用ZBED6基因敲除猪研究心脏发育的分子机制

doi:10.3864/j.issn.0578-1752.2018.07.016

摘要/Abstract

摘要： 【目的】ZBED6基因是一个调控肌肉生长发育的转录因子，为了探讨 ZBED6 在心肌生长发育中的调控机制，利用RNA Sequencing（RNA-Seq）技术比较ZBED6基因敲除巴马小型猪（ZBED6-KO)和同日龄正常巴马小型猪（ZBED6-WT)的心脏组织转录组，挖掘ZBED6基因的敲除对家猪心脏组织发育及基因表达的影响。【方法】利用t-test对ZBED6-KO猪和ZBED6-WT猪心脏组织大小的表型特征进行显著性分析，利用实时荧光定量PCR对ZBED6基因的靶基因IGF2的表达量进行定量。通过制作石蜡组织切片对心肌进行组织学分析，比较其组织结构的差异。提取ZBED6-KO猪和ZBED6-WT猪心脏组织的总RNA，以Illumina Hiseq 2000平台进行RNA-Seq分析。以猪Sus_scrofa10.2为参考序列，用转录组的标准流程筛选ZBED6-KO猪和ZBED6-WT猪心脏组织中的差异表达基因，对差异基因进行GO和IPA富集分析。随机选择9个差异表达基因，利用实时荧光定量PCR验证RNA-Seq结果的可靠性。【结果】ZBED6-KO猪心脏重以及IGF2表达量均显著高于ZBED6-WT猪（P＜0.05），与ZBED6-WT猪相比，ZBED6-KO猪肌纤维的宽度较宽，结缔组织较少，ZBED6基因的敲除对家猪心脏的生长发育有一定的促进作用；测序结果显示，各样本获得至少10 G的数据量，每个样本clean ratio、Q30 data均达到90%以上，其中62.8%-80.1%的reads能比对到猪的基因组上，表明测序饱和度良好，测序数据真实可靠；对测序数据进行分析，筛选到 184个差异基因，其中上调的基因114个，下调的基因70个，注释的141个，未注释的43个；差异基因层次聚类分析显示，ZBED6-KO组（x1、x3、x6）的3个个体表达模式相似，ZBED6-WT组（x2、x4、x5）的3个个体表达模式相似；GO和IPA富集分析得到13个显著的GO条目，33条显著的pathway通路，差异基因主要富集在免疫反应、肌肉发育和RhoA信号等相关的通路；qRT-PCR 检测9个差异表达基因的表达模式与RNA-Seq分析结果一致，证实了RNA-Seq结果的可靠性。【结论】首次以ZBED6-KO巴马小型猪为模型，利用RNA-Seq技术探究了ZBED6敲除对心脏发育和功能的影响，丰富了ZBED6 对心脏发育和功能的研究。

关键词: ZBED6, 基因敲除, RNA-Seq, 心肌发育, 差异表达基因

Abstract: 【Objective】The ZBED6 gene is a transcription factor that regulates muscle growth and development. In order to investigate the regulatory mechanism of ZBED6 in myocardium growth and development, this study was designed to compare the transcriptome of cardiac tissue obtained from Bama miniature pig between the ZBED6 knockout group (ZBED6-KO) and the wild type group (ZBED6-WT) and to find out the effect of ZBED6 gene knockout on the development and gene expression profile of pig heart tissue. 【Method】 The phenotypic characteristics of ZBED6-KO and ZBED6-WT pig tissues were analyzed by t-test and the expression level of the target gene IGF2 of the ZBED6 gene was quantified by real-time quantitative PCR (q-PCR). Comparing the differences in histological level of tissue structure were performed by paraffin section method. The total RNA was isolated from ZBED6-KO group and ZBED6-WT group. RNA-Seq analysis was performed on Illumina Hiseq 2000 platform. The differentially expressed genes between ZBED6-KO and ZBED6-WT heart tissues were screened by the method of RNA-Seq Pig Sus_scrofa10.2 was used as the reference sequence. Differentially expressed genes were enriched and analyzed with IPA software. 9 differentially expressed genes were randomly selected to verify the reproducibility of RNA-Seq results by q-PCR. 【Result】The heart of ZBED6-KO pig had a larger measured data in weight than ZBED6-WT, which also had a higher expression profile of IGF2 than the latter. Compared with ZBED6-WT pig, ZBED6-KO pig had wide muscle fiber width and less connective tissue. The results suggested that the knockout of ZBED6 gene could promote the growth and development of porcine heart. The sequencing results showed that at least 10 G data were obtained and the clean ratio, Q30 data were more than 90%, of which 62.8% -80.1% of the reads mapped to the pig genome. All of these indicated sequencing was well saturation and the sequencing data was reliable. By analyzing sequencing data, 184 different genes were indicated which contained 114 up-regulated genes and 70 down-regulated genes. Among the 184 genes, there were 141 intact function annotation genes and 43 non-annotated genes. Differential gene hierarchical clustering analysis showed that, the similar expression pattern was detected in ZBED6-KO group (x1, x3, x6), which was also happen to ZBED6-WT group (x2, x4, x5). GO and IPA enrichment analysis obtained 13 significant GO terms, 33 pathways. The difference gene was mainly enriched in the immune response, muscle development, RhoA signal and other related channels. The expression pattern of 9 differentially expressed genes was consistent with the results of RNA-Seq analysis. 【Conclusion】In this study, ZBED6-KO Bama miniature pig was first time to use as the model. We used RNA-Seq technique to explore the effects of ZBED6 gene knockout on cardiac development, which enriched the research of ZBED6 on myocardium development and function.

