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基于高通量测序的低温胁迫下赤桉miRNAs的挖掘与分析

张紫阳, 刘艳, 魏瑞研, 林元震

张紫阳, 刘艳, 魏瑞研, 等. 基于高通量测序的低温胁迫下赤桉miRNAs的挖掘与分析[J]. 华南农业大学学报, 2021, 42(3): 64-74. DOI: 10.7671/j.issn.1001-411X.202011009
引用本文: 张紫阳, 刘艳, 魏瑞研, 等. 基于高通量测序的低温胁迫下赤桉miRNAs的挖掘与分析[J]. 华南农业大学学报, 2021, 42(3): 64-74. DOI: 10.7671/j.issn.1001-411X.202011009
ZHANG Ziyang, LIU Yan, WEI Ruiyan, et al. Mining and analysis of miRNAs from Eucalyptus camaldulensis under low temperature stress based on high-throughput sequencing[J]. Journal of South China Agricultural University, 2021, 42(3): 64-74. DOI: 10.7671/j.issn.1001-411X.202011009
Citation: ZHANG Ziyang, LIU Yan, WEI Ruiyan, et al. Mining and analysis of miRNAs from Eucalyptus camaldulensis under low temperature stress based on high-throughput sequencing[J]. Journal of South China Agricultural University, 2021, 42(3): 64-74. DOI: 10.7671/j.issn.1001-411X.202011009

基于高通量测序的低温胁迫下赤桉miRNAs的挖掘与分析

基金项目: 广东省重点领域研发计划(2020B020215002);国家自然科学基金(31470673)
详细信息
    作者简介:

    张紫阳(1995—),男,硕士研究生,E-mail: xizzy@live.cn

    通讯作者:

    林元震(1979—),男,副教授,博士,E-mail: yzhlin@scau.edu.cn

  • 中图分类号: S718.46;Q943.2

Mining and analysis of miRNAs from Eucalyptus camaldulensis under low temperature stress based on high-throughput sequencing

  • 摘要:
    目的 

    预测、挖掘和分析涉及赤桉Eucalyptus camaldulensis低温胁迫应答的miRNA,为研究其调控赤桉低温胁迫应答的分子网络奠定基础。

    方法 

    采用高通量测序对低温处理组和对照(CK)组的赤桉组培苗茎尖进行小RNA测序。以miRBase21.0、Rfam14.1和巨桉E. grandis基因组为参考数据库,利用Bowtie、miREAP和miRDeep2等软件进行miRNA预测,使用RNAfold对预测到的miRNA前体进行二级结构的折叠;采用psRNATarget预测靶基因,通过DEGSeq包分析差异表达的miRNA,并对它们进行GO注释和KEGG富集分析。

    结果 

    在赤桉中,共预测到隶属于54个家族的392个已知miRNA和97个新miRNA;其中,CK组共预测到282个已知miRNA,65个新miRNA;低温处理组共预测到329个已知miRNA,51个新miRNA。挖掘到80个在低温处理下显著差异表达的miRNA,包括55个上调和25个下调。GO基因功能注释和KEGG富集分析的结果表明,这些差异表达miRNA可能通过参与代谢通路、次生代谢物的生物合成、细胞膜的改变、信号转导和生物调节等响应低温胁迫。此外,还挖掘到25个可能与ICE1-CBFs-COR通路有关的miRNA。

    结论 

    借助高通量测序和生物信息学软件初步得到了低温胁迫下差异表达的赤桉miRNA,为进一步分析这些miRNA在赤桉低温胁迫中的分子功能提供一些参考。

    Abstract:
    Objective 

    To predict, mine and analyze the miRNAs involved in low temperature stress response of Eucalyptus camaldulensis, and lay a foundation for further study of the molecular network of regulating low temperature stress response.

    Method 

    Small RNAs were sequenced by high-throughput sequencing using the shoot tips of the tissue cultured seedlings of E. camaldulensis from the low temperature treatment group and the control group (CK). The miRBase21.0, Rfam14.1 and E. grandis genome were taken as reference databases. Bowtie, miREAP as well as miRDeep2 software were used for miRNA prediction. RNAfold was used to fold the secondary structure of the predicted miRNA precursors. psRNATarget was used to predict target genes. The miRNAs with differential expression were analyzed through DEGSeq package, and GO annotation and KEGG enrichment analysis were further performed.

