Mining and analysis of miRNAs from Eucalyptus camaldulensis under low temperature stress based on high-throughput sequencing
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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:ObjectiveTo 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.
MethodSmall 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.
ResultA 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.
ConclusionThe 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.
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赤桉Eucalyptus camaldulensis是桃金娘科Myrtaceae桉属Eucalyptus比较广泛栽培的树种,原产于澳大利亚,与其他桉树相比,赤桉具有高抗寒性,是研究桉树抗寒分子机理的理想材料[1]。
miRNA是由microRNA基因所编码、II型RNA聚合酶(Pol II)和DCL1酶(Dicer-like 1)等加工形成的长度约18~24 nt的单链非编码RNA[2]。研究证实,miRNA通过对靶基因的剪切或抑制来实现转录后水平上的负调控,在植物的生长发育、形态建成和信号转导以及逆境胁迫响应中都发挥着重要的调控作用[3-5]。
低温是一种常见的环境因子,它限制植物只能在特定的地理位置和季节生长。植物为适应低温胁迫,在生理生化和分子水平上形成了一系列的抵御机制,包括瞬时Ca2+和脱落酸(ABA)含量的提高、植物体液组成的改变、抗氧化物含量的增加,以及一些渗透调节物质的集聚[6]。最早在拟南芥Arabidopsis thaliana中发现低温胁迫下miRNA的表达丰度会发生变化[7],之后许多研究报道了木本植物低温胁迫下差异表达的miRNA[4,8]。华南农业大学桉树育种课题组前期对赤桉耐寒关键性转录因子ICE1(Inducer of CBF expression 1)[9]、SIZ1(SAP and Miz)苏素化[10]和HOS1(High expression of osmotically responsive gene 1)泛素化[11]通路进行了一些研究,但在赤桉ICE1通路及其上下游中是否有miRNA参与调节,仍然未知。本研究拟通过高通量测序和生物信息学软件相结合的方式,挖掘、分析涉及低温胁迫应答的赤桉miRNA,以进一步弄清赤桉低温胁迫应答的完整分子调控网络,并为后续的桉树抗寒分子育种提供一些理论依据和技术支撑。
1. 材料与方法
1.1 植物材料与处理
赤桉无性系CV103无菌组培苗取自广东省森林植物种质创新与利用重点实验室。在MS培养基(pH=5.8)附加0.5 mg·L−1 6-BA、0.1 mg·L−1 NAA、30 g·L−1蔗糖和6 g·L−1琼脂,并接种赤桉无菌苗,25 ℃条件下16 h光照、8 h黑暗光周期培养3~4个月。低温处理组于4 ℃低温条件下处理24 h,对照组(CK)不进行处理。分别选取长势健壮无菌苗的茎尖组织,液氮速冻后磨碎,用Trizol(上海生工生物工程技术服务有限公司)提取总RNA。
1.2 Small RNA文库构建与测序
用Qubit 2.0 (Thermo, 美国)对总RNA进行定量,使用T4 RNA连接酶(NEB, 美国)将RNA 3′端和5′端与接头连接,随后用M-MuLV Reverse Transcriptase(NEB, 美国)合成cDNA单链,经PCR扩增后通过12 g·L−1聚丙烯酰胺凝胶电泳回收140~150 bp左右的PCR产物条带,通过质检后得到满足Illumina平台测序的文库,并委托上海生工生物工程技术服务有限公司进行高通量测序。
1.3 测序数据处理
测序下机的原始序列(Raw reads)经过去接头,去低质量、冗余序列等处理后得到纯净序列(Clean reads),使用Bowtie(参数为-v 1)将纯净序列匹配到巨桉E. grandis基因组上,得到的序列再与Rfam14.1数据库对比,过滤其他非编码RNA。
1.4 miRNA的预测
