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白桦木质部原生质体初生细胞壁再生过程转录组分析

郑杰 于颖 贺卓熙 路明远 孙宇 尹天龙 王超

郑杰, 于颖, 贺卓熙, 路明远, 孙宇, 尹天龙, 王超. 白桦木质部原生质体初生细胞壁再生过程转录组分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200376
引用本文: 郑杰, 于颖, 贺卓熙, 路明远, 孙宇, 尹天龙, 王超. 白桦木质部原生质体初生细胞壁再生过程转录组分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200376
Zheng Jie, Yu Ying, He Zhuoxi, Lu Mingyuan, Sun Yu, Yin Tianlong, Wang Chao. Transcriptome analysis of regeneration process of primary cell wall in xylem protoplast of Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200376
Citation: Zheng Jie, Yu Ying, He Zhuoxi, Lu Mingyuan, Sun Yu, Yin Tianlong, Wang Chao. Transcriptome analysis of regeneration process of primary cell wall in xylem protoplast of Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200376

白桦木质部原生质体初生细胞壁再生过程转录组分析

doi: 10.12171/j.1000-1522.20200376
基金项目: 东北林业大学大学生创新项目(201910225011)
详细信息
    作者简介:

    郑杰。主要研究方向:林木重要性状形成的分子机制。Email:jiezheng@nefu.edu.cn 地址:150040黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林学院

    责任作者:

    王超,教授。主要研究方向:林木重要性状形成的分子机制。Email:wangchao@nefu.edu.cn 地址:同上

  • 中图分类号: S718.43.

Transcriptome analysis of regeneration process of primary cell wall in xylem protoplast of Betula platyphylla

  • 摘要:   目的  木质部细胞壁的组成及特性是决定材性的重要因素,研究木质部细胞壁形成的分子调控机制对于木材改良具有重要意义。本研究探究了白桦木质部原生质体初生壁再生过程的分子调控机制,并鉴定出重要调控基因,旨在为林木材性性状研究提供数据和材料。  方法  分别以培养0 h和2 h的白桦木质部原生质体为材料,通过荧光增白剂染色观察初生壁再生过程。利用转录组分析技术研究初生壁再生前后的差异表达基因及其参与的调控途径,将检测到的差异表达基因在GO、KEGG、PlantTFDB数据库中进行比对分析。  结果  荧光显微镜观察结果显示:原生质体分离后不具有细胞壁,培养2 h再生初生细胞壁。以|log2(FC)| ≥ 1(FC为差异倍数)且q < 0.05为标准筛选差异基因,结果显示:相较于刚分离的原生质体,培养2 h的原生质体中检测到4 396个上调表达的基因,4 056个下调表达基因,总计8 452个差异表达基因。其中GO数据库共注释到10个显著上调条目,KEGG数据库注释到10个显著差异代谢通路,PlantTFDB数据库共注释到16个家族的360个差异表达转录因子。GO注释结果表明,DNA复制、细胞周期相关基因上调表达。KEGG注释结果表明,谷胱甘肽、α-亚麻酸等与抗逆代谢相关的基因下调表达,果胶脂酶相关基因上调表达。PlantTFDB注释结果表明,bHLH、NAC、MYB、bZIP等与细胞壁合成密切相关的转录因子均差异表达。  结论  培养2 h的木质部原生质体处于细胞壁再生及分裂准备状态,DNA复制、细胞周期、多糖合成代谢等相关基因在白桦木质部原生质体培养及初生细胞壁形成过程中起调控作用。

     

  • 图  1  白桦原生质体细胞壁再生荧光显微镜观察结果

    a. 培养0 h的原生质体;b. 培养0.5 h的原生质体;c. 培养1 h的原生质体;d. 培养2 h的原生质体。a, protoplasts cultured for 0 h; b, protoplasts cultured for 0.5 h; c, protoplasts cultured for 1 h; d, protoplasts cultured for 2 h.

    Figure  1.  Fluorescence microscope observation of cell wall regeneration in protoplasts of birch

    图  2  白桦木质部原生质体RNA提取结果

    C1 ~ C3. 培养0 h时的原生质体;T1 ~ T3. 培养2 h时的原生质体。C1 − C3, protoplast cultured for 0 h; T1 − T3, protoplast cultured for 2 h.

