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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水田耕整是水稻生产的重要环节，水田精准耕整有利于提高水肥利用率、减少杂草、提高水稻产量[1-4]。为满足水稻生产中“寸水不过田”的要求，华南农业大学成功研制出与乘坐式插秧机底盘配套的水田激光平地机，田间应用结果表明该平地机的平地精度和平整效果满足要求[5-10]；研制了不同形式的与轮式拖拉机配套的水田激光平地机，进一步提高了水田激光平地机的作业效率[11-12]。水田平整前需进行打浆作业，一般采用旋耕机或打浆机，人工手动操作易出现作业深度不稳定，甚至出现作业过深导致拖拉机过载熄火的现象，不仅影响作业质量，破坏水田原有硬底层造成水田硬底层凹凸不平[13]，还会影响拖拉机和机具的使用寿命。为此，研究人员通过在拖拉机三点悬挂机构上增加油缸，实现耕整机具高程和水平自动控制[14-16]，但增加的油缸难以通用于不同拖拉机。于志成等[17]设计了由缺口耙和尾翼拖板组成的水田复式整地机自动调平装置，但该装置调平转动中心位于悬架中部，限制了拖拉机PTO动力输入至旋耕机或打浆机。此外，水田打浆和水田平整通常是分开进行的，打浆机和平地机分别进地作业，拖拉机多次进田易破坏水田硬底层。在打浆机构后增加仿形平地装置的水田打浆平地技术是较好的解决方案之一，李明金[18]和Xu等[19]设计了一种水田搅浆机平地装置，可同时进行水田打浆和平地作业，但还是由人工凭经验操作拖拉机三点悬挂机构对水田进行仿形打浆，且平地铲不具备运土功能。为提高打浆平地作业质量和自动化水平，万松等[20]设计了调平自动控制的平地装置，仅对平地拖板的横滚角和俯仰角进行控制，平地拖板高程和旋耕机水平均未实现自动控制；杨青丰[21]设计了水田激光打浆平地系统，对打浆机的高程和调平实现了自动控制，但该机并没有平地铲，不具备平地功能。本研究拟设计一种激光控制的水田打浆平地机，平地铲由激光控制自动调节高程，且可自动调平打浆机和平地铲，实现一次进田完成水田打浆和平地作业，并进行田间试验验证，分析自动调平作业和平地作业精度。
1. 激光控制水田打浆平地机设计
1.1 整机设计
激光控制水田打浆平地机主要包括平地机构、打浆机构、自动调平机构、平地铲高程调节机构、液压系统和控制系统。如图1所示，平地机构位于打浆机构后方，平地机构和打浆机构均连接至自动调平机构。通过自动调平机构的调平油缸调节平地机构和打浆机构的倾斜角度，平地铲高程调节机构通过高程油缸调节平地机构。
![图 1 整机结构简图]()
图 1 整机结构简图1：打浆机构；2：倾角传感器；3：液压阀块及阀组；4：调平支撑架；5：调平油缸；6：前调平连接板；7：后调平连接板；8：高程油缸；9：上平地连接架；10：平地机构；11：平地拖板；12：下连接杆；13：控制器；14：直线电机；15：接收器升降杆安装座；16：接收器升降杆；17：激光接收器；18：调平销轴Figure 1. Sketch map of the beating-leveler1: Beating mechanism; 2: Roll angle sensor; 3: Hydraulic valve module; 4: Tilt adjustment bracket; 5: Tilt adjustment cylinder; 6: Front leveling connection plate; 7: Back leveling connection plate; 8: Elevation cylinder; 9: Upper grade connection bracket; 10: Leveling mechanism; 11: Tail plate; 12: Lower connection rod; 13: Controller; 14: Linear motor; 15: Mounting plate of lifting rod for laser receiver; 16: Lifting rod for laser receiver; 17: Laser receiver; 18: Leveling pin由于水田硬底层高低不平，轮式拖拉机的四轮陷深不一致，打浆平地机作业深度和水平倾角会不断变化。激光接收器感应激光发射器形成的参考平面激光信号，当平地铲低于设定深度时，控制器控制高程油缸伸长，提升平地铲至设定平面，当平地铲高于设定平面时则控制降低平地铲至设定平面；倾角传感器实时检测拖拉机车身横滚角度，控制器根据横滚角度控制调平油缸伸缩，调节打浆机和平地铲与拖拉机的相对角度保持在设定倾角[22]。通过对平地机构的高程和调平自动控制，打浆机构调平自动控制，实现打浆平地作业。
