初生及成年牦牛大脑皮质的转录组分析

doi:10.11843/j.issn.0366-6964.2023.11.016

摘要/Abstract

摘要： 旨在分析初生和成年牦牛大脑皮质转录组表达谱,筛选影响大脑皮质发育的候选基因。本研究选取初生 (1~7日龄(NYB),n=3)和成年(3~4岁 (AYB),n=3) 牦牛,放血处死,采集大脑皮质,利用Illumina HiSeq平台对转录组进行测序与分析,筛选得到差异表达基因,在此基础上对差异基因进行GO功能注释、KEGG富集分析和实时荧光定量PCR(qRT-PCR)方法验证。测序结果共分析得到了4 790个显著差异表达的基因(P<0.05),其中包括2 670个上调表达的差异基因,2 120个下调表达的差异基因。随机选取了9个差异基因进行qRT-PCR验证,表达趋势与转录组测序结果一致,表明测序结果可靠。GO功能注释发现其中占比较多的条目为与蛋白合成及ATP利用相关的转录调控、RNA聚合酶II正调控转录、蛋白结合及ATP结合;KEGG富集分析发现占比较多与大脑发育相关的通路为调控轴突发育及重塑的轴突导向通路,与神经胶质细胞增殖相关的MAPK信号通路,与突触传递相关的神经活性配体-受体相互作用、 PI3K-Akt、钙信号及磷脂酶D信号通路,与免疫防御相关的Toll样受体通路,与低氧适应调控相关的HIF信号通路。从富集通路中发现,Slit2、SEMA5A、BDNF、Rab3a、FGF18、HIF1α、HIF2α、NGF、VEGFA、CaMK2A、PLD1、TLR1、TLR4基因可能是影响牦牛大脑皮质发育的候选基因。上述结果提示,成年牦牛大脑皮质的突触发育、可塑性和传递、免疫防御可能更活跃,并且低氧适应性更强,这为进一步探讨牦牛大脑发育潜在分子机理提供了一定参考。

关键词: 牦牛, 大脑皮质, 初生, 成年, 转录组

Abstract: The aim of this study was to analyze the transcriptomic expression profile of the cerebral cortex in newborn and adult yaks, and screen the candidate genes affecting cerebral cortex development. The newborn yaks (1-7 days of age (NYB), n=3) and adult yaks (3-4 years of age (AYB), n=3) were selected and slaughtered, then the cerebral cortex was collected. Moreover, transcriptome sequencing and analysis were performed using Illumina HiSeq platform, and differen-tially expressed genes (DEGs)were screened. On this basis, the differential genes were performed by GO functional annotation and KEGG enrichment analysis, and verified by real-time fluorescence quantification PCR (qRT-PCR). The results showed 4 790 DEGs (P<0.05), including 2 670 up-regulated DEGs and 2 120 down-regulated DEGs. Nine differentially expressed genes were randomly selected for qRT-PCR verification, and their expression trend was consistent with the transcriptome sequencing results, indicating that the sequencing results were reliable. GO analysis showed that the most DEGs were enriched in regulation of transcription, positive regulation of transcription by RNA polymerase II, protein binding and ATP binding, which were related to protein synthesis and ATP utilization. Moreover, the most DEGs according to analysis based on KEGG database were mainly enriched in the axon guidance signaling pathway that regulate axon development and remodeling; MAPK signaling pathway which related to glial cell proliferation; in addition neuroactive ligand-receptor interactions, PI3K-AKT, calcium signaling and phospholipase D signaling pathways were related to synaptic transmission; and Toll-like receptor pathways were related to immune defense, and HIF signaling pathways related to hypoxic adaptive regulation. From the enrichment pathway, Slit2, SEMA5A, BDNF, Rab3a, FGF18, HIF1α, HIF2α, NGF, VEGFA, CaMK2A, PLD1, TLR1 and TLR4 might be candidate genes affecting the cerebral cortex development in yaks. These results suggest that synapse development, plasticity, transmission, immune defense may be more active in the cerebral cortex of adult yaks. And hypoxic adaptation of the cerebral cortex in adult yaks might be more perfect. It provided the reference for further exploring the potential molecular mechanism of brain development in yaks.

Key words: yak, cerebral cortex, newborn, adult, transcriptome

中图分类号:

S823.85

张倩, 崔燕, 余四九, 何俊峰, 潘阳阳, 王萌. 初生及成年牦牛大脑皮质的转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4605-4614.

