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Identification of MicroRNAs during Seed Germination and Its Response to PEG and NaCl Stresses

  • Corresponding author: Li Lu-bin, lilubin@126.com
  • Received Date: 2022-04-27
    Accepted Date: 2022-07-07
  • Objective To identify microRNAs (miRNAs) and reveal its expressional pattern in seed coat rupture stage of Moso bamboo seeds (Phyllostachys edulis) under different drought and salt stresses. Methods Polyethylene glycol (PEG6000) and NaCl were used to simulate drought and salinity stress, respectively. Small RNA libraries were separately built for Moso bamboo seeds germinated under H2O, 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl, and the seeds were all sampled at seed coat rupture stage. High throughput sequencing and bioinformatics analysis were used to explore the expressional pattern of miRNA. Results A total of 246 known miRNAs and 262 novel mature miRNAs were identified in this study. The most abundant miRNAs in seed coat rupture stage of Moso bamboo was miR166, followed by miR159, miR6478, miR319, etc. According to miRNA target prediction, MIR396 family owned the largest number of target genes, and ph02gene13935 (GAMYB) could to be regulated by 28 miRNAs of MIR159, MIR319 or MIR396; A total of 181 differentially expressed miRNAs (DEmiRNA) were identified in six comparison groups; Compared with control group, in 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl treatments, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a were differentially expressed respectively with highest expressional level in known miRNAs; The target genes of DEmiRNA were significantly enriched in different GO and KEGG pathways; Ten DEmiRNAs were verified by qPCR, and the overall trend of qPCR results was consistent with the sequencing data. Conclusion In seed coat rupture stage of moso bamboo, there exhibit high accumulation of known miRNAs such as miR159, miR6478, miR319 in all control and four treatment groups, which may play a conservative regulatory role in Moso bamboo seed germination. Compared with the control group, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a 1 are differentially expressed in 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl, respectively, which can respond to PEG or NaCl stress during seed coat rupture stage of Moso bamboo.
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Identification of MicroRNAs during Seed Germination and Its Response to PEG and NaCl Stresses

    Corresponding author: Li Lu-bin, lilubin@126.com
  • 1. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
  • 2. Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Floriculture Engineering Technology Research Centre, Beijing Botanical Garden, Beijing 100093, China
  • 3. College of Plant Science and Technology, Beijing Agriculture University, Beijing 100096, China

Abstract:  Objective To identify microRNAs (miRNAs) and reveal its expressional pattern in seed coat rupture stage of Moso bamboo seeds (Phyllostachys edulis) under different drought and salt stresses. Methods Polyethylene glycol (PEG6000) and NaCl were used to simulate drought and salinity stress, respectively. Small RNA libraries were separately built for Moso bamboo seeds germinated under H2O, 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl, and the seeds were all sampled at seed coat rupture stage. High throughput sequencing and bioinformatics analysis were used to explore the expressional pattern of miRNA. Results A total of 246 known miRNAs and 262 novel mature miRNAs were identified in this study. The most abundant miRNAs in seed coat rupture stage of Moso bamboo was miR166, followed by miR159, miR6478, miR319, etc. According to miRNA target prediction, MIR396 family owned the largest number of target genes, and ph02gene13935 (GAMYB) could to be regulated by 28 miRNAs of MIR159, MIR319 or MIR396; A total of 181 differentially expressed miRNAs (DEmiRNA) were identified in six comparison groups; Compared with control group, in 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl treatments, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a were differentially expressed respectively with highest expressional level in known miRNAs; The target genes of DEmiRNA were significantly enriched in different GO and KEGG pathways; Ten DEmiRNAs were verified by qPCR, and the overall trend of qPCR results was consistent with the sequencing data. Conclusion In seed coat rupture stage of moso bamboo, there exhibit high accumulation of known miRNAs such as miR159, miR6478, miR319 in all control and four treatment groups, which may play a conservative regulatory role in Moso bamboo seed germination. Compared with the control group, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a 1 are differentially expressed in 10% PEG, 15% PEG, 50 mmol·L−1 NaCl and 100 mmol·L−1 NaCl, respectively, which can respond to PEG or NaCl stress during seed coat rupture stage of Moso bamboo.

