基于第三代纳米孔测序技术的茶树全长转录组分析

王芬; 木仁; 张雯; 马媛; 郭治友; 崔宝禄; 李静

doi:10.19303/j.issn.1008-0384.2021.10.009

基于第三代纳米孔测序技术的茶树全长转录组分析

doi: 10.19303/j.issn.1008-0384.2021.10.009

黔南民族师范学院生物科学与农学院，贵州　都匀　558000

基金项目: 国家自然科学基金项目（31900486）；贵州省科技计划项目（黔科合基础[2019]1298）；贵州省教育厅项目（黔教合KY字[2018]420、[2016]020、2017347、（2015）337、QNYSKYPT2018007）；国中医药办科技函（[2018]132号、[2019]186号）；黔南民族师范学院科研创新团队（Qnsyk201605、QNYSKYTD2018011）；贵州省植物学重点支持学科开放基金（qnsyzw1809）；黔南民族师范学院项目（QNSY2018ZJ006、QNSY2018CG007、2018xjg0520、2019xjg0303）

详细信息

作者简介:
王芬（1984−），女，博士研究生，副教授，主要从事生物信息学教学及研究工作（E-mail: fenmin521@163.com）

中图分类号: Q 945
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出版历程
- 收稿日期: 2021-06-10
- 修回日期: 2021-07-21
- 网络出版日期: 2021-10-23
- 刊出日期: 2021-10-28

Full-length Transcriptomes of Tea Obtained by Oxford Nanopore Technology

Department of Life Science and Agriculture, Qiannan Normal University for Nationalities, Duyun, Guizhou　558000, China

摘要

摘要: 目的研究茶树的全长转录组测序，以期为解析控制茶叶质量形成的分子机制提供理论依据。方法以贵州都匀地区福鼎大白茶叶、根和茎为材料，采用牛津大学的纳米孔技术（Oxford Nanopore Technology, ONT）进行全长转录组测序；将茶树叶、根和茎进行基因表达量分析；为了验证转录组测序结果的可靠性，随机抽取4个基因进行实时荧光定量PCR（qRT-PCR）。结果（1）获得69 379条全长转录本、93 102个SSR、45 852个ORF、6 335个转录因子和2 229个lncRNA；（2）获得叶与根、叶与茎以及茎与根的差异转录本，叶与根的差异转录本注释到GO、KEGG和KOG数据库分别有9 646、2 825和7 349个，其中参与茶叶品质形成中茶叶滋味相关代谢途径的有各种氨基酸代谢、类黄酮生物合成和苯丙素生物合成等，与香气相关的代谢途径有泛醌和其他萜烯醌生物合成等；qRT-PCR结果显示其表达量与转录组相一致。结论筛选到2个差异基因（ONT.24127.2和TEA003892.1）与谷氨酸合成酶有关，在根部合成茶氨酸后再运输到叶部，进而影响茶叶的品质。研究结果为保障茶叶产量和质量打下良好基础，为后续良种选育和引种及开展后续分子生物学研究提供重要的数据支持。
- 茶树 /
- 福鼎大白茶 /
- 纳米孔技术 /
- 全长转录组
Abstract: Objective The full-length transcriptomes of tissues of the famed Camellia sinensis (L.) O. Kuntze cv. Fuding Dabaicha were obtained to facilitate cultivar selection in upgrading the Duyun Maojian tea industry and increasing farmers’ income in Guizhou province. Method The Oxford nanopore technology (ONT) was applied to secure the full-length transcriptomes in the leaves, roots, and stems of the tea plant. Gene expressions in the tissues were analyzed. For verification of the ONT results, expressions of 4 randomly selected genes were tested by qRT-PCR. Result The ONT sequencing rendered 69,379 full-length transcripts with approximately 93,102 SSRs, 45,852 ORFs, 6,335 transcription factors, and 2,229 lncRNAs. The differential expression transcripts (DETs) among the leaves, roots, and stems were identified. Between the leaves and roots, there were 9,646 DETs annotated to GO database, 2,825 to KEGG, and 7,348 to KOG. The metabolisms of various amino acids, flavonoid biosynthesis, and phenylpropanol biosynthesis were related to the taste that affects the quality of a tea. Whereas the metabolic pathways involving aroma formation might include biosynthesis of ubiquinone and other terpene quinone. The ONT transcriptomes agreed with the qRT-PCR test results. Conclusion Two differentially expressed genes, ONT.24127.2 and TEA003892.1, were identified to be related to glutamate synthase. Theanine, which is closely associated with tea quality, was synthesized in the roots, then transported to the leaves. The full-length transcriptomes on Fuding Dabaicha demonstrated the practical application of the information for tea cultivar selection.
- Tea plant /
- Fuding Dabaicha /
- Oxford nanopore technology /
- full-length transcriptome