ZHANG Qian, CUI Yan, YU Sijiu, HE Junfeng, PAN Yangyang, WANG Meng. Transcriptome Analysis of the Cerebral Cortex in Newborn and Adult Yaks (Bos grunniens)[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4605-4614.

参考文献 35

[1]	庄蕾,吴森.高通量测序技术在牦牛相关研究中应用[J].家畜生态学报,2022,43(3):77-82.ZHUANG L,WU S.Application of high-throughput sequencing technology in yak[J].Journal of Domestic Animal Ecology, 2022,43(3):77-82.(in Chinese)
[2]	李欣,李小俊,陈晓丽,等.转录组数据分析与功能基因挖掘[J].畜牧兽医学报,2019,50(3):474-484.LI X,LI X J,CHEN X L,et al.Transcriptomics analysis and functional genes mining[J].Acta Veterinaria et Zootechnica Sinica, 2019,50(3):474-484.(in Chinese)
[3]	CADWELL C R,BHADURI A,MOSTAJO-RADJI M A,et al.Development and arealization of the cerebral cortex[J].Neuron, 2019,103(6):980-1004.
[4]	GONZÁLEZ-VELASCO O,PAPY-GARCÍA D,LE DOUARON G,et al.Transcriptomic landscape,gene signatures and regulatory profile of aging in the human brain[J].Biochim Biophys Acta Gene Regul Mech,2020,1863(6):194491.
[5]	XIMERAKIS M,LIPNICK S L,INNES B T,et al.Single-cell transcriptomic profiling of the aging mouse brain[J].Nat Neurosci, 2019,22(10):1696-1708.
[6]	AYALEW W,CHU M,LIANG C N,et al.Adaptation mechanisms of yak (Bos grunniens) to high-altitude environmental stress[J]. Animals,2021,11(8):2344.
[7]	杨柏高,郝海生,杜卫华,等.牦牛高原适应研究进展[J].畜牧兽医学报,2023,54(1):12-23.YANG B G,HAO H S,DU W H,et al.Advances in research on plateau adaptation of yak[J].Acta Veterinaria et Zootechnica Sinica,2023,54(1):12-23.(in Chinese)
[8]	梁林.不同发育阶段大通牦牛大脑皮质组织学研究[D].西宁:青海大学,2012.LIANG L.Study on the histology of cerebral cortex in da tong yak at different developmental stages[D].Xining:Qinghai University, 2012.(in Chinese)
[9]	黄兴.牦牛大脑和小脑低氧适应性的转录组研究[D].成都:西南民族大学,2019.HUANG X.The transcriptome analysis of cerebrum and cerebellum reveals the well hypoxic adaptability of yak[D].Chengdu: Southwest Minzu University,2019. (in Chinese)
[10]	唐娇,夏果,刘益丽,等.不同年龄段牦牛瘤胃组织形态学与转录组研究[J].畜牧兽医学报,2022,53(11):3797-3810.TANG J,XIA G,LIU Y L,et al.The research of histomorphological and transcriptomic of yak rumen at different ages[J].Acta Veterinaria et Zootechnica Sinica,2022,53(11):3797-3810.(in Chinese)
[11]	傅芳,王利,字向东.麦洼牦牛不同生长阶段肝脏差异表达基因分析[J].兽类学报,2022,42(1):85-94.FU F,WANG L,ZI X D.Differential expression genes analysis of liver in Maiwa yak at different growth stages[J].Acta Theriologica Sinica,2022,42(1):85-94.(in Chinese)
[12]	傅芳,官久强,曲秀龙,等.不同海拔饲育的麦洼牦牛肺脏组织转录组学分析[J].兽类学报,2020,40(5):475-484.FU F,GUAN J Q,QU X L,et al.Transcriptomics analysis of lungs in yaks breeding at different altitudes[J].Acta Theriologica Sinica,2020,40(5):475-484.(in Chinese)
[13]	纪会,王会,柴志欣,等.牦牛不同年龄肌肉组织microRNA表达谱及生物信息学分析[J].畜牧兽医学报,2019,50(5):957-971.JI H,WANG H,CHAI Z X,et al.Differential expression profile and bioinformatics analysis of miRNAs in yak muscle tissue during development[J].Acta Veterinaria et Zootechnica Sinica,2019,50(5):957-971.(in Chinese)
[14]	KIM D,LANGMEAD B,SALZBERG S L.HISAT:a fast spliced aligner with low memory requirements[J].Nat Methods,2015, 12(4):357-360.