1.2 主要部件设计
1.2.1 调平机构
调平机构主要包括调平支撑架、调平连接组件、调平油缸和调平销轴等。如图1a所示，调平连接组件下端与打浆机构固定，左右摆动的调平连接组件与调平支撑架可转动式连接，通过调平油缸的伸缩带动调平连接组件、平地机构和打浆机构左右运动。
1.2.2 平地铲高程调节机构
平地铲高程调节机构主要包括上连接架、下连接架和高程油缸；上连接架与下连接架前端打浆机后侧相接，另一端与平地机构相接，上连接架和下连接杆等长且平行安装，保证平地机构垂直于地面升降；通过高程油缸伸缩实现平地铲的升降。
1.2.3 液压系统
水田打浆平地机的液压系统主要包括2条独立的液压油路，分别用于打浆平地机的调平控制和平地机构的高程控制；调平油缸的上端与调平支撑架悬臂梁一侧铰接，下端与打浆机构铰接，通过电磁换向阀控制调平油缸伸缩驱动打浆机构和平地机构绕连接点轴线相对于调平支撑架转动。高程油缸上端与打浆机铰接，下端与上连接架铰接，通过电磁换向阀控制高程油缸伸缩实现平地机构垂直地面升降。
1.2.4 控制系统
激光控制水田打浆平地机控制系统包括激光发射器、激光接收器、控制器和车身倾角传感器(图2)。根据激光接收器接收到的激光参考信号，控制器驱动电磁阀组调节高程油缸，自动调节平地铲作业深度；倾角传感器检测拖拉机横滚角，调平油缸直线位移传感器测量打浆机相对于拖拉机车身的转动角度，控制器控制电磁阀组调节调平油缸的伸缩，实现打浆机和平地铲的自动调平。
2. 材料与方法
2.1 试验设备与仪器
水田精准打浆平地机的打浆机构采用豪丰1GQN-180A型旋耕机，配套拖拉机为常发CFD804，试验中拖拉机动力输出轴转速选取档位760 r/min，打浆机转速370 r/min，试验平均作业速度0.38 m/s。采用2台姿态航向参考系统(AHRS，荷兰Xsens，MTi-100，角度动态测量精度0.3°)测量拖拉机和打浆平地机横滚角；2台AHRS分别安装于调平支撑架和平地铲上，同步测量拖拉机和打浆平地机横滚角，波特率115 200 bps，由40 Hz外部脉冲触发采样；此外，还包括MOXA Uport 1 450多串口数据采集卡、拓普康AT-B4水准仪、秒表和皮尺。
2.2 试验方法
试验地点为华南农业大学增城试验基地水田，面积约0.312 hm2，收获后的稻茬田经旱旋耕后泡水48 h。试验前，依田块形状进行网格划分，如图3所示，黑色实线为田埂，虚线交叉点为平整度采样点，并做好标识，采用水准仪测量平整度采样点高度值；作业过程中，采集AHRS测量拖拉机车身和打浆平地机的横滚角(图4)；作业完成后采用水准仪测量平整度采样点高度值。
![图 4 水田打浆平地机田间试验照片]()
图 4 水田打浆平地机田间试验照片1：拖拉机车身的AHRS; 2：打浆平地机的AHRSFigure 4. Pictures for field test of beating-leveler in paddy field1: AHRS on the tractor body; 2: AHRS on the beating-leveler2.3 数据处理
试验通过水准仪采集了50个平整度高度数据，采用所有高度数据的标准偏差(Sd)来定量描述水田平整度，方法参照文献[23]，采用采样点高度与期望高度的绝对差值不大于3 cm的采样点个数占总采样个数的百分比来描述田块高度的分布差异和特征，统计分析方法分别如式(1)和(2)所示。
$$ {S_d} = \sqrt {\sum\limits_{i = 1}^n {{{({{\rm{Z}}_i} - \overline Z )}^2}/(n - 1)} } \;\;,$$ (1) $$ P(\left| {{Z_i} - \overline Z } \right| \leqslant 0.03) = \frac{m}{n} \times 100\% \;\;,$$ (2) 式中，Zi为各平整度采样点相对高度值，m；
$\overline{Z}$ 为各平整度采样点相对高度的期望值，m；n为平整度采样点个数；m为采集点高度与其期望高度值的绝对差值不大于3 cm的采样点个数。3. 结果与分析
3.1 打浆平地机自动调平效果
通过2台AHRS分别采集的拖拉机车身横滚角和打浆平地机横滚角如图5所示。由图5可知，拖拉机横滚角在±4.5°内变化，标准偏差为1.86°，打浆平地机横滚角保持在±1°内变化，标准偏差为0.86°，结果说明调平自动控制系统能提高打浆平地机作业过程中水平角度的稳定性。
3.2 打浆平地作业平整度
