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

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

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
计量
- 文章访问数: 38
- HTML全文浏览量: 17
- PDF下载量: 3
- 被引次数: 0
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

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

School of Food and Pharmaceutical Engineering (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 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 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

HTML全文

图 1 试验材料

Figure 1. Experimental materials

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

图 7 花青素生物合成差异基因GO富集分析

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

下载: 全尺寸图片幻灯片

图 8 花青素生物合成差异基因KEGG富集

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

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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

下载: 导出CSV

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

下载: 导出CSV

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

下载: 导出CSV

参考文献(24)

[1]	刘富知, 黄建安, 付冬和, 等. 茶树上红紫色芽叶部分生化特性的研究 [J]. 湖南农业大学学报, 2000, 26(1):41−42,57. LIU F Z, HUANG J A, FU D H, et al. Biochemical properties of new shoots of tea trees [J]. Journal of Hunan Agricultural University, 2000, 26(1): 41−42,57. (in Chinese)
[2]	张凯凯, 苏鸿锋, 林泳恩, 等. 高花青素茶呈色与花青素积累机制研究进展 [J]. 食品安全质量检测学报, 2022, 13(11):3585−3592. doi: 10.3969/j.issn.2095-0381.2022.11.spaqzljcjs202211025 ZHANG K K, SU H F, LIN Y E, et al. Research progress on the mechanism of color generation and anthocyanin accumulation in anthocyanin-rich tea [J]. Journal of Food Safety & Quality, 2022, 13(11): 3585−3592. (in Chinese) doi: 10.3969/j.issn.2095-0381.2022.11.spaqzljcjs202211025
[3]	LAI Y S, LI S, TANG Q, et al. The dark-purple tea cultivar ‘ziyan’ accumulates a large amount of delphinidin-related anthocyanins [J]. Journal of Agricultural and Food Chemistry, 2016, 64(13): 2719−2726. doi: 10.1021/acs.jafc.5b04036
[4]	解东超. 紫娟茶中花青素及其在加工过程中变化规律研究[D]. 北京: 中国农业科学院, 2017. XIE D C. Research on anthocyanins of Zijuan and their varying patterns during the process[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese)
[5]	DA SILVA T B V, CASTILHO P A, SÁ-NAKANISHI A B, et al. The inhibitory action of purple tea on in vivo starch digestion compared to other Camellia sinensis teas[J]. Food Research International, 2021, 150(Pt A): 110781.
[6]	王萌, 常格, 王琦, 等. 4种园林树木叶片秋季变色期的呈色机理 [J]. 林业与生态科学, 2020, 35(1):93−98. WANG M, CHANG G, WANG Q, et al. The leaf color mechanism of four tree species with red leaves in autumn color-changing period [J]. Forestry and Ecological Sciences, 2020, 35(1): 93−98. (in Chinese)
[7]	夏溪, 龚睿, 张春英. 不同颜色锦绣杜鹃花瓣中花青素苷组成及呈色机制[J]. 江苏农业学报, 2022, 38(1): 207−213. XIA X, GONG R, ZHANG C Y. Anthocyanin composition and coloration mechanism in petals of Rhododendron pulchrum with different colors[J]. Jiangsu Journal of Agricultural Sciences, 2022, 38(1): 207−213. (in Chinese)
[8]	李健. ‘紫娟’茶树紫叶花青素积累机理的转录组分析[D]. 福州: 福建农林大学, 2016. LI J. Transcriptome analysis reveals the accumulation mechanism of anthocyanins in ’zijuan’ tea [Camellia Sinensis Var. Asssamica (Masters) Kitamura] leaves[D]. Fuzhou: Fujian Agriculture and Forestry University, 2016. (in Chinese)
[9]	向奕, 刘硕谦, 龚志华, 等. 茶树紫芽中花青素形成相关基因差异表达分析 [J]. 茶叶科学, 2018, 38(5):439−449. doi: 10.3969/j.issn.1000-369X.2018.05.001 XIANG Y, LIU S Q, GONG Z H, et al. Differential expression analysis of genes related to anthocyanin biosynthesis in purple buds of tea plant(Camellia sinensis) [J]. Journal of Tea Science, 2018, 38(5): 439−449. (in Chinese) doi: 10.3969/j.issn.1000-369X.2018.05.001
[10]	刘悦, 曲浩, 李友勇, 等. 红芽直立茶紫色叶形成机制的转录组分析 [J]. 茶叶通讯, 2019, 46(4):409−418. doi: 10.3969/j.issn.1009-525X.2019.04.006 LIU Y, QU H, LI Y Y, et al. Transcriptome analysis of the formation mechanism of purple leaf in Camellia assamica cv. with erect red bud [J]. Journal of Tea Communication, 2019, 46(4): 409−418. (in Chinese) doi: 10.3969/j.issn.1009-525X.2019.04.006