  • 毛竹(Phyllostachys edulis (Carr.) H. de Lehaie)属于禾本科(Gramineae)竹亚科(Bambusoideae)刚竹属(Phyllostachys Sieb. Et Zucc.),是一种多年生木本散生竹,广泛分布在46° N以南地区,是我国种植面积最广的竹种之一,具有重要的经济、生态和文化价值[1-2]。毛竹开花后产生典型颖果类果实,薄而干的膜质果皮与种皮紧密贴合,在种子生物学和苗木培育上毛竹颖果通常被称为种子[3]。毛竹种子萌发是实生苗造林的基础,通过毛竹种子萌发进行实生苗造林能降低运输成本,便于移栽,有利于提高造林整齐度、成活率和遗传多样性,延长竹林高产时间,且实生苗竹林萌笋多、竹鞭生长较快、竹材利用率更高[4-5],因此开展毛竹种子萌发的研究对于竹类资源的培育和应用等具有重要意义。

    随着气候变化的加剧,干旱和由土壤初级或次级盐碱化造成的盐胁迫已经成为全球性的问题,并对种子萌发、植物生长等造成严重影响[6-7]。干旱和盐胁迫抑制了毛竹种子的萌发,进而影响了毛竹实生苗的生长,限制了通过毛竹种子萌发进行实生苗培育和应用[8]。目前研究者逐步开展了干旱或盐胁迫对毛竹种子萌发率、种子活力、种子寿命和萌发后幼苗生长等生理方面的研究[9-12],但相关的分子调控机制却鲜有报道。

    MicroRNA(miRNA)是一类长度约为21~24 nt的小分子非编码RNA,能够通过互补配对抑制或降解靶基因进行转录后调控[13]。miRNA广泛参与了植物的生长发育[13-15],在种子萌发[16-18]和非生物胁迫响应[19]中具有重要的调控作用。在竹类植物中,研究者已经在组学水平开展了关于毛竹叶片[20]、花发育[21]、雄蕊发育[22]、茎秆快速生长[23]和竹笋发育[24]等的miRNA研究,但毛竹种子中miRNA的表达、及其在干旱和盐胁迫下萌发时的调控仍是未知的,因此本研究通过small RNA测序对毛竹种子露白阶段的miRNA进行系统分析,并探究其在不同PEG或NaCl胁迫下萌发时的表达模式,挖掘毛竹种子萌发阶段调控干旱或盐胁迫抗性潜在的关键miRNA,以期为后续的研究提供基础和参考。

    • 试验所用毛竹种子于2020年9月收集自广西壮族自治区桂林市灵川县,剥除外稃后挑选大小均一,颗粒饱满,色泽明亮的完整种子备用(图1-a)。种子的千粒质量为21.85 g ± 0.04 g,含水量为12.05% ± 0.35%。种子表面消毒后在培养皿中使用纸床法进行萌发[11]。聚乙二醇(PEG6000)是一种亲水性大分子物质,是模拟植物干旱胁迫的常用试剂之一,本研究使用PEG6000模拟干旱胁迫环境,同时使用氯化钠(NaCl)模拟盐胁迫。使用完全随机实验设计将种子分为5组(A-E),A组为对照组,B组、C组模拟干旱胁迫,D组、E组模拟盐胁迫。参考前期预实验的结果,分别在培养皿中加入H2O(A)、10% PEG(B)、15% PEG(C)、50 mmol·L−1 NaCl(D)和100 mmol·L−1 NaCl(E),每组萌发200粒种子,并重复3次。每天观察并记录种子萌发状态,在第4天选择生长状态一致且均到达种皮破裂阶段的种子(图1-b)。对于每个处理,在3个重复中各取5粒种子并将15粒种子混合取样后用于建库。取样后使用液氮速冻并置于−80 ℃保存。

      Figure 1.  Dry seeds (a) and sampled seeds germinated at seed coat rupture stage (b) of Moso bamboo (Phyllostachys edulis)

    • 使用植物多糖多酚RNA提取试剂盒(DP441,TIANGEN)提取样品总RNA。在建库时首先根据small RNA的结构特点将总RNA中small RNA加接头并进行纯化,随后进行反转录、PCR扩增和聚丙烯酰胺凝胶电泳,切胶回收目的条带即得到small RNA的cDNA文库。文库有效浓度合格后进行测序,使用Illumina高通量测序平台NovaSeq 6000进行单端测序,测序长度为50 bp。对获得的原始数据首先进行过滤,去除接头序列、低质量序列、含N比例>10% 的序列、5’接头污染的序列、没有3’接头序列和插入片段的序列;含连续的/T/G/C的序列;以及长度异常的序列。

    • 首先将过滤后的数据比对到miRbase数据库(V22)鉴定已知miRNA[25];将未比对至miRBase的数据比对至Rfam数据库中的ncRNA 序列(rRNA,tRNA,snRNA,snoRNA) 和参考基因组重复序列[26];随后对于上述均未比对中的clean reads,使用mirEvo和miRdeep2进行新miRNA预测[27-28]。对于已知和新的miRNA序列,将其前体序列比对至miRBase数据库进行microRNA家族的鉴定[25]