    Figure  2.  RNA extraction results from protoplasts of birch xylem

    图  3  对照组和实验组的聚类分析结果

    Figure  3.  Cluster analysis results of control group and experimental group

    图  4  培养2 h相对0 h原生质体内差异表达基因数量柱状图

    Figure  4.  Histogram of the number of DEGs in 2 h versus 0 h protoplasm

    图  5  培养2 h相对0 h原生质体内差异表达基因GO注释结果柱状图

    Figure  5.  GO annotation of differentially expressed genes in protoplasts after 2 h culture compared with just isolated protoplasts

    图  6  GO注释结果富集热图

    Figure  6.  GO annotation results enrich heat map

    图  7  差异转录因子家族分布柱状图

    Figure  7.  Histogram of differential transcription factor family distribution

    图  8  糖酵解(a)、磷酸戊糖(b)、戊糖及葡萄糖互变异构体(c)、木质素合成途径(d)苯丙烷代谢途径(e)途径差异示意图

    果糖-1,6-二磷酸. F16P;果糖-6-磷酸. F6P;葡萄糖-6-磷酸. G6P;5-磷酸核糖. Ribose-5P;5-磷酸脱氧核糖. 2-Deoxy-Ribose-5P;核糖. Ribose;脱氧核糖. 2-Deoxy-Ribose;1,4-α聚乳糖. Poly(1,4-α-D-galacturonide);1,4-α聚乳糖酯 Poly(1,4-α-D-galacturonate)(n);不饱和聚乳糖酯. Unsaturated digalacturonate;α-D-6-磷酸葡萄糖. α-D-G6P;α-D-1-磷酸葡萄糖. α-D-G1P;UDP-葡萄糖. UDP-glucose;纤维素. Cellulose;松柏醛. Coniferyl-aldehyde;豆香醛. 5-hydroxy-conferaldehyde;芥子醛. Sinapaldehyde;松柏醇. Coniferyl alcohol;豆香醇. 5-hydroxy-conferyl alcohol;芥子醇. Sinapoyl alcohol;松柏木质素. Cuaiacyl lignin;豆香木质素. 5-Hydorxy-guaiacyl lignin;芥子木质素. Syringyl lignin ;果糖二磷酸酶. FBP;果糖二磷酸醛缩酶. ALOD;核糖激酶. RbsK;果胶酯酶. Pectinseter;果胶裂解酶. Pectate lyase;UDP-葡萄糖转移酶. UDPG;UDP葡萄糖醛酸转移酶. UDPGT;月桂醇脱氢酶. Cinnamyl-alcihol dehydrogenase;过氧化物酶. Peroxidase. 红色文本代表基因的上调;绿色文本代表基因的下调;其他文本颜色无特殊含义。Red text refers to gene up-regulation, green text refers to gene down-regulation, there is no special meaning for text in other colors.

    Figure  8.  Glycolysis pathway(a), pentose and phosphate pathway(b), pentose and glucuronate interconversions(c), cellulose synthesis(d), phenylpropanoid biosynthesis(e)

    表  1  Clean reads与参考基因组比对结果

    Table  1.   Clean reads and the sequence alignment results with reference genome

    组别
    Group
    过滤后序列总数
    Total clean reads
    比对成功率
    Mapping rate/%
    基因总数
    Total gene number
    Q30/%
    C144 905 83692.0419 83593.36
    C241 241 93092.2619 76493.44
    C345 786 95092.2819 88593.22
    T147 178 64691.3720 44293.53
    T244 340 70291.1320 31093.22
    T341 809 66091.0220 28193.32
    注:Q30为过滤后碱基质量值,表示碱基错误率小于0.001的测序碱基占测序总数的比例。Notes: Q30 is the base quality value after filtering, indicating the proportion of sequenced bases with a base error rate less than 0.001 in the total number of sequenced bases.
    下载: 导出CSV

    表  2  KEGG通路注释结果及富集分析统计表

    Table  2.   Results of KEGG notions and the significantly enriched KEGG pathway statistics