HTML全文

图 1 开放阅读框、转录因子、长链非编码RNA和转录本表达量分析

注：A，ORF编码区氨基酸序列长度分布；B，转录因子种类分布；C，Cnci、Cpc、Pfam、和Cpat四种方法预测的lncRNA韦恩图；D，叶根和茎转录本表达量箱线图。

Figure 1. Analyses on ORF, transcription factor, lncRNA, and transcript expression

Note: A: Distribution of lengths of amino acid sequence in ORF coding region; B: Distribution of transcription factor types; C: Venn graph of lncRNA predicted by 4 methods; D: CPM boxplot of transcripts of leaf, root, and stem.

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图 2 叶和根的差异表达转录本GO注释

注：1：代谢过程；2：细胞过程；3：单组织过程；4：生物调节；5：应激反应；6：定位；7：细胞成分组织或生物合成；8：发育过程；9：多细胞生物过程；10：信号；11：生殖；12：生殖过程；13：多组织过程；14：解毒作用；15：生长；16：免疫系统过程；17：节律性过程；18：运动；19：细胞杀伤性；20：生物附着；21：行为；22：细胞；23：细胞组件；24：细胞膜；25：细胞器；26：细胞膜组件；27：细胞器组件；28：大分子复合物；29：细胞膜内腔；30：胞外区；31：细胞连接；32：共质体；33：超分子复合物；34：病毒粒子；35：病毒粒子组件；36：胞外区组件；37：拟核；38：催化活性；39：结合；40：转运活性；41：结构分子活性；42：核酸结合转录因子活性；43：分子功能调节因子；44：信号转导活性；45：抗氧化活性；46：电子载体活性；47：转录因子活性，蛋白结合；48：分子转导活性；49：营养库活性；50：蛋白标签；51：金属伴侣活性；52：翻译调控活性。

Figure 2. GO annotation of DETs between leaf and root

Note: 1: Metabolic process; 2: Cellular process; 3: Single-organism process; 4: Biological regulation; 5: Response to stimulus; 6: Localization; 7: Cellular component organization or biogenesis; 8: Developmental process; 9: Multicellular organismal process; 10: Signaling; 11: Reproduction; 12: Reproductive process; 13: Multi-organism process; 14: Detoxification; 15: Growth; 16: Immune system process; 17: Rhythmic process; 18: Locomotion; 19: Cell-killing; 20: Biological adhesion; 21: Behavior; 22: Cell; 23: Cell part; 24: Membrane; 25: Organelle; 26: Membrane part; 27: Organelle part; 28: Macromolecular complex; 29: Membrane-enclosed lumen; 30: Extracellular region; 31: Cell junction; 32: Symplast; 33: Supramolecular complex; 34: Virion; 35: Virion part; 36: Extracellular region part; 37: Nucleoid; 38: Catalytic activity; 39: Binding; 40: Transporter activity; 41: Structural molecule activity; 42: Nucleic acid binding transcription factor activity; 43: Molecular function regulator; 44: Signal transducer activity; 45: Antioxidant activity; 46: Electron carrier activity; 47: Transcription factor activity, protein-binding; 48: Molecular transducer activity; 49: Nutrient reservoir activity; 50: Protein tag; 51: Metallochaperone activity; 52: Translation regulator activity.

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图 3 叶与根差异表达转录本KEGG功能分类

注：1：吞噬体；2：内吞作用；3：过氧化物酶体；4：植物激素信号转导；5：核糖体；6：RNA降解；7：内质网蛋白加工；8：氨酰tRNA生物合成；9：谷胱甘肽代谢；10：光合作用；11：光合生物的固碳作用；12：乙醛酸和二羧酸代谢；13：淀粉和蔗糖代谢；14：苯丙素生物合成；15：氨基酸生物合成；16：碳代谢；17：色氨酸代谢；18：苯丙氨酸、络氨酸和色氨酸生物合成；19：络氨酸代谢；20：缬氨酸、亮氨酸和异亮氨酸降解；21：磷酸肌醇代谢；22：莨菪碱、哌啶和吡啶生物碱的生物合成；23：苯丙氨酸代谢；24：硫代谢；25：氧化磷酸化；26：泛醌和其他萜类醌生物合成；27：类胡萝卜素生物合成；28：抗坏血酸和醛酸代谢；29：戊糖和葡萄糖醛酸转换；30：氮代谢；31：半乳糖代谢；32：果糖和甘露糖代谢；33：氰基氨基酸代谢；34：甘油磷脂代谢；35：α亚麻酸代谢；36：嘧啶代谢；37：脂肪酸降解；38：磷酸戊糖途径；39：类黄酮生物合成；40：萜类化合物生物合成；41：脂肪酸代谢；42：嘌呤代谢；43：卟啉和叶绿素代谢；44：丙酮酸代谢；45：甘氨酸、丝氨酸和苏氨酸代谢；46：光合作用天线蛋白；47：氨基糖和核苷酸糖代谢；48：糖酵解/葡萄糖异生作用；49：半胱氨酸和蛋氨酸代谢；50：植物病原菌互作。

