] JEZ J M,FLYNN T G,PENNING T M.A new nomenclature for the aldo-keto reductase superfamily[J].Biochemical Pharmacology,1997,54(6):639-647.
[23] PEROZICH J,NICHOLAS H,WANG B C,et al.Relationships within the aldehyde dehydrogenase extended family[J].Protein Science,1999,8(1):137-146.
[24] XIAO S,DENG Y,SHEN N,et al.Curc-mPEG454,a PEGylated curcumin derivative,as a multi-target anti-fibrotic prodrug[J].International Immunopharmacology,2021,101(Pt A):108166.
[25] SNYDER J M,ZHONG G,HOGARTH C,et al.Knockout of Cyp26a1 and Cyp26b1 during postnatal life causes reduced lifespan,dermatitis,splenomegaly,and systemic inflammation in mice[J].FASEB Journal,2020,34(12):15788-15804.
[26] KRIVOSPITSKAYA O,ELMABSOUT A A,SUNDMAN E,et al.A CYP26B1 polymorphism enhances retinoic acid catabolism and may aggravate atherosclerosis[J].Molecular Medicine,2012,18:712-718.
[27] BELYAEVA O V,JOHNSON M P,KEDISHVILI N Y.Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase[J].Journal of Biological Chemistry,2008,283(29):20299-20308.
[28] KAO Y H,LEE P H,CHIU T C,et al.Transcriptome analysis reveals a positive role for nerve growth factor in retinol metabolism in primary rat hepatocytes[J].Cytokine,2018,107:74-78.
[29] NAPOLI J L.Interactions of retinoid binding proteins and enzymes in retinoid metabolism[J].Biochim Biophys Acta,1999,1440(2-3):139-162.
[30] DRIESSEN C,WINKENS H J,HOFFMANN K,et al.Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters[J].Molecular and Cellular Biology,2000,20(12):4275-4287.
[31] SHANG E,LAI K,PACKER A I,et al.Targeted disruption of the mouse cis-retinol dehydrogenase gene:Visual and nonvisual functions[J].Journal of Lipid Research,2002,43(4):590-597.
[32] YAMAMOTO H,SIMON A,ERIKSSON U,et al.Mutations in the gene encoding 11-cis retinol dehydrogenase cause delayed dark adaptation and fundus albipunctatus[J].Nature Genetics,1999,22(2):188-191.
[33] ZHANG Q Y,DUNBAR D,KAMINSKY L.Human cytochrome P-450 metabolism of retinals to retinoic acids[J].Drug Metabolism and Disposition,2000,28(3):292-297.
[34] CHOUDHARY D,JANSSON I,STOILOV I,et al.Metabolism of retinoids and arachidonic acid by human and mouse cytochrome P450 1b1[J].Drug Metabolism and Disposition,2004,32(8):840-847.
[35] LAMPEN A,MEYER S,ARNHOLD T,et al.Metabolism of vitamin A and its active metabolite all-trans-retinoic acid in small intestinal enterocytes[J].Journal of Pharmacology and Experimental Therapeutics,2000,295(3):979-985.
[36] ELOVAARA E,VAINIO H,PFAFFLI P,et al.Effects of intermittent styrene inhalation,ethanol intake and their combination on drug biotransformation in rat liver and kidneys[J].Medical Biology,1979,57(5):321-327.