Key words: ZBED6, gene knockout, RNA-Seq, myocardial development, differentially expressed genes

王丹丹，唐雨婷，马月辉，王立刚，潘登科，蒋琳. 用ZBED6基因敲除猪研究心脏发育的分子机制[J]. 中国农业科学, 2018, 51(7): 1390-1400.

WANG DanDan, TANG YuTing, MA YueHui, WANG LiGang, PAN DengKe, JIANG Lin. Studying the Molecular Mechanism of Heart Development by Using ZBED6 Gene Knockout Pig[J]. Scientia Agricultura Sinica, 2018, 51(7): 1390-1400.

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参考文献

[1] XIA J, YUAN J, XIN L, ZHANG Y, KONG S, CHEN Y, YANG S, LI K. Transcriptome analysis on the inflammatory cell infiltration of nonalcoholic steatohepatitis in bama minipigs induced by a long-term high-fat, high-sucrose diet. PLoS One, 2014, 9: e113724.

[2] MARKLJUNG E, JIANG L, JAFFE JD, MIKKELSEN TS, WALLERMAN O, LARHAMMAR M, ZHANG X, WANG L, SAENZ-VASH V, GNIRKE A, LINDROTH AM, BARRES R, YAN J, STROMBERG S, DE S, PONTEN F, LANDER E S, CARR S A, ZIERATH JR, KULLANDER K, WADELIUS C, LINDBLAD-TOH K, ANDERSSON G, HJALM G, ANDERSSON L. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth. PLoS Biology, 2009, 7: e1000256.

[3] VAN LAERE A S, COPPIETERS W, GEORGES M. Characterization of the bovine pseudoautosomal boundary: Documenting the evolutionary history of mammalian sex chromosomes. Genome Researxh, 2008, 18:1884.

[4] WANG X, JIANG L, WALLERMAN O, ENGSTROM U, AMEUR A, GUPTA RK, QI Y, ANDERSSON L, WELSH N. Transcription factor ZBED6 affects gene expression, proliferation, and cell death in pancreatic beta cells. Proceedings of The National Academy of Sciences of The United States of America, 2013, 110: 15997.

[5] JEON J T, CARLBORG O, TORNSTEN A, GIUFFRA E, AMARGER V, CHARDON P, ANDERSSON-EKLUND L, ANDERSSON K, HANSSON I, LUNDSTROM K, ANDERSSON L. A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus. Nature Genetics, 1999, 21:157.

[6] BUTTER F, KAPPEI D, BUCHHOLZ F, VERMEULEN M, MANN M. A domesticated transposon mediates the effects of a single- nucleotide polymorphism responsible for enhanced muscle growth. EMBO Reports, 2010, 11:305.

[7] HUANG Y Z, SUN Y J, LI M X, WANG J, SUN J J, ZHANG C L, JIA Y T, CHEN H. Evaluation of the causality of the zinc finger BED-type containing 6 gene (ZBED6) for six important growth traits in Nanyang beef cattle. Animal Genetics, 2015, 46:225.

[8] HUANG Y Z, SUN Y J, ZHAN Z Y, LI M X, WANG J, XUE J, LAN X Y, LEI C Z, ZHANG C L, CHEN H. Expression, SNP identification, linkage disequilibrium, and haplotype association analysis of the growth suppressor gene ZBED6 in Qinchuan beef cattle. Animal Biotechnology, 2014, 25:35.

[9] HUANG Y Z, ZHAN Z Y, SUN Y J, WANG J, LI M X, LAN X Y, LEI C Z, ZHANG C L, CHEN H. Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle. Genome, 2013, 56:327.