打浆平地作业前后采集的田面数据如图6所示，图6a为打浆平地试验前田面平整度三维图，图6b为打浆平地作业后田面平整度三维图，由图6可以看出打浆平地作业后田面平整度明显提高。
![图 6 打浆平地作业前后的田面平整度(田面等高图)]()
图 6 打浆平地作业前后的田面平整度(田面等高图)Figure 6. Field surface flatness before and after beating and leveling operation (field surface contour map)打浆平地平整度统计分析结果表明，打浆平地作业后田面最大高差从作业前的17.7 cm降低到6.7 cm，Sd从作业前的4.08 cm下降到1.75 cm，绝对差值不大于3 cm的平整度采样点由作业前的62%提高到82%以上，表明激光控制水田打浆平地机作业后可明显改善田面平整情况。
图7为打浆平地作业试验中某行自西向东数据的变化曲线图，由图7可知，作业后田面平整度波动较小，对2条曲线采用最小二乘法分别进行直线拟合，如图7中各对应虚线所示，从作业前田面高度拟合直线可以看出田面西边整体高于东边，经作业后，田面高度拟合直线基本水平，说明平地铲将高处泥土搬运至低处，为打浆作业提供了良好的田面平整度。
![图 7 作业前后某行数据采集点高度变化]()
图 7 作业前后某行数据采集点高度变化Figure 7. Height changes of one row of sampling points before and after operation4. 结论
1)设计了平地铲安装于打浆机构后方的水田打浆平地机，自动控制打浆机的调平，对平地铲的高程和调平实现自动控制，实现了一次进田完成水田打浆作业和平地作业。
2)水田打浆平地作业过程中，拖拉机横滚角范围为±4.5°内，打浆平地机的横滚角保持在±1°内，标准偏差为0.86°，表明调平自动控制系统明显提高了水田打浆平地机的水平稳定性。
3)水田打浆平地作业结果表明，打浆平地作业后田面最大高差从作业前的17.7 cm降低到6.7 cm，Sd从作业前的4.08 cm下降到1.75 cm，绝对差值不大于3 cm的平整度采样点由作业前的62%提高到82%以上，表明激光控制水田打浆平地机打浆平地作业后可以明显改善田面平整情况。
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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
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图 3 赤桉预测miRNA长度分布
Figure 3. The length distribution of the predicted miRNAs in Eucalyptus camaldulensis
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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
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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
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图 6 赤桉差异表达miRNA火山图
Figure 6. Volcanic map of differentially expressed miRNAs in Eucalyptus camaldulensis
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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
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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 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
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 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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