MSTN基因编辑牛肌肉转录组测序及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.07.001

摘要/Abstract

摘要： 为探究肌生长抑制素（myostatin，MSTN）基因对牛肌肉发育的具体调控机制，本研究选取同一牛场健康鲁西黄牛10头，其中通过转基因技术得到的基因编辑牛MSTN^-/-和同种非转基因野生型牛各5头。分别采集两组牛腿臀肌肉样品，利用IIlumina HiSeq高通量测序技术进行转录组测序分析，通过生物信息学方法比较两组样本间的差异表达基因，并进行GO和KEGG富集分析，最后利用实时荧光定量PCR验证转录组测序数据。结果显示，基因编辑型牛和野生型牛之间共检测到18 071个基因。在log₂|FoldChange|≥ 1.48条件下，筛选出406个差异表达基因，其中347个显著上调，59个显著下调。GO功能富集分析显示，MSTN基因编辑后显著影响915个功能类别（P<0.05），差异基因主要参与结合、生物系统调节、免疫系统等相关功能。KEGG通路富集分析结果共涉及211个通路，差异基因主要富集在细胞黏附分子、趋化因子信号通路、细胞因子互作等信号通路上，进一步从中筛选出可能参与细胞生长、肌肉发育的差异基因（CD14、KIT、CSF1R、FBP1、DUSP4、ULBP21、PRKCB、SPN、CHAD、SRC）。实时荧光定量PCR检测结果显示，所选差异基因表达水平与转录组表达水平一致，证明测序结果的可靠性。本研究结果表明，MSTN基因发挥作用后可以介导多个下游基因表达，从而影响相关信号通路及生物学过程；同时，所筛选出的差异表达基因可作为进一步研究骨骼肌调控机制的候选靶标。

关键词: 肌生长抑制素; 转录组测序; 基因筛选; 实时荧光定量PCR

Abstract: To explore the specific regulation mechanism of myostatin (MSTN) gene on muscle development of bovine,a total of ten healthy bovine were selected from the same farm,including five MSTN^-/- gene-edited bovine and five wild-type bovine,and extracted the RNA of bovine muscle tissue sample.Collecting the calf-hip muscle samples of two groups,and the transcriptome analysis were sequenced by Illumina high-throughput sequencing technology.Bioinformatics method was used to compare the differentially expressed genes between two groups,and Go and KEGG enrichment analysis were performed.Real-time quantitative PCR was used to verify the transcriptome sequencing data.A total of 18 071 genes were obtained from the gene-edited bovine and wild-type bovine.Under the conditions of log₂|FoldChange|≥ 1.48,a total of 406 significantly differentially expressed genes were screened,in which 347 genes were up-regulated and 59 genes were down-regulated.GO functional analysis results showed that total 915 functional categories were effected after MSTN gene editing (P<0.05).The differentially expressed genes were mainly involved in the binding,regulation of biological process,immune system process and other related functions.KEGG functional enrichment analysis results indicated that differential genes were involved in total of 211 pathways,mainly concentrated in cell adhesion molecule,chemokine,cytokine interaction and other signaling pathways.The differentially expressed genes might be related to cell growth and muscle development were further screened out,including CD14,KIT,CSF1R,FBP1,DUSP4,ULBP21,PRKCB,SPN,CHAD and SRC genes.Real-time quantitative PCR results showed that the expression levels of the differentially expressed genes were basically consistent with the transcriptome expression,indicating the reliability of sequencing results.The results of this study indicated that MSTN gene could mediate the expression of multiple downstream genes after exert its role,effecting the relevant signaling pathways and biological processes.The screened differentially expressed genes in this study could be used as candidate targets for further study of the regulation mechanism of skeletal muscle.

Key words: myostatin; transcriptome sequencing; gene screening; Real-time quantitative PCR

中图分类号:

苗曼宁, 郭益文, 胡德宝, 曾雨晗, 李新, 张林林, 丁向彬, 郭宏. MSTN基因编辑牛肌肉转录组测序及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2271-2281.

MIAO Manning, GUO Yiwen, HU Debao, ZENG Yuhan, LI Xin, ZHANG Linlin, DING Xiangbin, GUO Hong. Transcriptome Sequencing and Bioinformatics Analysis of MSTN Gene-edited Bovine Muscles[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2271-2281.

