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紫芽六堡茶花青素合成相关基因的转录组测序及表达分析

梁燕妮 魏诗琴 乔如颖 梁剑锋 谭花桂

梁燕妮,魏诗琴,乔如颖,等. 紫芽六堡茶花青素合成相关基因的转录组测序及表达分析 [J]. 福建农业学报,2024,39(X):1−11
引用本文: 梁燕妮,魏诗琴,乔如颖,等. 紫芽六堡茶花青素合成相关基因的转录组测序及表达分析 [J]. 福建农业学报,2024,39(X):1−11
LIANG Y N, WEI S Q, QIAO R Y, et al. Transcriptome analysis of anthocyanin synthesis related genes in Purple bud Liupao tea Plant [J]. Fujian Journal of Agricultural Sciences,2024,39(X):1−11
Citation: LIANG Y N, WEI S Q, QIAO R Y, et al. Transcriptome analysis of anthocyanin synthesis related genes in Purple bud Liupao tea Plant [J]. Fujian Journal of Agricultural Sciences,2024,39(X):1−11

紫芽六堡茶花青素合成相关基因的转录组测序及表达分析

基金项目: 广西自然科学基金面上项目(2023GXNSFAA026221);广西高校中青年教师基础能力提升项目(2021KY0680);广西大学生创新创业项目
详细信息
    作者简介:

    梁燕妮(1980 —),女,硕士,高级实验师,主要从事植物分子生物学相关研究,E-mail:371200021@qq.com

  • 中图分类号: S571.1

Transcriptome analysis of anthocyanin synthesis related genes in Purple bud Liupao tea Plant

  • 摘要: [目的 ] 紫芽六堡茶(Camellia sinensis var. sinensis cv. ‘Liupao’)是六堡群体种茶树中芽叶呈紫色且富含花青素的特异品系,研究紫芽六堡茶花青素合成相关基因可为深入了解紫芽六堡茶花青素积累的分子机制奠定基础,为高花青素六堡茶树的分子育种提供遗传资源。  方法  利用盐酸乙醇分别提取紫芽和绿芽六堡茶芽中花青素,测定其含量;通过Illumina Hiseq 2500高通量测序平台对紫芽和绿芽六堡茶进行转录组测序,分析花青素合成相关基因的表达水平,找出差异表达基因,进一步进行GO功能分析和KEGG富集分析;并通过荧光定量PCR对转录组测序结果进行验证。  结果  紫芽六堡茶花青素含量是绿芽的7倍;转录组测序共获得165570条Unigene,平均长度1450 bp;对测序结果进行分析,筛选出与紫芽六堡茶花青素生物合成通路相关的基因243条,进一步筛选出43个差异显著表达基因,这些基因共编码14个关键酶,包括苯丙氨酸氨裂解酶(Phenylalanine ammonialyase, PAL)、查尔酮合成酶(Chalcone synthase, CHS)、查尔酮异构酶(Chalcone isomerase, CHI)、肉桂酸4-羟化酶(Cinnamate acid 4-hydroxylase, C4H)、花色素苷还原酶(Anthocyanidin reductase, ANR)、4-香豆酸CoA连接酶(4-coumarate-Co A ligase, 4CL)、乙酰辅酶A羧化酶(Acetyl-CoA carboxylase, ACC)、黄酮醇合成酶(Flavonol synthase, FLS)、黄酮3′,5′-羟化酶(Flavonoid-3′,5′-hydroxylase, F3'5'H)、黄酮3′-羟化酶(Flavonoid 3'-hydroxylase, F3'H)、黄酮3-羟化酶(Flavanone-3-hydroxylase, F3H)、二氢黄酮醇还原酶(dihydroflavonol 4-reductase, DFR)、花青素成合酶(Anthocyanidin synthase,ANS)、无色花青素还原酶(Leucoanthocyanidin reductase, LAR)。  结论  编码花青素生物合成的14个关键酶基因中有34个基因在紫芽六堡茶中上调表达,推测这些基因在紫芽花青素积累中起重要作用。
  • 图  1  试验材料