[15]	ANDERS S,PYL P T,HUBER W.HTSeq—a Python framework to work with high-throughput sequencing data[J]. Bioinformatics, 2015,31(2):166-169.
[16]	PERTEA M,PERTEA G M,ANTONESCU C M,et al.StringTie enables improved reconstruction of a transcriptome from RNA-seq reads[J].Nat Biotechnol,2015,33(3):290-295.
[17]	LOVE M I,HUBER W,ANDERS S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J].Genome Biol,2014,15(12):550.
[18]	WU T Z,HU E Q,XU S B,et al.ClusterProfiler 4.0:A universal enrichment tool for interpreting omics data[J]. Innovation, 2021,2(3):100141.
[19]	KUMAR A,GIBBS J R,BEILINA A,et al.Age-associated changes in gene expression in human brain and isolated neurons[J].Neurobiol Aging,2013,34(4):1199-1209.
[20]	LIANG X,LIU X M,LU F X,et al.HIF1α signaling in the endogenous protective responses after neonatal brain hypoxia-ischemia[J].Dev Neurosci,2018,40(5-6):617-626.
[21]	WANG L,ZHOU K,FU Z,et al.Brain development and Akt signaling:the crossroads of signaling pathway and neurodevelopmental diseases[J].J Mol Neurosci,2017,61(3):379-384.
[22]	ZHANG Z,YAO L,YANG J H,et al.PI3K/Akt and HIF-1 signaling pathway in hypoxia-ischemia (Review)[J].Mol Med Rep, 2018,18(4):3547-3554.
[23]	YUASA-KAWADA J,KINOSHITA-KAWADA M,TSUBOI Y,et al.Neuronal guidance genes in health and diseases[J].Protein Cell,2023,14(4):238-261.
[24]	YASUDA R,HAYASHI Y,HELL J W.CaMKII:a central molecular organizer of synaptic plasticity,learning and memory[J].Nat Rev Neurosci,2022,23(11):666-682.
[25]	BARBER C N,GOLDSCHMIDT H L,LILLEY B,et al.Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina[J].J Lipid Res,2022,63(8):100247.
[26]	WANG X C,YU D M,WANG H Y,et al.Rab3 and synaptotagmin proteins in the regulation of vesicle fusion and neurotransmitter release[J].Life Sci,2022,309:120995.
[27]	IROEGBU J D,IJOMONE O K,FEMI-AKINLOSOTU O M,et al.ERK/MAPK signalling in the developing brain:Perturbations and consequences[J].Neurosci Biobehav Rev,2021,131:792-805.
[28]	CARULLI D,DE WINTER F,VERHAAGEN J.Semaphorins in adult nervous system plasticity and disease[J].Front Synaptic Neurosci,2021,13:672891.
[29]	TONG M F,JUN T,NIE Y Z,et al.The role of the slit/robo signaling pathway[J].J Cancer,2019,10(12):2694-2705.
[30]	KLIMASCHEWSKI L,CLAUS P.Fibroblast growth factor signalling in the diseased nervous system[J].Mol Neurobiol,2021, 58(8):3884-3902.
[31]	ARÉVALO J C,DEOGRACIAS R.Mechanisms controlling the expression and secretion of BDNF[J].Biomolecules, 2023, 13(5):789.
[32]	WITTKO-SCHNEIDER I M,SCHNEIDER F T,PLATE K H.Brain homeostasis:VEGF receptor 1 and 2- two unequal brothers in mind[J].Cell Mol Life Sci,2013,70(10):1705-1725.
[33]	ROCCO M L,SOLIGO M,MANNI L,et al.Nerve growth factor:early studies and recent clinical trials[J].Curr Neuropharmacol, 2018,16(10):1455-1465.
[34]	AHMADABAD R A,MIRZAASGARI Z,GORJI A,et al.Toll-like receptor signaling pathways:Novel therapeutic targets for cerebrovascular disorders[J].Int J Mol Sci,2021,22(11):6153.
[35]	OSTROWSKI R P,ZHANG J H.The insights into molecular pathways of hypoxia-inducible factor in the brain[J].J Neurosci Res,2020,98(1):57-76.