以miRBase21.0库中植物和巨桉[12]的miRNA序列为参考源,使用blastn软件预测已知miRNA。具体参数为:-task blastn-short-word_size 16-evalue 0.01。将错配数≤1、e<0.01的序列作为潜在miRNA。同时,将这些潜在的miRNA 用Bowtie(参数为 -v 2)匹配回巨桉基因组后,通过miREAP和RNAfold软件预测所有已知miRNA及其二级结构。未匹配上的序列,继续使用miRDeep2(v2.0.0.8)[13]软件预测新miRNA,采用默认参数。
对不同长度miRNA的首位碱基进行统计并绘制直方图,分析赤桉miRNA碱基的偏好性。本文除了miRNA二级结构采用RNAfold软件生成,其他所有图形均使用R语言绘制[14]。
1.5 差异表达miRNA的挖掘与分析
低温处理下赤桉差异表达的miRNA采用Bioconductor中的DEGSeq[15]包分析。首先将2组数据使用TPM进行归一化处理,归一化表达量=(Readcount
$ \times $ 1 000 000)/libsize,其中:Readcount为miRNA序列数目,libsize为样品所有miRNA 序列数目之和。设置DEGSeq的算法为MARS,进行差异分析。定义表达量变化2倍以上且P <0.001的为显著差异表达miRNA。1.6 miRNA靶基因预测与富集分析
使用psRNATarget[16]软件对miRNA进行靶基因预测,采用默认参数。同时对差异表达miRNA的靶基因分别进行Gene ontology[17] ( http://www.geneontology.org/)和KEGG[18] ( http://www.genome.jp/kegg/)富集分析。GO注释使用在线网站agriGO v2.0[19] ( http://systemsbiology.cau.edu.cn/agriGOv2/index.php),并定义P<0.05为显著差异的类别。KEGG使用KOBAS3.0[20]软件,该软件以KEGG通路为单位,应用超几何检验进行分析:
$$ p=1-\displaystyle\sum\limits_{i=0}^{m-1}\dfrac{\left(\dfrac{M}{i}\right)\left(\dfrac{N-M}{n-i}\right)}{\left(\dfrac{N}{n}\right)},$$ (1) 式中,p为KEGG通路富集概率,i为求和中的特定项,N为所有基因中具有通路注释的基因数目,n为N中候选靶基因的数目,M为所有基因中注释为某特定通路的基因数目,m为注释为某特定通路的候选靶基因数目。同时,以拟南芥的低温胁迫响应网络和华南农业大学桉树育种课题组前期研究为参考,进一步挖掘涉及ICE1-CBFs-COR通路的miRNA。
2. 结果与分析
2.1 测序数据分析
对照组和低温处理组的小RNA高通量测序分别得到 29725524和31104120条原始序列,过滤掉低质量、两端接头和重复序列后,最后分别得到15813414和14456077条纯净序列。质控数据显示,低温处理组的Q30为96.47%,对照组的Q30为96.46%,结果可靠性高。此外,纯净序列的长度统计结果(图1)显示:2组小RNA长度的分布特征基本一致,以21 nt分布最多,其次是20和24 nt。
2.2 赤桉已知miRNA和新miRNA的预测
以RFam数据库对纯净序列进行注释,结果如表1所示,除了其他类别外,核糖体RNA(rRNA)占比最高,在对照和低温处理组中分别占比30.27%和26.08%,而核仁小RNA (snoRNA)占比最少,分别占比0.21%和0.17%。过滤掉rRNA、snRNA、snoRNA和tRNA序列后,以巨桉和miRBase21中所有植物的成熟miRNA为参考组,进行miRNA预测,结果如图2所示:2组数据一共预测到392个已知miRNAs(隶属于54个家族),其中对照组282个,低温处理组329个,它们共同的有219个;预测到97个新miRNA,其中对照组65个,低温处理组51个,它们共同的有19个。在预测到的所有miRNA中,长度均以21 nt为主,其次,已知miRNA为20和22 nt,新miRNA为24 nt(图3)。图4展示了部分预测miRNA前体的二级结构。
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 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miRNAFigure 4. The secondary structure of some predicted miRNAs in Eucalyptus camaldulensisA: 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 miRNAsmiRNA是由DCL1酶剪切得到的,DCL1酶在剪切pre-miRNA时,对其5′端第1个碱基具有强烈的偏好性,尤其体现在对尿嘧啶(U)的偏好上。miRNA首位碱基偏好性结果表明,对照和低温处理组均呈现出相似规律,具体为:长度为18 nt的miRNA其主要为腺嘌呤(A),19~22 nt的miRNA其主要为尿嘧啶(U),而23~26 nt的miRNA其以腺嘌呤(A)或鸟嘌呤(G)为主(图5)。
