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泰州鹅开产前后肝脏组织转录组对比分析

张蕾 章敬旗 章玮月 糜娟 何文杰 王健

张蕾,章敬旗,章玮月,等. 泰州鹅开产前后肝脏组织转录组对比分析 [J]. 福建农业学报,2020,35(9):987−996 doi: 10.19303/j.issn.1008-0384.2020.09.010
引用本文: 张蕾,章敬旗,章玮月,等. 泰州鹅开产前后肝脏组织转录组对比分析 [J]. 福建农业学报,2020,35(9):987−996 doi: 10.19303/j.issn.1008-0384.2020.09.010
ZHANG L, ZHANG J Q, ZHANG W Y, et al. RNA-Seq Analysis on Liver of Taizhou Geese before and after Egg-laying [J]. Fujian Journal of Agricultural Sciences,2020,35(9):987−996 doi: 10.19303/j.issn.1008-0384.2020.09.010
Citation: ZHANG L, ZHANG J Q, ZHANG W Y, et al. RNA-Seq Analysis on Liver of Taizhou Geese before and after Egg-laying [J]. Fujian Journal of Agricultural Sciences,2020,35(9):987−996 doi: 10.19303/j.issn.1008-0384.2020.09.010

泰州鹅开产前后肝脏组织转录组对比分析

doi: 10.19303/j.issn.1008-0384.2020.09.010
基金项目: 江苏省农业科技自主创新资金项目(CX[18]1004);泰州市科技支撑计划(农业)项目(TN201802)
详细信息
    作者简介:

    张蕾(1987−),女,博士研究生,研究方向:动物遗传育种与繁殖(E-mail:leizhang@jsahvc.edu.cn

    通讯作者:

    王健(1974−),男,教授,研究方向:动物遗传育种与繁殖(E-mail:568418583@qq.com

  • 中图分类号: S 835

RNA-Seq Analysis on Liver of Taizhou Geese before and after Egg-laying

  • 摘要:   目的  进一步完善泰州鹅基因组结构信息,探索泰州鹅开产前、后肝脏的调控机制。  方法  利用RNA-Seq技术对开产前、后泰州鹅肝脏组织转录组进行分析,利用qRT-PCR对部分RNA-Seq数据进行验证;利用GO富集分析和KEGG通路注释对差异表达基因功能进行解析。  结果  测序获得的初始数据(raw reads)进行系列质控筛选,6个泰州鹅开产前、后肝脏组织获得73 596 894~79 837 756个高质量数据(clean reads),结合FPKM值定量估计所检测的基因表达值,筛选出202个差异基因,其中上调差异表达基因100个,下调差异表达基因102个,经GO功能注释后富集到42个生物功能类别;KEGG分析筛选到63条差异信号通路,其中5条显著富集信号通路为药物代谢-细胞色素P450、氮代谢、戊糖和葡萄糖醛酸相互转化、视黄醇代谢、类固醇激素生物合成通路,这些信号通路功能分析显示与脂质代谢以及生殖过程密切相关。  结论  开产前后泰州鹅肝脏组织RNA-Seq分析表明,泰州鹅开产前后肝脏组织中的差异基因多集中于脂质代谢以及生殖过程,研究结果为进一步研究泰州鹅开产期的肝脏调控机制以及加速泰州鹅地方种群的进一步选育提供了新的数据支撑。
  • 图  1  转录组数据比对基因区域分布统计

    注:1~6:样品编号;不同颜色代表所在基因的不同区域(外显子、内含子以及基因间区),区域大小代表具有某一覆盖度范围的转录组数据所占比例。

    Figure  1.  Transcriptome data vs. regional distribution of genes

    Note: 1~6: sample codes; different colors in pie chart represent different locations of genes (exon, intron and intergenic); area size represents transcriptome datum coverage.

    图  2  差异表达基因火山图

    注:差异表达火山图中的每一个点表示一个基因。蓝色代表下调表达基因,黄色代表上调表达基因,灰色代表无显著性差异表达的基因。

    Figure  2.  Volcano plot of differentially expressed genes

    Note: each dot represents a gene. Blue dot: significantly downregulated genes; yellow dot: significantly upregulated genes; gray dot: genes with no differentiated expression.

