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

doi:10.19303/j.issn.1008-0384.2024.06.010

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

doi: 10.19303/j.issn.1008-0384.2024.06.010

梧州学院食品与制药工程学院（六堡茶现代产业学院），广西　梧州　543002

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（2024年）

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
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出版历程
- 收稿日期: 2023-07-13
- 修回日期: 2023-11-10
- 网络出版日期: 2024-05-17
- 刊出日期: 2024-06-28

Transcriptome Analysis on Anthocyanin Synthesis-related Genes in Liupao Tea Plants

School of Food and Pharmaceutical Engineering, Wuzhou University (Guangxi Liupao Tea Modern Industry College),Wuzhou, Guangxi　543002, China

摘要

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。
- 紫芽 /
- 六堡茶 /
- 花青素 /
- 转录组 /
- 差异表达基因
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 Liupao 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 of 1450 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.
- Purple buds /
- Liupao Tea /
- anthocyanin /
- transcriptome /
- differentially expressed genes

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图 1 试验材料

Figure 1. Experimental materials

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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. Anthocyanin contents in Liupao Tea plants with purple or green buds

**: extremely significant at 0.01 level.

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图 3 绿芽vs紫芽差异基因散点图

Figure 3. Scatter plot of differential genes in two varieties of Liupao Tea plants

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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.

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图 7 花青素生物合成差异基因GO富集分析

1~20分别为金属离子结合、催化活性、氧化还原酶活性、转移酶活性、作用于配体的氧化还原酶活性、铁离子结合、曲红素酸盐、连接酶活性、单加氧酶活性、柚皮素-查尔酮合酶活性、苯丙氨酸解氨酶活性、乙酰辅酶a羧化酶活性、分子内裂解酶活性、查尔酮异构酶活性、双加氧酶活性、类黄酮生物合成过程、生物合成过程、肉桂酸生物合成过程、生物合成过程、肉桂酸生物合成过程、L-苯丙氨酸分解代谢过程、脂肪酸生物合成。

Figure 7. GO-enrichment analysis on differential genes associated with anthocyanin biosynthesis

1–20 was metal ion binding, catalytic activity, oxidoreductase activity, transferase activity, oxidoreductase activity acting on paired donors, iron ion binding,heme binding, ligase activity, monooxygenase activity, naringenin-chalcone synthase activity, phenylalanine ammonia-lyase activity, acetyl-CoA carboxylase activity, intramolecular-lyase activity, chalcone isomerase activity, dioxygenase activity, flavonoid biosynthetic process, biosynthetic process, cinnamic acid biosynthetic process, L-phenylalanine catabolic process,fatty acid biosynthetic process, respectively.

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图 8 花青素生物合成差异基因KEGG富集

1~12分别为类黄酮生物合成、苯丙氨酸代谢、苯丙素的生物合成、泛素和其他萜类醌的生物合成、丙酸代谢、黄酮和黄酮醇的生物合成、脂肪酸生物合成、类二苯乙烯生物合成、脂肪酸代谢、丙酮酸代谢、半胱氨酸和蛋氨酸的代谢、昼夜节律植物。

Figure 8. KEGG enrichment on differential genes associated with anthocyanin biosynthesis

1–12 was flavonoid biosynthesis, phenylalanine metabolism, phenylpropanoid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, propanoate metabolism, flavone and flavonol biosynthesis, fatty acid biosynthesis, stilbenoid biosynthesis, fatty acid metabolism, pyruvate metabolism, cysteine and methionine metabolism, circadian rhythm – plant, respectively.

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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 on RNA-Seq data

*: significant difference at P＜0.05; **: extremely significant difference at P＜0.01.

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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/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

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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/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

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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 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

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