[11]	许丽颖, 刘斗南, 刘月. 不同茶树品种叶片的花青素研究进展 [J]. 福建茶叶, 2019, 41(3):4−5. doi: 10.3969/j.issn.1005-2291.2019.03.003 XU L Y, LIU D N, LIU Y. Research progress of anthocyanins in leaves of different tea varieties [J]. Tea in Fujian, 2019, 41(3): 4−5. (in Chinese) doi: 10.3969/j.issn.1005-2291.2019.03.003
[12]	ZHENG X L, TIAN S P. Effect of oxalic acid on control of postharvest browning of litchi fruit [J]. Food Chemistry, 2006, 96(4): 519−523. doi: 10.1016/j.foodchem.2005.02.049
[13]	WANG L K, FENG Z X, WANG X, et al. DEGseq: An R package for identifying differentially expressed genes from RNA-seq data [J]. Bioinformatics, 2010, 26(1): 136−138. doi: 10.1093/bioinformatics/btp612
[14]	CHEN C J, CHEN H, HE Y H, et al. TBtools, a Toolkit for Biologists integrating various biological data handling tools with a user-friendly interface [J]. BioRxiv, 2018, 13: 1194−1202.
[15]	YAN F, ZHU Y C, ZHAO Y N, et al. De novo transcriptome sequencing and analysis of salt-, alkali-, and drought-responsive genes in Sophora alopecuroides [J]. BMC Genomics, 2020, 21(1): 423. doi: 10.1186/s12864-020-06823-4
[16]	吴华玲, 乔小燕, 李家贤, 等. “红紫芽” 茶树新品系的生物学特性研究 [J]. 热带作物学报, 2011, 32(6):1009−1015. doi: 10.3969/j.issn.1000-2561.2011.06.005 WU H L, QIAO X Y, LI J X, et al. Biological characters of new tea germplasms with reddishviolet shoots [J]. Chinese Journal of Tropical Crops, 2011, 32(6): 1009−1015. (in Chinese) doi: 10.3969/j.issn.1000-2561.2011.06.005
[17]	FREYRE R, UZDEVENES C, GU L W, et al. Genetics and anthocyanin analysis of flower color in Mexican Petunia [J]. Journal of the American Society for Horticultural Science, 2015, 140(1): 45−49. doi: 10.21273/JASHS.140.1.45
[18]	蒋会兵, 夏丽飞, 田易萍, 等. 基于转录组测序的紫芽茶树花青素合成相关基因分析 [J]. 植物遗传资源学报, 2018, 19(5):967−978. JIANG H B, XIA L F, TIAN Y P, et al. Transcriptome analysis of anthocyanin synthesis related genes in purple bud tea plant [J]. Journal of Plant Genetic Resources, 2018, 19(5): 967−978. (in Chinese)
[19]	周琼琼, 孙威江. 茶树芽叶紫化的生理生化分析及其关键酶基因的表达 [J]. 生物技术通报, 2015, 31(1):86−91. ZHOU Q Q, SUN W J. Physiological and biochemical analysis of young shoot purple-related and gene expression of key enzymes in tea plant(Camellia sinensis) [J]. Biotechnology Bulletin, 2015, 31(1): 86−91. (in Chinese)
[20]	周天山, 王新超, 余有本, 等. 紫芽茶树类黄酮生物合成关键酶基因表达与总儿茶素、花青素含量相关性分析 [J]. 作物学报, 2016, 42(4):525−531. doi: 10.3724/SP.J.1006.2016.00525 ZHOU T S, WANG X C, YU Y B, et al. Correlation analysis between total catechins(or anthocyanins) and expression levels of genes involved in flavonoids biosynthesis in tea plant with purple leaf [J]. Acta Agronomica Sinica, 2016, 42(4): 525−531. (in Chinese) doi: 10.3724/SP.J.1006.2016.00525
[21]	范晶, 黄明远, 吴苗苗, 等. 山茶属三个F3H基因的分子特性、系统进化及蛋白结构差异分析 [J]. 基因组学与应用生物学, 2016, 35(5):1195−1205. FAN J, HUANG M Y, WU M M, et al. Comparison of molecular characteristics, phylogeny and structure of three Camellia F3H genes [J]. Genomics and Applied Biology, 2016, 35(5): 1195−1205. (in Chinese)
[22]	MEI Y, XIE H, LIU S R, et al. Metabolites and transcriptional profiling analysis reveal the molecular mechanisms of the anthocyanin metabolism in the "Zijuan" tea plant (Camellia sinensis var. assamica) [J]. Journal of Agricultural and Food Chemistry, 2021, 69(1): 414−427. doi: 10.1021/acs.jafc.0c06439
[23]	TIAN J, HAN Z Y, ZHANG J, et al. The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapples [J]. Scientific Reports, 2015, 5: 12228. doi: 10.1038/srep12228
[24]	宋中邦, 李文正, 高玉龙, 等. 烟草FLS基因表达抑制对类黄酮代谢的影响 [J]. 基因组学与应用生物学, 2016, 35(12):3501−3506. SONG Z B, LI W Z, GAO Y L, et al. Suppression of tobacco FLS genes' expression and its effect on flavonoid metabolism [J]. Genomics and Applied Biology, 2016, 35(12): 3501−3506. (in Chinese)