    通路名称
    Pathway name
    Map编号
    Map No.
    p
    p value
    q
    q value
    上调数
    Number of up-regulation
    下调数
    Number of down-regulation
    谷胱甘肽代谢 Glutathione metabolism
    map00480 0.000 002 450 0.000 238 8 30
    α-亚麻酸代谢
    Alpha-Linolenic acid metabolism
    map00592 0.000 029 600 0.001 353 6 17
    DNA复制 DNA replication map03030 0.000 332 000 0.011 156 18 1
    光合作用天线蛋白
    Photosynthesis - antenna proteins
    map00196 0.001 147 000 0.026 197 7 0
    黄酮代谢 Flavonoid biosynthesis map00941 0.001 623 000 0.027 359 9 5
    戊糖及葡萄糖糖醛互变异构体
    Pentose and glucuronate interconversions
    map00040 0.001 997 000 0.027 359 14 8
    光合作用 Photosynthesis map00195 0.001 945 000 0.027 359 13 0
    卟啉和叶绿素代谢
    Porphyrin and chlorophyll metabolism
    map00860 0.001 666 000 0.027 359 14 3
    牛磺酸和压及亚牛磺酸代谢
    Taurine and hypotaurine metabolism
    map00430 0.002 319 000 0.028 882 2 5
    黄酮及黄酮合成
    Flavone and flavanol biosynthesis
    map00944 0.004 234 000 0.04 8340 6 1
    下载: 导出CSV

    表  3  原生质体细胞壁再生过程中相关差异基因统计

    Table  3.   DEGs in the process of cell wall regeneration

    细胞壁发育相关代谢过程
    Metabolic processes associated with
    cell wall development
    差异表达基因编码的酶
    Enzyme cluster of DEGs encoding
    差异基因总数(上调数 + 下调数)
    Total DEGs number (up-regulation
    number +down-regulation number)
    葡萄糖及葡萄糖酰代谢
    Glucose and glucosyl metabolic
    UDP葡萄糖醛酸转移酶 UDP-glucosyl transferase (UDPGT) 37(13 + 24)
    UDP葡萄糖转移酶 UDP-UDP glucose transferase(UDPG) 3(3 + 0)
    果糖二磷酸酶 Fructose diphosphatase (FBP) 1(1 + 0)
    果糖二磷酸醛缩酶 (ALOD) 1(1 + 0)
    羰基转移酶家族 Glycosyl transferase family 12(0 + 12)
    糖基羟化酶家族 Glycosyl hydrolases family . 9(9 + 0)
    内质网二糖结合 Di-glucose binding within endoplasmic reticulum 5(2 + 3)
    3-β葡萄糖苷酶 3-beta-glucosidase 3(3 + 0)
    1,3-葡聚糖合成酶 1,3-beta-glucan synthase 1(1 + 0)
    6-磷酸葡萄糖羟化酶 6-phosphogluconate dehydrogenase 1(0 + 1)
    6-磷酸葡萄糖异构酶 Glucosamine-6-phosphate isomerases 1(1 + 0)
    3-磷酸甘油醛脱氢酶 Glyceraldehyde 3-phosphate dehydrogenase 1(1 + 0)
    木糖代谢
    Xylose metabolic
    β-1,4-木糖基转移酶 Beta-1,4-xylosyltransferase 1(1 + 0)
    β-1,2-木糖基转移酶 Beta-1,2-xylosyltransferase 1(1 + 0)
    半乳糖代谢
    Galactose metabolic
    细胞壁相关受体激酶半乳糖醛酸结合
    Wall-associated receptor kinase galacturonan-binding
    10(4 + 6)
    Galactosyltransferase 5(3 + 2)
    聚半乳糖醛酸酶 Polygalacturonase 5(2 + 3)
    聚半乳糖醛酸酶抑制剂 Polygalacturonase inhibitor 3(0 + 3)
    糖基水解酶 Glycosyl hydrolases 2(0 + 2)
    磷酸戊糖代谢
    Pentose phosphate metabolic
    核糖激酶 Ribokinase (RbsK) 3(3 + 0)
    6-磷酸葡萄糖脱氢酶 Phosphogluconic dehydrogenase (G6PD) 2(2 + 0)
    果胶代谢
    Pectic metabolism
    果胶酯酶 Pectinester 6(0 + 6)
    果胶裂解酶 Pectate lyase 7(7 + 0)
    苯丙素生物合成
    Phenylpropanoid biosynthesis
    月桂醇脱氢酶 Cinnamyl-alcihol dehydrogenase (CAD) 2(0 + 2)
    过氧化物酶 Peroxidase (POD) 6(6 + 0)
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-11-29
  • 录用日期:  2022-05-09
  • 修回日期:  2021-02-08
  • 网络出版日期:  2022-05-13

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