    Figure  1.  Experimental Materials

    图  2  紫芽和绿芽六堡茶花青素含量比较

    **表示差异极显著(P<0.01)。

    Figure  2.  Comparison of tea anthocyanin content between purple bud and green bud

    ** means extremely significant (P<0.01).

    图  3  绿芽VS紫芽差异基因散点图

    Figure  3.  Gene scatter plot of green bud VS purple bud differences

    图  6  紫芽VS绿芽差异基因表达量热图

    G-1、G-2、G-3为紫芽的3个重复,F-1、F-2、F-3为绿芽的3个重复。颜色越红表达量越高,颜色越蓝,表达量越低。

    Figure  6.  Heatmap of differential genes expression in Purple bud VS green bud

    G-1, G-2, and G-3 represent the three samples of the purple bud. F-1, F-2, and F-3 represent the three samples of the green bud. Redder color indicates higher expression, bluer color and lower expression..

    图  7  花青素生物合成差异基因GO富集分析

    Figure  7.  GO-enrichment analysis of differential genes in anthocyanin biosynthesis

    图  8  花青素生物合成差异基因KEGG富集

    Figure  8.  KEGG enrichment of differential genes in anthocyanin biosynthesis

    图  9  差异基因荧光定量PCR验证

    *表示差异显著 (P<0.05),**表示差异极显著 (P<0.01)

    Figure  9.  qRT-PCR Validation of RNA-Seq data

    * indicates significant difference (P<0.05), ** indicates very significant difference (P<0.01)

    表  1  荧光定量 PCR 引物序列

    Table  1.   Primers for real-time quantitative PCR

    引物名称
    Primer name
    基因ID
    Gene ID
    引物序列(5’-3’)
    Primer sequence(5’-3’)
    序列长度
    Sequence length/bp
    PIP Unigene82719 F:TTGTGGTGCTGCTGTTGTCA
    R:TGGCGGAGAAGACGGTGTA
    158
    psaG Unigene19457 F:CGAGCCAAAGAGTATGTGAGTC
    R:AGTAAGCAACGATGTGTCCAAT
    103
    purH Unigene31003 F:GAAGCAGCCGACTGTTGGA
    R:CCACCAGCATTAACCTCAGAGA
    144
    CYP75A Unigene14189 F:TGATAAGCCGAAGCCTTGTGT
    R:CGATTGCGTGGATGGACT
    112
    CHI Unigene41427 F:ACAATGTCTCCGTCGCAGTCA
    R:AGCCGTGAACTTGATGAACTTG
    161
    PAL CL10001.Contig25 F:GCAAAGCCCAACCAAGTAGG
    R:GCCGAGCAACACAACCAAG
    114
    4CL Unigene2852 F:TGCTGTTGTGCTCGCTGAG
    R:AGTGACCACCATCGGATTCTTC
    158
    ANS Unigene69431 F:AACCGCAAGTACAAGAGCATTC
    R:CTCCTCCTCCGTCACTGTCT
    138
    LAR CL6821.Contig1 F:ATGACTGTGTTGGAATCTGTGT
    R:CTAGCTTCGGCGATGAACTG
    92
    CsEF CsEF1 F:TTGGACAAGCTCAAGGCTGAACG
    R:ATGGCCAGGAGCATCAATGACAGT
    109
    下载: 导出CSV

    表  2  有效数据统计

    Table  2.   Effective data statistics

    样本
    Sample
    过滤前的序列数
    Raw reads /M
    过滤后的序列数
    Clean reads/M
    过滤后的序列占比
    Clean reads ratio/%
    过滤后的碱基数
    Clean bases/Gb
    Q20数量
    Q20 Number/M
    Q20占比
    Q20 ratio/%
    Q30数量
    Q30 Number/M
    Q30占比
    Q30 Ratio/%
    紫芽G-1 43.82 42.2 96.31 6.33 42.43 96.83 40.26 91.88
    紫芽G-2 43.82 42.42 96.79 6.36 42.52 97.03 40.45 92.31
    紫芽G-3 43.82 42.41 96.79 6.36 42.5 96.99 40.41 92.22
    绿芽F-1 45.57 43.59 95.66 6.54 44.17 96.94 42.01 92.19
    绿芽F-2 43.82 42.61 97.23 6.39 42.50 96.98 40.41 92.24
    绿芽F-3 43.82 42.54 97.08 6.38 42.46 96.89 40.32 92.02
    下载: 导出CSV