    Result 

    A total of 392 known miRNAs and 97 novel miRNAs belonging to 54 families were predicted in E. camaldulensis. The 282 known miRNAs and 65 novel miRNAs were predicted in CK, while 329 known miRNAs and 51 novel miRNAs were predicted in the low temperature treatment group. At the same time, 80 significantly differentially expressed miRNAs in low temperature treatment group were mined, including 55 up-regulated and 25 down-regulated. The results of GO annotation and KEGG enrichment analysis indicated that these differentially expressed miRNAs might respond to low temperature stress by participating in metabolic pathways, biosynthesis of secondary metabolites, cell membrane changes, signal transduction, and biological regulation. In addition, we found 25 miRNAs that might be associated with the ICE1-CBFs-COR pathway.

    Conclusion 

    The differentially expressed miRNAs are initially obtained by high-throughput sequencing and bioinformatics software under low temperature stress, which can provide some references for further analysis of the molecular functions of these miRNAs in E. camaldulensis under low temperature stress.

  • 图  1   赤桉小RNA纯净序列的长度分布图

    Figure  1.   Length distribution of clean reads of small RNA in Eucalyptus camaldulensis

    图  2   赤桉miRNA维恩图

    Figure  2.   Venn diagram of miRNAs in Eucalyptus camaldulensis

    图  3   赤桉预测miRNA长度分布

    Figure  3.   The length distribution of the predicted miRNAs in Eucalyptus camaldulensis

    图  4   赤桉部分预测miRNA前体二级结构

    A: eca-miR164b-3p, B: eca-miR390b-5p, C: eca-miR395f-3p, D: eca-miR-n40, E: eca-miR-n45, F: eca-miR-n51; A~C: 已知miRNA, D~F: 新miRNA

    Figure  4.   The secondary structure of some predicted miRNAs in Eucalyptus camaldulensis

    A: eca-miR164b-3p, B: eca-miR390b-5p, C: eca-miR395f-3p, D: eca-miR-n40, E: eca-miR-n45, F: eca-miR-n51; A−C: Known miRNAs; D−F: Novel miRNAs

    图  5   赤桉不同长度miRNA首位碱基偏好性

    Figure  5.   Bias of the first base of different length miRNAs in Eucalyptus camaldulensis

    图  6   赤桉差异表达miRNA火山图

    Figure  6.   Volcanic map of differentially expressed miRNAs in Eucalyptus camaldulensis

    图  7   赤桉差异表达miRNA靶基因的GO注释

    Figure  7.   GO annotation of the target genes of differentially expressed miRNAs in Eucalyptus camaldulensis

    图  8   赤桉差异表达miRNA靶基因KEGG富集分析

    Figure  8.   KEGG pathway analysis of the target genes of differentially expressed miRNAs in Eucalyptus camaldulensis

    表  1   赤桉小RNA分类统计

    Table  1   Classification statistics of small RNAs in Eucalyptus camaldulensis

    种类
    Type
    对照 CK 低温处理 Low temperature treatment
    数量 Count 占比/% Percentage 数量 Count 占比/% Percentage
    核糖体RNA rRNA 4 787 452 30.27 3 769 781 26.08
    核内小RNA snRNA 55 640 0.35 32 977 0.23
    核仁小RNA snoRNA 33 402 0.21 24 413 0.17
    转运RNA tRNA 828 159 5.24 368 918 2.55
    其他 Other 10 108 761 63.93 10 259 988 70.97
    总计 Total 15 813 414 100.00 14 456 077 100.00
    下载: 导出CSV