    • 为了探究毛竹种子萌发露白阶段miRNA的表达,首先统计各样本中已知和新miRNA的readscount,进行TPM(Transcripts per million)归一化转化(TPM = readscount × 107/library size)。随后使用edgeR软件进行差异表达分析,以鉴定露白阶段毛竹种子响应干旱或盐胁迫的miRNA[29],差异表达miRNA筛选条件为|log2foldchange|≥1且p<0.05。

    • 为了探究毛竹萌发种子中miRNA的潜在功能,对本研究中已知和新miRNA的靶基因进行预测,并进行靶基因的KEGG和GO功能富集;同时通过对差异表达miRNA靶基因的功能富集,进一步分析毛竹萌发种子中miRNA响应干旱或盐胁迫的潜在通路或途径。使用软件TargetFinder和psRNAtarget进行miRNA靶基因的预测,并对预测结果取交集[30-31]。使用OmicShare在线平台((https://www.omicshare.com/tools))进行靶基因KEGG和GO功能富集,富集显著性阈值为p<0.05[32-33]

    • 总RNA提取方法同1.2,反转录使用miRcute增强型miRNA cDNA第一链合成试剂盒(KR211,TIANGEN),荧光定量检测使用增强型荧光定量试剂盒(FP411,TIANGEN)。荧光定量正向引物如表1所示,反向引物使用试剂盒通用引物,内参引物为U6[34]。荧光定量检测仪器为qTOWER3.0 (Analytik jene),每个基因进行三次生物学重复和三次技术重复,采用2−△△CT方法计算相对表达水平[35]

      miRNA编号     
      miRNA ID     
      引物方向
      Primer direction
      引物序列       
      Sequence (5′-3′)       
      phe-miR3630-3p F UGUGGGAAUCUCUCUGAUGCUU
      phe-miR159a.1 F UUUGGAUUGAAGGGAGCUCUG
      phe-miR168a-5p F UCGCUUGGUGCAGAUCGGGAC
      phe-miR171e-5p F UGUUGGCUCGGCUCACUCAGA
      phe-miR396e-5p F UCCACAGGCUUUCUUGAACUG
      phe-miR894 F CGUUUCACGUCGGGUUCACC
      phe-miR6478 F CCGACCUUAGCUCAGUUGGUG
      novel_14 F CUGAGAGGUAGAGAACGGGAUG
      novel_311 F UCGUGUGCUGAACUUUAUUGA
      novel_5 F AUCUCAGCUGUUGAUUCCAUGAUC
      u6 F GGACATCCGATAAAATTGGAACGATACAG

      Table 1.  Primers used for quantitative real time PCR of Differential expressed miRNA

    2.   结果与分析
    • small RNA测序结果表明,每个文库产出数据约0.9~1.8 GB,过滤后每个样本中分别有12 984 751条(A,H2O)、14 982 423条(B,10% PEG)、13 401 719条(C,15% PEG)、13 995 450条(D,50 mmol·L−1 NaCl)和11 452 796条(E,100 mmol·L−1 NaCl)cleanReads(表2)。cleanreads的Q20值约为98%,Q30值均≥93%,与毛竹参考基因组的比对率≥88.42%,表明测序数据质量较好。具体数据产出及比对情况如表2所示。

      样品名称
      Sample
      原始读序
      Raw Reads
      Q20值
      Q20/%
      Q30值
      Q30/%
      GC含量
      GC Content/%
      过滤后读序数
      Clean Reads
      比对sRNA数量
      Mapped sRNA
      A29 379 98398945412 984 75111 504 336 (88.60%)
      B37 101 40398935414 982 42313 326 206 (88.95%)
      C32 426 28698945413 401 71911 915 649 (88.91%)
      D33 041 22298945413 995 45012 374 157 (88.42%)
      E19 605 42198935311 452 79610 198 110 (89.04%)

      Table 2.  Statistics of sequence data in small RNA libraries

    • 对small RNA数据库中miRNA进行比对和预测,结果显示每个样品中有0.08%~0.11%的序列被注释为已知miRNA,0.1%~0.11%的序列被预测为novel miRNA。对miRNA长度进行统计发现本研究成熟miRNA长度主要分布在21 nt和24 nt(图2)。本研究共鉴定miRNA成熟体序列508条,前体序列845条,其中已知miRNA成熟体序列数量为246,前体序列数量为574,鉴定出novel miRNA成熟体数量共有262条,前体序列数量为271。

      Figure 2.  Length distribution of known and novel miRNA

      对miRNA前体序列进行家族分析,共鉴定到包含514条前体序列的45个家族,包括MIR159、MIR166、MIR156、MIR408、MIR399、MIR530等,其中有10条新miRNA前体序列也被鉴定属于已知家族,如novel_57属于MIR408,novel_14和novel_66属于MIR169_2。统计每个家族包含的前体成员数目表明,本研究毛竹萌发种子中最大的miRNA家族为MIR159,包含54个成员,其次为MIR166(48)、MIR156(46)、MIR167_1(37)、MIR396(32)等(详细数据未列出)。