    图  3  差异表达基因热图聚类分析

    注:图中1~6列代表不同的样品,不同行代表不同的基因。颜色代表了基因在样品中的表达量FPKM的对数值,蓝色代表下调表达基因,红色代表上调表达基因。

    Figure  3.  Heat map of differentially expressed genes

    Note: Columns 1–6: individual samples; different rows represent differentially expressed genes. Color scales as log2 (FPKM); blue: significantly downregulated genes; red: significantly upregulated genes.

    图  4  开产前后泰州鹅肝脏组织相关基因表达检测

    Figure  4.  Expressions of related genes in liver of Taizhou geese before and after start of egg-laying

    图  5  差异表达基因GO注释

    注:细胞组分:胞外区、细胞、膜、细胞连接处、膜封闭腔、大分子复合物、细胞器、胞外区部分、细胞器部分、病毒离子部分、膜部分、突触部分、细胞部分、突触,胞上、分子复合物;生物过程:免疫系统过程、行为、代谢过程、细胞增殖、细胞过程、生殖过程、生物黏附、信号传导、多细胞组织过程、发育过程、生长、运动、节律过程、刺激反应、定位、多生物过程、生物调节、细胞成分组织或生物发生;分子功能:催化活性、信号转导活性、结构分子活性、转运体活性、结合、翻译调节活性、分子转导活性、分子功能调节、转录、调节活性。

    Figure  5.  GO annotation on differentially expressed genes

    Note:Cellular component: extracellular region, cell, membrane, cell junction, membrane-enclosed lumen, macromolecular complex, organelle, extracellular region part, organelle part, virion part, membrane part, synapse part, cell part, synapse, supramo, lecular complex;Biological process:immune system process, behavior, metabolic process, cell proliferation, cellular process, reproductive process, biological adhesion, signaling, multicellular organismal process, developmental process, growth, locomotion, rhythmic process, response to stimulus, localization, multi-organism process, biological regulation, cellular component organization or biogenesis;Molecular function:catalytic activity, signal transducer activity, structural molecule activity, transporter activity, binding, translation regulator activity, molecular transducer activity, molecular function regulator, transcription, regulator activity.

    图  6  KEGG富集分析

    Figure  6.  KEGG enrichment on differentially expressed genes

    表  1  qRT-PCR引物序列、退火温度以及目的片段大小

    Table  1.   Primer sequences, annealing temperature, and predicted length for qRT-PCR

    基因
    Gene
    引物序列(5′-3′)
    Sequence (5′-3′)
    退火
    温度
    Tm/℃
    产物
    大小
    Size/bp
    CHRNA9 F:CAGCCAAAGGGAAATATGCTC 60 180
    R:TTTATCCGTGTCTTCCACTGGTC
    MRAP F:ATTCACTCTTCTCTCAGTTACGTC 58 135
    R:AACATGAGGAGTAACTGGTACATG
    EPSTI1 F:GGAGGAAATGAAGCAAGAACAAC 60 160
    R:TCCTCTGTTCATTTTGATGTGCTC
    TLDC2 F:AGCCCTGGAGCCTGCTGTAC 60 152
    R:GCCTCAGTGTCACGGATGAG
    SLC13A5 F:GTCTTCACCGAATGCACCAG 61 128
    R:AGCATGAAGGCAAAGGAAGCAC
    β-actin F:CAGCCATCTTTCTTGGGTAT 60 164
    R:CTGTGATCTCCTTCTGCATCC
    下载: 导出CSV