    表  2   赤桉ICE1-CBFs-COR通路相关miRNA

    Table  2   The miRNAs associated with ICE1-CBFs-COR pathway in Eucalyptus camaldulensis

    miRNA1) 长度/nt
    Length
    序列(5′→3′)
    Sequence
    靶基因 
    Target gene 
    基因ID 
    Gene ID 
    蛋白质特征 
    Protein characteristic 
    eca-miR-n33 21 ACGGAAUUGUUCGAGCCGACU ICE1 Eucgr.G01938 转录因子 Transcription factor
    eca-miR171g-3p 19 UGAGCCGGACCAAUAUCAC MPK6 Eucgr.L00026 蛋白激酶 Protein kinase
    eca-miR171j-3p 22 GAUGAGCCGGACCAAUAUCACG MPK6 Eucgr.L00026 蛋白激酶 Protein kinase
    eca-miR5780b-5p 23 UCCAGUCUCUGAUCAAUUUUGAC OST1 Eucgr.E00345 蛋白激酶 Protein kinase
    eca-miR390b-5p 21 GGCGCUAUCCCUCCUGAGCUU OST1 Eucgr.I00977 蛋白激酶 Protein kinase
    eca-miR-n51↓ 21 GAAUGUCUCCAAUCUGCCCGA OST1 Eucgr.H04745 蛋白激酶 Protein kinase
    eca-miR-n60 20 AGCUCAUCCAUCUGUAAGAG OST1 Eucgr.D02135 蛋白激酶 Protein kinase
    BZR1 Eucgr.H01239 转录因子 Transcription factor
    eca-miR156m-3p 20 UGCUCUCUCUCUUCUGUCAA BZR1 Eucgr.F01541 转录因子 Transcription factor
    eca-miR156o-3p 20 UGCUCUCUAUCUUCUGUCAA SOC1 Eucgr.A02846 转录因子 Transcription factor
    eca-miR156j-5p 21 UUGACAGAAGAGAGAGAGCAC SOC1 Eucgr.D02427 转录因子 Transcription factor
    下载: 导出CSV
    续表 2 Continued table 2
    miRNA1) 长度/nt
    Length
    序列(5′→3′)
    Sequence
    靶基因 
    Target gene 
    基因ID 
    Gene ID 
    蛋白质特征 
    Protein characteristic 
    eca-miR159k-3p 19 UUUGGAUUGAAUGGAGUCU SOC1 Eucgr.K00208 转录因子 Transcription factor
    eca-miR94a-3p 21 UCCCGGGAACAGAAUCAUUAC EIN3 Eucgr.J00631 转录因子 Transcription factor
    eca-miR845c-3p 20 CCUACAAUUGGUAUCAGAGC PIF3 Eucgr.B01825 转录因子 Transcription factor
    eca-miR-n38 21 AGGUGAAUUCUUAUAGAUCCA PIF3 Eucgr.B01825 转录因子 Transcription factor
    eca-miR482f-3p 21 UCUUUCCUAUUCCUCCAUUCC SIZ1 Eucgr.B02470 E3苏素化连接酶 E3 SUMO ligase
    eca-miR23a-5p 25 UGAGAGUGAGUGUAGAGUAGGGAAU HOS1 Eucgr.E00402 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR-n2 21 GCUCCCCAAACUGACUACCAA HOS1 Eucgr.E00402 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR-n41↓ 22 UCGGAAGUCUUUGAGGGAGAGA EBF1 Eucgr.C01723 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR862a-5p↓ 21 AGUUUCCUUGAAGACAUCCAA EBF1 Eucgr.C01524 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR845a-5p 20 AGCUCUGAUACCAAUUGUUG EBF1 Eucgr.C02778 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR396a-3p 21 AAGCUCAAGAAAGCUGUGGGA EBF1 Eucgr.C02778 E3泛素化连接酶 E3 ubiquitin ligase
    eca-miR7782a-5p 19 AGUGGUAUCAGAGCAGGUU BTF3 Eucgr.K02308 NAC蛋白β亚基 β-subunit of NAC protein)
    eca-miR7782b-5p 23 AGUGGUAUCAGAGCAGGUCGUCG BTF3 Eucgr.K02308 NAC蛋白β亚基 β-subunit of NAC protein)
    eca-miR827b-5p 22 UUUUGUUGAUGGCCAUCUAAUC CAMTA3 Eucgr.H04783 转录激活子 Transcription activator
    eca-miR164b-3p 20 UGGAGAAGCAGGGCACGUAA PhyB Eucgr.A00380 光感受器 Photoreceptor
     1)“↓”表示在4 ℃低温处理24 h后显著下调
     1)“↓” shows significant down-regulation after 4 ℃ low temperature treatment for 24 h
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-11-10
  • 网络出版日期:  2023-05-17
  • 刊出日期:  2021-05-09

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