[10] HUANG Y Z, ZHANG L Z, LAI X S, LI M X, SUN Y J, LI C J, LAN X Y, LEI C Z, ZHANG C L, ZHAO X, CHEN H. Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoteractivity and DNA methylation in myoblasts. Scientific Reports, 2014, 4:4570.

[11] HUANG Y Z, LI MX, WANG J, ZHAN Z Y, SUN Y J, SUN J J, LI C J, LAN X Y, LEI C Z, ZHANG C L, CHEN H. A 5'-regulatory region and two coding region polymorphisms modulate promoter activity and gene expression of the growth suppressor gene ZBED6 in cattle. PLoS One, 2013, 8: e79744.

[12] 马燕. 绵羊ZBED6基因的遗传变异及其与胴体性状的相关性研究[D]. 石河子: 石河子大学, 2016.

MA Y. Effects of ZBED6 variations on carcass traits in sheep[D] Shihezi: Shihezi University, 2016. (in Chinese)

[13] 杨帆, 胡长敏, 丁明星. 7例犬乳腺肿瘤的病理组织学分析. 畜牧与兽医, 2012(s2):122-125.

YANG F, HU C M, DING M X. The pathological histological analysis of 7 canine breast tumors. Animal Husbandry and Veterinarian, 2012(s2):122-125. (in Chinese)

[14] 董坤哲. 藏猪高海拔环境适应性分子机制探讨[D]. 北京: 中国农业科学院, 2015.

DONG K Z. Discussion on the adaptive molecular mechanism of high altitude environment in Tibetan pig[D]. Beijing : Chinese Academy of Agricultural Sciences, 2015. (in Chinese)

[15] TRAPNELL C, PACHTER L, SALZBERG S L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 2009, 25:1105.

[16] TRAPNELL C, WILLIAMS BA, PERTEA G, MORTAZAVI A, KWAN G, VAN BAREN M J, SALZBERG S L, WOLD B J, PACHTER L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 2010, 28:511.

[17] TRAPNELL C, ROBERTS A, GOFF L, PERTEA G, KIM D, KELLEY D R, PIMENTEL H, SALZBERG S L, RINN J L, PACHTER L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols, 2012, 7: 562.

[18] REIMAND J, ARAK T, ADLER P, KOLBERG L, REISBERG S, PETERSON H, VILO J. g:Profiler-a web server for functional interpretation of gene lists (2016 update). Nucleic Acids Research, 2016, 44: W83.

[19] CONG L, RAN FA, COX D, LIN S, BARRETTO R, HABIB N, HSU PD, WU X, JIANG W, MARRAFFINI LA, ZHANG F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339:819.

[20] HAI T, TENG F, GUO R, LI W, ZHOU Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 2014, 24:372.

[21] ZHOU X, XIN J, FAN N, ZOU Q, HUANG J, OUYANG Z, ZHAO Y, ZHAO B, LIU Z, LAI S, YI X, GUO L, ESTEBAN MA, ZENG Y, YANG H, LAI L. Generation of CRISPR/Cas9-mediated gene- targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72:1175.

[22] JIANG L, WALLERMAN O, YOUNIS S, RUBIN CJ, GILBERT ER, SUNDSTROM E, GHAZAL A, ZHANG X, WANG L, MIKKELSEN TS, ANDERSSON G, ANDERSSON L. ZBED6 modulates the transcription of myogenic genes in mouse myoblast cells. PLoS One 2014, 9: e94187.

[23] ARBER S, CARONI P. Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Development, 1996;10:289.

[24] ARBER S, HUNTER JJ, ROSS JJ, HONGO M, SANSIG G, BORG J, PERRIARD JC, CHIEN KR, CARONI P. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell, 1997, 88:393.

[25] KNOLL R, KOSTIN S, KLEDE S, SAVVATIS K, KLINGE L, STEHLE I, GUNKEL S, KOTTER S, BABICZ K, SOHNS M, MIOCIC S, DIDIE M, KNOLL G, ZIMMERMANN WH, THELEN P, BICKEBOLLER H, MAIER LS, SCHAPER W, SCHAPER J, KRAFT T, TSCHOPE C, LINKE W A, CHIEN K R. A common MLP (muscle LIM protein) variant is associated with cardiomyopathy. Circulation Research,2010, 106:695.

[26] KONG Y, FLICK M J, KUDLA A J, KONIECZNY S F. Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Molecular CellBiology, 1997, 17:4750.