2.3 差异表达miRNA分析
以TPM标准化后的序列数为源数据,使用DEGSeq包分析对照组和低温处理组2个样品间差异表达的miRNA。共筛选出80个显著差异表达的miRNA(图6),包括55个显著上调(含10个新miRNA)和25个显著下调的miRNA(含16个新miRNA)。上调的miRNA归属于miR395、miR167、miR399、miR8282、miR477、miR156、miR171和miR482(eca-miR482a-3p, eca-miR482d-5p)等8个家族,而下调的miRNA归属于miR862、miR482(eca-miR482b-5p)、miR6300、miR397和miR10506等5个家族。
2.4 靶基因预测和基因富集分析
默认参数下,psRNATarget软件共预测到17 614个靶基因,其中低温胁迫下差异表达miRNA的靶基因有 5983个。这些差异表达miRNA的靶基因GO注释结果(图7)显示,定位到87个功能群中,包括分子功能(Molecular function, MF)49个、生物学过程(Biological process, BP)31个和细胞组成(Cell component)7个。这些靶基因中最多的一类是分子功能,主要集中在结合作用;在生物学过程中,主要集中在单一生物过程、生物调节和信号转导;在细胞成分中,靶基因主要与细胞膜有关。KEGG富集分析结果表明,这些靶基因参与的通路有23条,其中代谢通路(Metabolic pathway)和次生代谢物的生物合成(Biosynthesis of secondary metabolite)最多,分别占到了36.7%和17.8%(图8)。
2.5 赤桉ICE1-CBFs-COR通路相关miRNA的筛选
ICE1-CBFs-COR通路是植物低温胁迫应答的重要信号转导途径[21],本试验进一步筛选到25个与该通路相关的miRNA(表2)。这些靶基因主要编码蛋白激酶、E3泛素化连接酶和转录因子,具体包含:ICE1,直接调控ICE1的MPK6(Mitogen-activated protein kinase 6)、OST1(Open stomata 1)、HOS1、SIZ1,与调控ICE1下游的CBF1~CBF3有关的BTF3(Basic transcription factor 3)、CAMTA3(Calmodulin-binding transcription activator 3)、BZR1(Brassinazole-resistant 1)、EIN3(Ethylene in sensitive 3)、PIF3(Phytochrome-interacting factor 3)和SOC1(Suppressor of constans overexpression 1)等。其中,仅eca-miR-n60有2个靶基因,其余均有1个靶基因;多个miRNA靶向同一个基因,比如eca-miR5780b-5p、eca-miR390b-5p、eca-miR-n51和eca-miR-n60均靶向OST1。这些miRNA长度主要为20~21 nt,包括8个保守型miRNA(miR156、miR159、miR164、miR171和miR482等家族)、10个非保守型miRNA以及7个新miRNA。此外,有3个miRNA(eca-miR-n51,eca-miR-n41和eca-miR862a-5p)经过4 ℃低温处理24 h后显著差异表达,其中,eca-miR-n51靶向OST1,而eca-miR-n41和eca-miR862a-5p均靶向EBF1,其余的miRNA则差异不显著。
表 2 赤桉ICE1-CBFs-COR通路相关miRNATable 2. The miRNAs associated with ICE1-CBFs-COR pathway in Eucalyptus camaldulensismiRNA1) 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 characteristiceca-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 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 characteristiceca-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 h3. 讨论与结论
