Transcriptome Analysis on Anthocyanin Synthesis-related Genes in Liupao Tea Plants
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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。 Abstract:Objective Liupao Tea (Camellia sinensis var. sinensis cv. Liupao) was studied for the genes associated with anthocyanin synthesis for target breeding. Methods Anthocyanin were extracted with ethanol hydrochloride from Liubao Tea plants bearing purple or green buds. Transcriptome sequencing was performed using Illumina Hiseq 2500 high-throughput platform to identify differentially expressed genes, determine expression of the genes related to anthocyanin synthesis, and conduct GO and KEGG enrichment analyses on the extracts. The sequencing results were subsequently verified by fluorescence quantification PCR.Results The young leaves on the tea plants borne with purple buds contained sevenfold higher anthocyanin than those on the plants with green buds. The transcriptome of the genes of the purple buds had 165570 unigenes with an average length of1450 bp. Of them, 243 related to the anthocyanin biosynthesis pathway, and 43 significantly differentially expressed between the two types of plants. The 43 differentiated genes encoded 14 key enzymes, i.e., phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), cinnamate acid 4-hydroxylase (C4H), anthocyanidin reductase (ANR), 4-coumarate-CoA ligase (4CL), acetyl-CoA carboxylase (ACC), flavonol synthase (FLS), flavonoid-3′,5′-hydroxylase (F3′5′H), flavonoid 3′-hydroxylase (F3′H), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and leucoanthocyanidin reductase (LAR).Conclusion Thirty-four genes significantly upregulated in Liupao Tea plants with purple buds were found to be associated with 14 key enzymes encoding anthocyanin biosynthesis. They were speculated to play an important role in the bud color differentiation between the two varieties. -
Key words:
- Purple buds /
- Liupao Tea /
- anthocyanin /
- transcriptome /
- differentially expressed genes
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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 expressions of genes in two varieties of Liupao Tea plants
G-1, G-2, and G-3: 3 samples of tea plants with purple buds; F-1, F-2, and F-3: 3 samples of tea plants with green buds; deeper red color indicates higher expression; deeper blue, lower expression.
表 1 荧光定量 PCR 引物序列
Table 1. Primers for qRT-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/bpPIP 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 表 2 转录组测序有效数据统计
Table 2. Statistics on effective data
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/GbQ20数量
Q20 number/MQ20占比
Q20 ratio/%Q30数量
Q30 number/MQ30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 表 3 紫芽六堡茶花青素合成相关的差异显著基因
Table 3. Significantly differential genes associated with 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
RegulatedPAL 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 -
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