[27] KNOLL R, HOSHIJIMA M, HOFFMAN H M, PERSON V, LORENZEN-SCHMIDT I, BANG ML, HAYASHI T, SHIGA N, YASUKAWA H, SCHAPER W, MCKENNA W, YOKOYAMA M, SCHORK N J, OMENS J H, MCCULLOCH AD, KIMURA A, GREGORIO C C, POLLER W, SCHAPER J, SCHULTHEISS H P, CHIEN K R. The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy. Cell, 2002, 111:943.

[28] GEIER C, GEHMLICH K, EHLER E, HASSFELD S, PERROT A, HAYESS K, CARDIM N, WENZEL K, ERDMANN B, KRACKHARDT F, POSCH M G, OSTERZIEL K J, BUBLAK A, NAGELE H, SCHEFFOLD T, DIETZ R, CHIEN K R, SPULER S, FURST D O, NURNBERG P, OZCELIK C. Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy. Human Molecular Genetics, 2008, 17:2753.

[29] MA K K, BANAS K, DE BOLD A J. Determinants of inducible brain natriuretic peptide promoter activity. Regulatory Peptides, 2005, 128:169.

[30] POTTER L R. “Corination” of the proANP converting enzyme. Cell Metabolism, 2005, 1:88.

[31] 张凤, 周广海, 王尧, 王欣农, 曹景宇, 文今福. 心房钠尿肽与心肌肥大的关系. 中华高血压杂志 2008, 16(8): 763-765.

ZHANG F, ZHOU G H, WANG Y, WANG X N, CAO J Y, WEN J F. Relationship between atrial natriuretic peptide and myocardial hypertrophy. Chinese Journal of Hypertension, 2008, 16(8): 763-765. (in Chinese)

[32] BURRIDGE K, WENNERBERG K. Rho and Rac take center stage. Cell, 2004, 116:167.

[33] TAKEUCHI S, KAWASHIMA S, RIKITAKE Y, UEYAMA T, INOUE N, HIRATA K, YOKOYAMA M. Cerivastatin suppresses lipopolysaccharide-induced ICAM-1 expression through inhibition of Rho GTPase in BAEC. Biochemical and Biophysical Research Communications, 2000, 269:97.

[34] TIMSON D J. Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase. FEBS Journal, 2005, 272:6170.

[35] HEASMAN S J, RIDLEY A J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nature Reviews Molecular Cell Biology, 2008, 9:690.

[36] RIDLEY A J. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends in Cell Biology, 2006, 16:522.

[37] CHANDRASHEKAR R, SALEM O, KRIZOVA H, MCFEETERS R, ADAMS P D. A switch I mutant of Cdc42 exhibits less conformational freedom. Biochemistry-Us, 2011, 50: 6196.

[38] MELENDEZ J, GROGG M, ZHENG Y. Signaling role of Cdc42 in regulating mammalian physiology. Journal of Biological Chemistry, 2011, 286:2375.

[39] 文丽丹. 心肌特异性敲除Cdc42对血管紧张素Ⅱ诱导心肌肥大的保护作用[D]. 南昌: 南昌大学, 2015.

WEN L D. Myocardial specificity of angiotensin knockout Cdc42 Ⅱ induced myocardial hypertrophy of protection[D]. Nanchang: Nanchang University, 2015. (in Chinese)

[40] HOVANESSIAN A G. Interferon-induced and double-stranded RNA- activated enzymes: a specific protein kinase and 2',5'-oligoadenylate synthetases. Journal of Interferon and Cytokine Research, 1991, 11:199.

[41] SILVERMAN R H. Viral encounters with 2', 5'-oligoadenylate synthetase and RNase L during the interferon antiviral response. Journal of Virology, 2007, 81:12720.

[42] 刘维婷. 重组猪源OAS1抗日本脑炎病毒的特性研究[D]. 南京: 南京农业大学, 2013.

LIU W T. Characteristics of recombinant pig source OAS1 anti - Japanese encephalitis virus[D]. Nanjing: Nanjing Agricultural University, 2013. (in Chinese)

[43] MAYER N J, RUBIN S A. Molecular and cellular prospects for repair, augmentation, and replacement of the failing heart. American Heart Journal, 1997, 134:577.

[44] CARNIEL E, TAYLOR M R, SINAGRA G, DI LENARDA A, KU L, FAIN P R, BOUCEK M M, CAVANAUGH J, MIOCIC S, SLAVOV D, GRAW S L, FEIGER J, ZHU X Z, DAO D, FERGUSON D A, BRISTOW M R, MESTRONI L. Alpha-myosin heavy chain: a sarcomeric gene associated with dilated and hypertrophic phenotypes of cardiomyopathy. Circulation, 2005, 112:54.