    • 毛竹萌发种子中表达量前5的已知miRNA分别为phe-miR166a-3p、phe-miR159a.1、phe-miR319a-3p.2-3p、phe-miR6478、phe-miR156a-5p,表达量前5的新miRNA分别为novel_1、novel_199、novel_311、novel_241、novel_2,其中novel_1在5个样本所有miRNA中表达量均为最高。表达量前10的新miRNA折叠最小自由能值为−187.0~−13.8 KJ·mol−1表3表4)。

      同源序列名称 
      Name 
      序列  
      Sequence  
      表达量 (TPM Counts)
      A B C D E
      phe-miR166a-3p UCGGACCAGGCUUCAUUCCCC 74 864 57 593 64 907 73 677 66 116
      phe-miR159a.1 UUUGGAUUGAAGGGAGCUCUG 54 736 42 178 41 405 45 018 31 525
      phr-miR319a-3p.2-3p UUGGACUGAAGGGUGCUCCC 27 992 25 959 28 454 25 962 27 126
      phe-miR6478 CCGACCUUAGCUCAGUUGGUG 25 651 48 135 24 454 21 842 28 992
      phe-miR156a-5p UGACAGAAGAGAGUGAGCAC 17 694 16 099 15 274 23 739 28 492
      phe-miR167d-5p UGAAGCUGCCAGCAUGAUCUG 17 288 28 696 19 426 18 345 16 262
      phe-miR396e-5p CGUUUCACGUCGGGUUCACC 12 919 15 012 12 456 10 017 18 662
      phe-miR894 UCCACAGGCUUUCUUGAACUG 11 796 17 105 14 627 10 106 5 332
      phe-miR166k-3p UCGGACCAGGCUUCAAUCCCU 8 925 6 641 5 866 11 706 14 463
      phe-miR319a UUGGACUGAAGGGAGCUCCCU 8 707 6 761 6 437 7 528 7 898

      Table 3.  Sequences abundance and homologues of top ten predicted conserved miRNA in five libraries

      名称 
      Name 
      序列  
      Sequence  
      表达量 (TPM counts)最小折叠自由能
      Hairpin mfe
      ABCDE
      novel_1 CUUCACUCUUCAGACUUUGAG 174 786 172 858 173 542 135 291 189 416 −46.6
      novel_199 CGAUUAGUCGGCAAGUCGGGCGACU 17 413 23 705 16 760 12 951 15 796 −44.7
      novel_311 UCGUGUGCUGAACUUUAUUGA 15 135 31 513 36 072 22 702 16 496 −13.4
      novel_241 UUACCGGUCUUUAAACUUUUUAU 14 792 17 990 26 054 17 486 11 764 −175.9
      novel_2 CCGGUCUAUAGAUUUUCAAAGUG 8 332 10 464 14 170 13 544 9 664 −115.7
      novel_3 UCUCGGAUCAGGCUUCGUUCC 7 084 5 594 5 180 5 424 10 664 −39.4
      novel_287 AUCGCGAUCUUGACAACCUUGACU 5 586 6 600 8 761 9 543 7 165 −27.5
      novel_5 AUCUCAGCUGUUGAUUCCAUGAUC 4 993 3 381 4 723 6 698 2 299 −32.8
      novel_9 CCUUCGAUCGUAUGAUGUGUCACG 4 494 4 306 4 457 4 742 2 799 −98.6
      novel_87 UGUAGGAUCUCUGAUGACGGU 3 776 5 031 5 371 3 082 6 765 −187.0

      Table 4.  Sequences abundance and homologues of top ten predicted novel miRNA in five libraries

      对每个样本中表达水平前10的已知miRNA和新miRNA的进行TPM值统计,结果表明排名前10的已知miRNA在每个样本总miRNA中的读序丰度(total miRNA reads)占45%~51.8%,排名前10的新miRNA在每个样本总miRNA中的读序丰度中占23.1%~29.5%。PEG和NaCl胁迫下这些miRNA在毛竹露白阶段种子表达水平均为最高,推测其在毛竹种子萌发的调控中可能具有保守的重要作用。

    • 通过差异表达分析,本研究共鉴定181个差异miRNA,其中有84个上调表达,97个下调表达(图3 -a)。与对照组(A)相比,10% PEG(B)、15% PEG(C)、50 mmol·L−1 NaCl(D)、100 mmol·L−1 NaCl(E)4种胁迫下分别有20、26、41和24个差异表达miRNA(DEmiRNA),此外在B-C和D-E比较组分别有16和54个DEmiRNA(图3 -b)。