    表  3  紫芽六堡茶花青素合成相关的显著差异基因

    Table  3.   Significant different genes involved in anthocyanin synthesis

    基因名称
    Name
    基因 ID
    Gene_ID
    绿芽中相对表达量
    Green bud expression
    紫芽中相对表达量
    Purple bud expression
    差异倍数
    Differential multiple
    表达调控
    Regulated
    PAL CL10001.Contig10 0.040 16.373 8.428 上调 Up
    PAL CL10001.Contig19 1.926 20.020 3.284 上调 Up
    PAL CL10001.Contig9 0.443 8.380 4.222 上调 Up
    CHS CL13438.Contig10 0.000 83.240 9.947 上调 Up
    CHS CL13438.Contig16 0.313 42.316 5.994 上调 Up
    CHS CL13438.Contig4 1.263 122.906 5.154 上调 Up
    CHS Unigene23474 0.576 31.160 5.285 上调 Up
    CHS Unigene9932 0.526 19.780 4.808 上调 Up
    CHI CL1850.Contig6 0.796 6.346 2.948 上调 Up
    CHI CL5568.Contig1 85.453 284.253 1.708 上调 Up
    CHI Unigene41427 14.090 76.673 2.341 上调 Up
    CHI Unigene72176 0.050 8.013 5.537 上调 Up
    DFR Unigene25358 1.156 0.053 −4.396 下调 Down
    DFR Unigene8872 0.883 0.080 −3.165 下调 Down
    DFR Unigene8644 0.000 1.800 7.766 上调 Up
    DFR CL13149.Contig6 0.030 1.043 5.484 上调 Up
    C4H CL1010.Contig15 1.133 5.286 2.164 上调 Up
    C4H CL10269.Contig2 0.000 12.236 7.711 上调 Up
    C4H CL5289.Contig2 158.473 444.453 1.463 上调 Up
    ANR CL1162.Contig11 0.033 31.566 9.650 上调 Up
    ANR CL1162.Contig14 8.700 422.806 5.531 上调 Up
    ANR CL1162.Contig2 0.070 23.650 7.396 上调 Up
    4CL Unigene18597 0.676 22.903 4.901 上调 Up
    4CL Unigene5041 1.440 54.046 5.109 上调 Up
    4CL Unigene79704 13.510 37.350 1.440 上调 Up
    4CL Unigene9176 0.186 82.623 8.500 上调 Up
    ACC CL10106.Contig13 0.606 10.120 3.698 上调 Up
    ACC Unigene6677 0.670 6.476 3.095 上调 Up
    ACC CL10106.Contig22 6.416 17.563 1.432 上调 Up
    F3'5'H Unigene14189 2.396 302.993 6.088 上调 Up
    LAR CL52.Contig1 2.650 0.000 −6.676 下调 Down
    LAR CL6821.Contig1 188.293 2.716 −6.078 下调 Down
    LAR Unigene12558 3.456 0.190 −3.935 下调 Down
    ANS CL9955.Contig1 0.040 2.073 4.915 上调 Up
    ANS Unigene16647 0.106 6.580 5.672 上调 Up
    ANS Unigene5088 0.330 2.420 2.666 上调 Up
    FLS Unigene62039 1.790 0.210 −3.455 下调 Down
    FLS Unigene16307 5.530 1.143 −2.245 下调 Down
    FLS CL4659.Contig3 5.803 0.410 −3.785 下调 Down
    FLS CL3149.Contig1 158.973 0.676 −7.731 下调 Down
    F3'H CL1322.Contig3 0.393 25.246 5.605 上调 Up
    F3H Unigene23406 220.476 558.353 1.301 上调 Up
    F3H Unigene45907 0.286 39.816 4.832 上调 Up
    下载: 导出CSV
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  • 收稿日期:  2023-07-13
  • 修回日期:  2023-11-10
  • 网络出版日期:  2024-05-17

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