    表  2  样本数据质量预处理评价分析

    Table  2.   Quality evaluation and analysis on data preprocessing

    样品编号
    No.
    Raw读数
    Raw reads
    Raw基数
    Raw bases
    净读数
    Clean reads
    净读数比例
    Clean ratio/%
    碱基质量
    Q30/%
    1 81 525 658 12 228 848 700 79 443 928 97.45 95.46
    2 76 140 902 11 421 135 300 74 434 560 97.76 95.43
    3 81 984 186 12 297 627 900 79 837 756 97.38 95.62
    4 77 907 180 11 686 077 000 76 091 514 97.67 95.64
    5 75 754 528 11 363 179 200 73 596 894 97.15 95.82
    6 77 403 998 11 610 599 700 75 702 218 97.80 95.41
    注:Raw读数:每个样本的测序raw reads的总数;Raw基数:每个样本测序所获碱基的总数;净读数:过滤后的测序数据;净读数比例:过滤后高质量序列数占原始下机序列数的比例;碱基质量:过滤后,总序列中质量值大于30(错误率小于0.1%)的碱基数的比例。
    Note:raw reads: the number of raw reads; raw bases: the number of bases counted on raw reads; clean reads: the number of clean reads after preprocessing; clean ratio: the ratio of bases counted on clean reads; Q30: the ratio of clean bases whose quality threshold over 30.
    下载: 导出CSV

    表  3  转录组数据与参考基因组比对结果统计

    Table  3.   Results of clean reads on sample vs. reference genome

    样品编号
    No.
    总读数
    Total reads
    总覆盖数
    Total mapped
    覆盖率
    Mapped ratio/%
    多重覆盖数
    MultiMap reads
    多重覆盖率
    MultiMap ratio/%
    179 443 92870 396 34788.612 257 9502.84
    274 434 56067 324 68090.452 308 6663.10
    376 091 51467 957 32289.311 993 7882.62
    473 596 89466 002 06389.681 916 8372.60
    575 702 21867 433 93689.081 967 3032.60
    679 837 75670 832 38888.722 001 7102.51
    注:总读数:测序序列经过测序数据过滤后的数量(Clean reads);总覆盖数:能定位到基因组上的测序序列的数量;覆盖率:在参考序列上能够比对位置的测序序列的百分比;多重覆盖数:比对到基因组多个位置的序列数;多重覆盖率:比对到基因组多个位置的序列数的百分比。
    Note:total reads: the number of clean reads; total mapped: the number of clean reads that align with reference genome; mapped ratio: the percentage of clean reads that align with reference genome; multiMap reads: the number of clean reads that align with reference genome at multiple positions; multiMap ratio: the percentage of clean reads that align with reference genome at multiple positions.
    下载: 导出CSV

    表  4  泰州鹅开产前、后肝脏组织上调/下调差异最大的前10位基因

    Table  4.   Top 10 upregulated and downregulated genes in Taizhou geese before and after start of egg-laying

    基因名称
    Gene ID
    开产前表达量
    Before laying
    开产后表达量
    Laying group
    相对表达量
    Log2FC
    上调/下调
    Up/Down
    基因描述
    Gene description
    gene10578 150.00 83.89 −2.52 下调 Down CHRNA9
    gene14778 10698.00 6660.66 −2.07 下调 Down SLC13A5
    gene20392 1892.67 1136.15 −2.06 下调 Down RGS9BP
    gene14476 1484.33 893.87 −1.94 下调 Down MRAP
    gene6885 94.00 509.69 2.51 上调 Up EPSTI1
    gene3244 10.33 58.45 2.51 上调 Up MYO18B
    gene4794 292.00 1268.87 2.37 上调 Up LOC106032697
    gene16287 20.67 85.35 2.15 上调 Up TLDC2
    gene9668 89.33 397.50 2.09 上调 Up LOC106038139
    gene12640 22.67 130.54 1.99 上调 Up TMEM132E
    下载: 导出CSV

    表  5  泰州鹅开产前、后肝脏组织富集显著性最显著的前5条通路

    Table  5.   Top 5 enriched pathways in liver of Taizhou geese before and after start of egg-laying

    通路名称
    Pathway name
    通路编号
    Pathway ID
    富集基因数目
    Count
    校正后P
    P value
    Drug metabolism - cytochrome P450
    药物代谢-细胞色素P450信号通路
    map0098230.01
    Nitrogen metabolism
    氮代谢信号通路
    map0091050.02
    Pentose and glucuronate interconversions
    戊糖和葡萄糖醛酸相互转化信号通路
    map0004080.03
    Retinol metabolism
    视黄醇代谢信号通路
    map0083030.01
    Steroid hormone biosynthesis
    类固醇激素生物合成信号通路
    map0014060.01
    下载: 导出CSV
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  • 收稿日期:  2020-04-21
  • 修回日期:  2020-07-23
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