      Figure 3.  Statistic of DEmiRNAs

      依据miRNA的差异表达情况和表达水平,对本研究具有高丰度且显著差异表达的miRNA进行聚焦。与对照组相比,10% PEG、15% PEG、50 mmol·L−1 NaCl、100 mmol·L−1 NaCl中表达水平最高的DEmiRNA分别为novel_14、novel_311、novel_14、phe-miR159a.1,表达水平最高的已知DEmiRNA分别为phe-miR171e-5p、phe-miR3630-3p、phe-miR171e-5p和phe-miR159a.1,这些miRNA在毛竹种子萌发露白阶段的大量积累,同时在PEG或NaCl胁迫下差异表达,可能参与了种子萌发露白阶段miRNA对干旱或盐胁迫的调控。

    • 通过Targetfinder预测,共获得505个miRNA的31 729个靶基因,psRNAtarget预测结果中501个miRNA可以靶向22 215个基因。对两个软件的预测结果取交集后共有17 666对miRNA-靶基因关系,包含489个miRNA 和8 812个靶基因。预测的miRNA-靶基因关系中,novel_153的靶基因数目最多,有188个靶基因,其次是phe-miR396b(167个)、phe-miR396e-5p(161个)、phe-miR396h(141个)、phe-miR164b(138个)。对同一miRNA家族成员的靶基因数目进行统计,靶基因数目前十的家族中MIR396家族靶基因数目最多。此外,根据预测结果8 812个靶基因中每个基因能够受到1~28个miRNA靶向,表明在毛竹种子萌发过程存在复杂的miRNA调控网络。具有miRNA-gene关系最多的基因中,排名前20的基因有7个属于GAMYBGibberellin MYB)家族,12个属于SPLSquamosa promoter-binding-like)家族,推测GAMYBSPL基因家族在毛竹萌发种子miRNA调控网络中可能具有重要意义。

    • 根据靶基因预测结果,A-B、A-C、A-D、A-E、B-C、D-E六个比较组差异表达miRNA分别有609、548、1 011、812、460和1 866个靶基因。GO富集结果显示6个组合差异表达miRNA的靶基因分别显著富集在520(A-B)、516(A-C)、620(A-D)、446(A-E)、483(B-C)、610(D-E)个条目中,总计1 989个涉及细胞成分、生物学过程和分子功能的条目,推测miRNA可以通过这些靶基因广泛地参与不同的生物学过程或分子功能等调控。表5为不同比较组GO富集前10的生物学过程条目,与对照组相比,10% PEG(B1)、15% PEG(C1)、50 mmol·L−1 NaCl(D1)、100 mmol·L−1 NaCl(E1)胁迫下DEmiRNA靶基因富集最显著的生物学过程分别为跨膜转运(GO:0055085)、DNA复制正向调控(GO:0045740)、发育过程(GO:0032502)、海藻糖生物合成(GO:0005992),推测这些生物学过程对于miRNA调控PEG和NaCl胁迫下的种子萌发具有重要作用。