参考文献

[1] MCPHERRON A C,LEE S J.Double muscling in cattle due to mutations in the myostatin gene[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(23):12457-12461.
[2] SMITH T P L,LOPEZ-CORRALES N L,KAPPES S M,et al.Myostatin maps to the interval containing the bovine MH locus[J].Mammalian Genome,1997,10:742-744.
[3] LEE C Y,HU S Y,GONG H Y,et al.Suppression of myostatin with vector-based RNA interference causes adouble-muscle effect in transgenic zebrafish[J].Biochemical and Biophysical Reserch Communications,2009,387(4):766-771.
[4] 魏著英,白春玲,李光鹏.牛肌肉生长抑制素基因突变的遗传效应与育种应用[J].生物技术进展,2018,8(3):197-205. WEI Z Y,BAI C L,LI G P.Genetic effect and breeding application of myostatin gene mutation in beef cattle[J].Current Biotechnology,2018,8(3):197-205.(in Chinese)
[5] MCPHERRON A C,LEE S J.Suppression of body fat accumulation in myostatin-deficient mice[J].Journal of Clinical Investigation,2002,109(5):595-601.
[6] 闫允君,卢霞,孟宪红,等.基于转录组分析对中国对虾myostatin基因调控的肌肉生长相关基因的筛选[J].渔业科学进展,2020,11:1-10. YAN Y J,LU X,MENG X H,et al.Screening of genes related to muscle growth under the myostatin regulation by RNA-Seq in Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2020,11:1-10.(in Chinese)
[7] 杨阳,杨琦,董然然,等.基于转录组测序筛选鼠伤寒沙门菌hfq基因缺失菌的差异表达基因[J].畜牧兽医学报,2019,50(12):2578-2584. YANG Y,YANG Q,DONG R R,et al.Screening of differentially expressed genes of Salmonella Typhimurium hfq gene deletion bacteria basedon transcriptome sequencing[J].Acta Veterinaria et Zootechnica Sinica,2019,50(12):2578-2584.(in Chinese)
[8] SHAH A,SILVERSTEIN P S,SINGH D P,et al.Involvement of metabotropic glutamate receptor 5,Akt/PI3K signaling and NF-κB pathway in methamphetamine-mediated increase in IL-6 and IL-8 expression in astrocytes[J].Neuroinflammation,2012,9(1):52.
[9] BHASKAR P T,HAY N.The two TORCs and Akt[J].Developmental Cell,2007,12(4):487-502.
[10] PAN B L,TONG Z W,LI S D,et al.Decreased microRNA-182-5p helps alendronate promote osteoblast proliferation and differentiation in osteoporosis via the Rap1/MAPK pathway[J].Bioscience Reports,2018,38(6):1-16.
[11] KONG F,LI Z,PARKS W M,et al.Cyclic mechanical reinforcement of integrin-ligand interactions[J].Molecular Cell,2013,49(6):1060-1068.
[12] 崔银江,李少华.E-钙黏附素的研究进展[J].中国矫形外科杂志,2009,17(13):1001-1003. CUI Y J,LI S H.Study of E-cadherin[J].Orthopedic Journal of China,2009,17(13):1001-1003.(in Chinese)
[13] 王成,武书庚,张海军,等.肉仔鸡卫星细胞氧化应激时MAPK信号通路[J].中国农业科学,2010,43(20):4286-4294. WANG C,WU S H,ZHANG H J,et al.Study on oxidative stress-activated MAPK signaling pathway in broilers breast muscle satellite cells[J].Scientia Agricultura Sinica,2010,43(20):4286-4294.(in Chinese)
[14] WANG Z,WANG Z,PANG Y,et al.Fibronectin type Ⅲ domain-containing 4 promotes the migration and differentiation of bovine skeletal muscle-derived satellite cells via focal adhesion kinase[J].Cell Adhesion & Migration,2020,14(1):153-164.
[15] LI T,QUAN H,ZHANG H,et al.Silencing cyclophilin A improves insulin secretion,reduces cell apoptosis,and alleviates inflammation as well as oxidant stress in high glucose-induced pancreatic β-cells via MAPK/NF-κB signaling pathway[J].Bioengineered,2020,11(1):1047-1057.