本试验通过高通量测序共预测到隶属于54个家族的392个已知miRNA和97个新miRNA,并通过软件预测到17614个靶基因。与对照组相比,低温处理组在4 ℃处理24 h后,预测到80个显著差异表达的miRNA,包括55个上调miRNA和25个下调miRNA。GO基因功能注释表明,低温胁迫下miRNA的靶基因主要与结合作用、单一生物过程、生物调节和信号转导以及细胞膜有关;KEGG富集分析表明,差异表达miRNA靶基因可能在代谢通路和次生代谢物的生物合成中起关键作用。同时,本试验还分析了可能参与到赤桉ICE1-CBFs-COR通路有关的miRNA。
在miRNA预测过程中,发现Lin等[12]的巨桉成熟miRNA序列存在一些序列无法匹配到巨桉基因组的现象,因此我们将Lin等[12]的miRNA序列与其他植物的miRNA整合后,作为miRNA参考组,再与测序下机后的纯净序列匹配,得到潜在的已知miRNA。随后,将这些潜在已知miRNA匹配回巨桉基因组,再使用miREAP和RNAFold软件寻找前体序列。但是,由于赤桉和巨桉属于近缘种,基于巨桉基因组预测的miRNA有可能存在假阳性,后续仍需试验进一步验证。
在植物中,行使重要功能的miRNA,往往是保守的。本试验中,miR156、miR167、miR395和miR399家族的多个成员在低温处理后均显著差异表达。Zhou等[22]指出,miR156家族广泛响应植物低温胁迫,比如低温胁迫会诱导杨树miR156表达量下调[23-24],而甘蔗抗寒品种‘FN39’在低温胁迫下miR156表达量上调[25]。研究发现,miR167和ARF(Auxin response factors)有关,并且miR167通过调控生长素水平抵御冷胁迫[26]。薄维平等[27]报道,低温胁迫会诱导木薯miR395的上调表达,其中miR395abcd的上调有利于减轻木薯的低温伤害。Gao等[28]发现在番茄中过表达拟南芥ath-miR399d可以提高番茄的耐寒性。除了这些保守型miRNA,本试验中其他显著差异表达miRNA在其他植物低温胁迫中也有过类似报道[29]。
ICE1作为关键性抗寒转录因子,其主导的ICE1-CBFs-COR途径是植物响应低温胁迫的重要通路。在该通路中,ICE1通过与MYB15[30]或HOS1[31]的互作来负调控CBFs及其下游基因,但其通过与SIZ1[32]或OST1[33]的互作来实现正调控。最近,Li等[34]报道MPK3/MPK6可与ICE1相互作用并将其磷酸化,降低ICE1的稳定性及其转录活性,从而负调控拟南芥CBF表达及其抗寒力。本试验进一步筛选到25个与该通路相关的miRNA,其中,eca-miR-n33靶向ICE1,可正调控ICE1-CBFs-COR通路的miRNA有靶向OST1的eca-miR5780b-5p、eca-miR390b-5p、eca-miR-n51和eca-miR-n60以及靶向SIZ1的eca-miR482f-3p,而负调控该通路的miRNA有靶向HOS1的eca-miR23a-5p和eca-miR-n2以及靶向MPK6的eca-miR171g-3p和eca-miR171j-3p。此外,Li等[35]报道BZR1可以正调控CBF1/2,增强植物抵御低温胁迫的能力。Jiang等[36]报道与光形态建成有关的转录因子PIF3可与CBF启动子直接结合,实现对低温胁迫的负调控;而EBF1(EIN3-BINDING F-BOX 1)可直接靶向PIF3进行26S蛋白酶体介导的降解,提高植物的抗寒力。综上可知,上述的这些miRNA可为后续深入研究其参与调控赤桉低温胁迫应答的分子网络奠定基础。
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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
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 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 characteristiceca-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 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 characteristiceca-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 -
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