      比较组
      Comparison Groups
      GO编号
      GO ID
      GO条目  
      GO Term  
      基因数量
      Gene Number
      P
      P-Value
      A-B GO:0055085 跨膜转运(transmembrane transport) 233 2.49E-11
      A-B GO:0098656 阴离子跨膜转运(anion transmembrane transport) 49 1.12E-07
      A-B GO:0006820 阴离子转运(anion transport) 75 1.48E-07
      A-B GO:1903825 有机酸跨膜转运(organic acid transmembrane transport) 41 1.83E-07
      A-B GO:1905039 羧酸跨膜转运(carboxylic acid transmembrane transport) 41 1.83E-07
      A-B GO:0003333 氨基酸跨膜转运(amino acid transmembrane transport) 38 2.49E-07
      A-B GO:0006865 氨基酸转运(amino acid transport) 38 6.65E-07
      A-B GO:0006811 离子转运(ion transport) 169 6.90E-07
      A-B GO:0006885 PH调节(regulation of pH) 25 1.14E-06
      A-B GO:0055067 单价无机阳离子稳态(monovalent inorganic cation homeostasis) 26 1.21E-06
      A-C GO:0045740 DNA复制的正调控positive regulation of DNA replication 7 4.04E-07
      A-C GO:0034644 细胞对紫外线的反应(cellular response to UV) 6 1.23E-06
      A-C GO:0019985 跨损伤合成(translesion synthesis) 7 1.40E-06
      A-C GO:0000731 参与DNA修复的DNA合成(DNA synthesis involved in DNA repair) 8 1.57E-06
      A-C GO:0051052 DNA代谢过程的调节(regulation of DNA metabolic process) 14 1.99E-06
      A-C GO:0072593 活性氧代谢过程(reactive oxygen species metabolic process) 36 2.97E-06
      A-C GO:0055074 钙离子稳态(calcium ion homeostasis) 5 3.06E-06
      A-C GO:0071482 细胞对光刺激的反应(cellular response to light stimulus) 13 3.24E-06
      A-C GO:0006979 对氧化应激的反应(response to oxidative stress) 41 5.55E-06
      A-C GO:0051054 DNA代谢的正调控(positive regulation of DNA metabolic process) 7 6.17E-06
      A-D GO:0032502 发育过程(developmental process) 288 9.35E-09
      A-D GO:0080022 初生根发育(primary root development) 11 6.87E-08
      A-D GO:0044767 单一有机体发育过程(single-organism developmental process) 266 1.59E-06
      A-D GO:0016567 蛋白质泛素化(protein ubiquitination) 104 2.24E-06
      A-D GO:0009657 质体组织(plastid organization) 46 2.80E-06
      A-D GO:0070647 通过小蛋白结合或去除来修饰蛋白质
      (protein modification by small protein conjugation or removal)
      128 3.51E-06
      A-D GO:0046271 苯丙烷代谢生物过程(phenylpropanoid catabolic process) 21 6.65E-06
      A-D GO:0046274 木质素分解代谢过程(lignin catabolic process) 21 6.65E-06
      A-E GO:0005992 海藻糖生物合成过程(trehalose biosynthetic process) 19 1.87E-06
      A-E GO:0071478 细胞对辐射的响应(cellular response to radiation) 22 2.64E-06
      A-E GO:0071214 细胞对非生物胁迫的响应(cellular response to abiotic stimulus) 22 4.18E-06
      A-E GO:0044767 单一有机体发育过程(single-organism developmental process) 214 8.24E-06
      A-E GO:0032502 发育过程(developmental process) 220 9.83E-06
      B-C GO:0055085 跨膜转运(transmembrane transport) 230 1.21E-14
      B-C GO:0006811 离子转运(ion transport) 173 1.54E-10
      B-C GO:0006820 阴离子转运(anion transport) 78 2.58E-10
      B-C GO:1903825 有机酸跨膜转运(organic acid transmembrane transport) 42 4.73E-09
      B-C GO:1905039 羧酸跨膜转运(carboxylic acid transmembrane transport) 42 4.73E-09
      B-C GO:0003333 氨基酸跨膜转运(amino acid transmembrane transport) 39 7.16E-09
      B-C GO:0006865 氨基酸转运(amino acid transport) 39 2.11E-08
      B-C GO:0006810 转运(transport) 424 2.33E-08
      B-C GO:0051234 定位的建成(establishment of localization) 427 5.56E-08
      B-C GO:0051179 定位(localization) 436 9.61E-08
      D-E GO:0080022 初生根发育(primary root development) 12 1.52E-09
      D-E GO:0006642 甘油三酯动员(triglyceride mobilization) 7 7.18E-06
      D-E GO:0009658 叶绿体组织(chloroplast organization) 35 7.27E-06
      D-E GO:0048506 分生组织相变时间调控(regulation of timing of meristematic phase transition) 9 8.33E-06

      Table 5.  Top 10 significantly enriched GO terms for DEmiRNA target genes in different comparision groups

      KEGG富集结果如表6所示,苯丙烷生物合成途径在A-B、A-C、A-E三个组合中被显著富集,推测苯丙烷生物合成途径对于毛竹种子萌发露白阶段miRNA响应PEG和NaCl胁迫可能具有重要意义,但毛竹种子萌发过程miRNA与苯丙烷合成途径关键基因的靶向关系,及其是否共同调控干旱或盐胁迫下的种子萌发需要进一步的研究和验证。此外,果糖和甘露糖代谢、玉米素生物合成途径仅在盐胁迫下(A-D和A-E)显著富集,油菜素内酯生物合成和脂肪酸降解途径仅在A-B中显著富集,表明在毛竹种子露白阶段miRNA响应10% PEG、15% PEG、50 mmol·L−1 NaCl、100 mmol·L−1 NaCl胁迫的调控通路也存在差异。