[16] 卜凡.耐力运动经AMPK介导的自噬维持老年小鼠骨骼肌卫星细胞干细胞属性[D].重庆:重庆医科大学,2019. BU F.Endurance exercise maintains the stem cell properties of muscle satellite cells in older mice through AMPK-mediated autophagy[D].Chongqing:Chongqing Medical University,2019.(in Chinese)
[17] 甘伟.AMPK/FOXO3信号通路调控鸭成肌细胞凋亡与分化的机制研究[D].雅安:四川农业大学,2016. GAN W.The regulation mechanism of AMPK/FOXO3 signal pathway in the apoptosis and differentiation of duck myoblast[D].Ya'an:Sichuan Agricultural University,2016.(in Chinese)
[18] 魏文燕,陈建英.非编码RNA调控巨噬细胞极化的研究进展[J].山东医药,2018,58(29):90-94. WEI W Y,CHEN J Y.Progress of the regulation of macrophage polarization by noncoding RNA[J].Shandong Medical Journal,2018,58(29):90-94.(in Chinese)
[19] WANG D,MALARKANNAN S.Transcriptional regulation of natural killer cell development and functions[J].Cancers (Basel),2020,12(6):1591.
[20] JIA S,YANG X,YANG X,et al.microRNA-210 protects against periodontitis through targeting HIF-3α and inhibiting p38MAPK/NF-κB pathway[J].Artificial Cells Nanomedicine and Biotechnology,2020,48(1):129-136.
[21] CHEN S,LI B.miR-128-3p post-transcriptionally inhibits WISP1 to suppress apoptosis and inflammation in human articular chondrocytes via the PI3K/Akt/NF-κB signaling pathway[J].Cell Transplant,2020,29:1-13.
[22] PAONE C,RODRIGUES N,ITTNER E,et al.The Tyrosine kinase Pyk2 contributes to complement-mediated phagocytosis in murine macrophages[J].Innate Immunity,2016,8(5):437-451.
[23] KUROKAWA M,SATO F,ARAMAKI S,et al.Monitor of the myostatin autocrine actionduring differentiation of embryonic chicken myoblasts into myotubes:Effect of IGF-Ⅰ[J].Molecular and Cellular Biochemistry,2009,331(1-2):193-199.
[24] YANG W,ZHANG Y,LI Y,et al.Myostatin induces cyclin D degradation to cause cell cycle arrest through a PI3K/Akt/GSK-3beta pathway and is antagonized by IGF-1[J].Biological Chemistry,2007,282(6):3799-3808.
[25] FREUDENBERG J A,CHEN D,CHEN W T.Stable suppression of membrane type 1 matrix metalloproteinase impairs MMP-2 activation,cell invasion,and tumorigenesis[J].Cellular,Molecular and Tumor Biology,2004,64(7):529-530.
[26] XU Z,ZHU X,SU L,et al.A high-throughput assay for screening natural products that boost NK cell-mediated killing of cancer cells[J].Pharmaceutical Biology,2020,58(1):357-366.
[27] GRABERT K,SEHGAL A,IRVINE K M,et al.A transgenic line that reports CSF1R protein expression provides a definitive marker for the mouse mononuclear phagocyte system[J].Immunology,2020,205(11):3154-3166.
[28] 李娜.鸭MITF、KIT基因SNP与屠宰、肉质性状的关联分析[D].武汉:华中农业大学,2017. LI N.Association analysis on duck MITF and KIT gene SNP with slaughter and meat quality traits[D].Wuhan:Huazhong Agricultural University,2017.
[29] HADDOCK A N,LABUZAN S A,HAYNES A E,et al.Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity[J].American Journal of Physiology-Cell Physiology,2019,316(4):567-581.
[30] HUANG P,ZHANG X,PAN M,et al.Effect of activated protein kinase C beta type mediated phosphorylation of signal transducer and activator of transcription 4 on proliferation and phenotypic transformation of vascular smooth muscle cells after vascular injury induced by Nano-SiO₂[J].Journal of Nanoscience Nanotechnology,2020,20(12):7385-7397.