      比较组
      Comparision Groups
      KEGG途径  
      KEGG Pathway  
      途径ID
      Pathway ID
      基因数量
      Gene Number
      P
      P-Value
      A-B 糖基磷脂酰肌醇(GPI)-锚定生物合成
      (Glycosylphosphatidylinositol(GPI)-anchor biosynthesis)
      ko00563 10 0.0007093
      脂肪酸延伸(Fatty acid elongation) ko00062 18 0.0019812
      2-羰基甲酸代谢(2-Oxocarboxylic acid metabolism) ko01210 22 0.0027901
      氨基酸生物合成(Biosynthesis of amino acids) ko01230 69 0.0034177
      精氨酸生物合成(Arginine biosynthesis) ko00220 14 0.0042814
      单环菌素生物合成(Monobactam biosynthesis) ko00261 6 0.0076043
      亚油酸代谢(Linoleic acid metabolism) ko00591 7 0.0077439
      苯丙烷生物合成(Phenylpropanoid biosynthesis) ko00940 48 0.0107222
      黄酮和黄酮醇生物合成(Flavone and flavonol biosynthesis) ko00944 3 0.0135929
      真核生物中的核糖体生物发生(Ribosome biogenesis in eukaryotes) ko03008 28 0.0150491
      植物昼夜节律(Circadian rhythm - plant) ko04712 14 0.0202961
      油菜素内酯生物合成(Brassinosteroid biosynthesis) ko00905 5 0.0312562
      脂肪酸降解(Fatty acid degradation) ko00071 13 0.0483513
      A-C 苯丙烷生物合成(Phenylpropanoid biosynthesis) ko00940 38 7.98E-06
      蛋白质输出(Protein export) ko03060 16 3.14E-04
      植物昼夜节律(Circadian rhythm - plant) ko04712 11 1.99E-03
      DNA复制(DNA replication) ko03030 14 3.38E-03
      RNA聚合酶(RNA polymerase) ko03020 8 2.56E-02
      A-D 磷酸肌醇代谢(Inositol phosphate metabolism) ko00562 39 1.93E-06
      异喹啉生物碱的生物合成(Isoquinoline alkaloid biosynthesis) ko00950 16 1.59E-04
      果糖和甘露糖代谢(Fructose and mannose metabolism) ko00051 33 3.64E-03
      自噬-其他真核生物(Autophagy - other eukaryotes) ko04136 15 4.63E-03
      类胡萝卜素生物合成(Carotenoid biosynthesis) ko00906 16 9.62E-03
      酮体的合成与降解(Synthesis and degradation of ketone bodies) ko00072 5 2.43E-02
      磷脂酰肌醇信号系统(Phosphatidylinositol signaling system) ko04070 28 2.50E-02
      A-D 维生素B6代谢(Vitamin B6 metabolism) ko00750 8 2.85E-02
      赖氨酸降解(Lysine degradation) ko00310 17 2.89E-02
      玉米素生物合成(Zeatin biosynthesis) ko00908 10 3.33E-02
      醚脂质代谢(Ether lipid metabolism) ko00565 13 4.62E-02
      A-E 醚脂质代谢(Ether lipid metabolism) ko00565 16 0.0002059
      非同源末端连接(Non-homologous end-joining) ko03450 7 0.0005795
      淀粉和蔗糖代谢(Starch and sucrose metabolism) ko00500 51 0.0008573
      类胡萝卜素生物合成(Carotenoid biosynthesis) ko00906 15 0.0017051
      甘油磷脂代谢(Glycerophospholipid metabolism) ko00564 35 0.0038386
      苯丙烷生物合成(Phenylpropanoid biosynthesis) ko00940 55 0.0044864
      玉米素生物合成(Zeatin biosynthesis) ko00908 9 0.0162331
      果糖和甘露糖代谢(Fructose and mannose metabolism) ko00051 23 0.0360763
      B-C 脂肪酸延伸(Fatty acid elongation) ko00062 18 0.0008336
      黄酮合黄酮醇生物合成(Flavone and flavonol biosynthesis) ko00944 3 0.0109971
      亚油酸代谢(Linoleic acid metabolism) ko00591 6 0.0198863
      其他聚糖降解(Other glycan degradation) ko00511 7 0.0487309
      2-羰基甲酸代谢(2-Oxocarboxylic acid metabolism) ko01210 17 0.0411353
      D-E 磷酸肌醇代谢(Inositol phosphate metabolism) ko00562 38 1.79E-06
      磷脂酰肌醇信号系统(Phosphatidylinositol signaling system) ko04070 31 2.35E-03
      异喹啉生物碱的生物合成(Isoquinoline alkaloid biosynthesis) ko00950 12 9.65E-03
      膦酸酯和次膦酸酯代谢(Phosphonate and phosphinate metabolism) ko00440 6 8.31E-03
      鞘糖脂生物合成-乳糖和新乳糖系列
      (Glycosphingolipid biosynthesis - lacto and neolacto series)
      ko00601 2 1.27E-02
      RNA转运(RNA transport) ko03013 59 1.57E-02
      组氨酸代谢(Histidine metabolism) ko00340 9 2.64E-02
      氮代谢(Nitrogen metabolism) ko00910 15 2.09E-02
      β-丙氨酸代谢(beta-Alanine metabolism) ko00410 17 2.67E-02
      叶酸合成(Folate biosynthesis) ko00790 9 4.69E-02

      Table 6.  Significantly enriched KEGG pathways for DEmiRNA target genes in different comparison groups

    • 随机选取10个在不同样本间存在差异表达的miRNA进行qPCR验证,miRNA的表达量在不同处理组之间被上调或下调,例如phe-miR6478在10% PEG胁迫下比对照组上调1.45倍,phe-miR3630-3p在100 mmol·L−1 NaCl胁迫下上调1.33倍,phe-miR171e-5p在10% PEG、15% PEG、50 mmol·L−1 NaCl、100 mmol·L−1 NaCl胁迫下分别下调0.23、0.50、0.56和0.61倍(图4)。miRNA荧光定量结果和small RNA测序数据在表达趋势上整体一致,表明测序数据的可靠性。

      Figure 4.  qPCR validation for DEmiRNAs

    3.   讨论
    • 在本研究中miR166、miR159、miR319、miR156、miR396、miR167、miR168、miR894、miR160等在对照组和不同浓度的PEG和NaCl胁迫条件下均具有较高表达水平,推测这些miRNA可能在毛竹种子萌发中有重要作用;其中miR159[36]、miR156[37]、miR396[38]、miR160[17]已经被证明参与了种子萌发调控。在大麦(Hordeum vulgare L.)萌发种子中,miR156、miR166、miR167、miR168等也具有高表达水平,同时miR5071被大量积累,而在本研究中未检测到miR5071的表达,表明不同物种间miRNA对种子萌发的调控具有特异性[39]。在已有研究中miR402[40]、miR163[41]和miR417[42]被证明能够参与种子萌发期的干旱或盐胁迫响应,而在本研究对照和处理组中均未检测到miR402、miR163和miR417的表达,推测这些miRNA在本研究中可能不是响应干旱或盐胁迫的关键miRNA,其分布和表达可能与物种有关。

      与对照组相比,本研究中miRNA主要在单一胁迫下显著差异表达,仅有2个miRNA能够同时响应两种胁迫,表明miRNA在10% PEG、15% PEG、50 mmol·L−1 NaCl、100 mmol·L−1 NaCl四种胁迫下的调控存在差异。本研究丰度较高且显著差异表达的miRNA中,phe-miR171e-5p能够响应10% PEG和50 mmol·L−1 NaCl胁迫,phe-miR3630-3p在15% PEG胁迫下显著上调表达,phe-miR159a.1在100 mmol·L−1 NaCl胁迫下差异表达,推测这3个miRNA对于毛竹种子萌发响应PEG或NaCl胁迫可能具有重要意义。

      前人的研究中,PEG胁迫下甘蓝型油菜(Brassica napus L.)萌发种子中的miR171比对照组显著下调表达[43];此外在拟南芥(Arabidopsis thaliana (L.) Heynh.)中过表达桑树(Morus alba L.)mno-miR171可提高转基因植株在NaCl和甘露醇胁迫下的种子发芽率[44],这与本研究phe-miR171e-5p能够响应PEG和NaCl胁迫是一致的。miR171主要通过靶向GRAS家族基因影响GA和ABA信号通路,参与胚胎发生潜能的维持[45-47]、花药发育、芽分枝和复叶形态的调控[48]、顶端优势的调控[49]等,其是否通过GRAS家族参与毛竹种子萌发过程中对干旱和盐胁迫的调控需要进一步研究。在苜蓿(Medicago sativa L.)中,miR3630被鉴定为响应干旱胁迫的miRNA[50],本研究中phe-miR3630-3p在15% PEG胁迫下显著上调表达,但目前关于miR3630的研究较少,本研究为miR3630响应干旱胁迫提供了新的证据。此外,phe-miR159a.1在A-E比较组显著下调。拟南芥中miR159通过介导GAMYB家族MYB101MYB33的转录本切割,参与种子萌发过程糊粉层细胞的细胞程序性死亡[18],推测miR159对于100 mmol·L−1 NaCl胁迫下毛竹种子的萌发也可能具有重要的调控作用。

    4.   结论
    • 本研究首次系统鉴定了毛竹萌发种子中的miRNA,并对其在PEG和NaCl胁迫下毛竹萌发露白阶段种子中的表达模式进行研究,探究了响应不同PEG和NaCl胁迫的差异表达miRNA。下一步将对本研究中涉及的重要miRNA进行靶基因的验证,并对其调控机制和调控功能进行深入研究和探讨。

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