Figure 3. KEGG function annotation of DETs between leaf and root

Note: 1: Phagosome; 2: Endocytosis; 3: Peroxisome; 4: Plant hormone signal transduction; 5: Ribosome; 6: RNA degradation; 7: Protein processing in endoplasmic reticulum; 8: Aminoacyl-tRNA biosynthesis; 9: Glutathione metabolism; 10: Photosynthesis; 11: Carbon fixation in photosynthetic organisms; 12: Glyoxylate and dicarboxylate metabolism; 13: Starch and sucrose metabolism; 14: Phenylpropanoid biosynthesis; 15: Biosynthesis of amino acids; 16: Carbon metabolism; 17: Tryptophan metabolism; 18: Phenylalanine, tyrosine and tryptophan biosysnthesis; 19: Tyrosine metabolism; 20: Valine, leucine and isoleucine degradation; 21: Inositol phosphate metabolism; 22: Tropane, piperidine and pyridine alkaloid biosynthesis; 23: Phenylalanine metabolism; 24: Sulfur metabolism; 25: Oxidative phosphorylation; 26: Ubiquinone and other terpenoid-quinone biosynthesis; 27: Carotenoid biosynthesis; 28: Ascorbate and aldarate metabolism; 29: Pentose and glucuronate interconversions; 30: Nitrogen metabolism; 31: Galactose metabolism; 32: Fructose and mannose metabolism; 33: Cyanoamino acid metabolism; 34: Glycerophospholipid metabolism; 35: Alpha-Linolenic acid metabolism; 36: Pyrimidine metabolism; 37: Fatty acid degradation; 38: Pentose phosphate pathway; 39: Flavonoid biosynthesis; 40: Terpenoid backbone biosynthesis; 41: Fatty acid metabolism; 42: Purine metabolism; 43: Porphyrin and chlorophyll metabolism; 44: Pyruvate metabolism; 45: Glycine, serine and threonine metabolism; 46: Photosynthesis-antenna proteins; 47: Amino sugar and nucleotide sugar metabolism; 48: Glycolysis/gluconeogenesis; 49: Cysteine and methionine metabolism; 50: Plant-pathogen interaction.

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图 4 转录组测序结果与qRT-PCR表达量比对

Figure 4. Comparison between RNA-seq and qRT-PCR

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表 1 qRT-PCR引物

Table 1. qRT-PCR primer

引物名称 The name of primer	引物序列（5′-3′） The sequence of primer（5′-3′）
ONT.4041-1-F	TTTGTGGAATGCCCTTTA
ONT.4041-1-R	GGAAGAATCAATACTGTCAAG
ONT.8670-1-F	AGGATTGGATGGCGTGTG
ONT.8670-1-R	GACTCTGCTTGGCTTCTTCA
TEA019914.1_gene-1-F	AAGTTGTTGCTGATTGATG
TEA019914.1_gene-1-R	GGTAATGAAGATGGAGTGT
TEA007016.1_gene-1-F	GCTCATCAGTCAGGTTAT
TEA007016.1_gene-1-R	GAGGCTGTCTTGTAGTAG
GE651107.1 EST1434-1-F（内参基因）	CCCTCTCTCCCTTTATCACTCA
GE651107.1 EST1434-1-R（内参基因）	CCACCAACCGACCAATCC

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表 2 Clean data数据

Table 2. Clean data

样品名 Sample ID	Reads数 The number of total Reads	Clean data平均长度 Mean length of clean data	Clean data N50值 N50 of clean data	全长序列数 Full length sequence number	全长序列占比 Full length sequence proportion	比对到参考转录组Reads个数 The number of mapped reads	比对到参考转录组Reads数目在全长序列中占的百分比 Mapped rates
L01	6 576 976	1 364	1 503	4 215 412	64.17%	5 609 570	85.29%
L02	6 741 658	1 380	1 526	4 819 053	71.55%	6 072 332	90.07%
L03	6 273 400	1 393	1 553	4 209 568	67.16%	5 496 900	87.62%
R01	7 421 517	1 400	1 605	5 078 770	68.59%	6 132 473	82.63%
R02	5 908 300	1 345	1 514	3 879 967	65.74%	5 049 284	85.46%
R03	6 290 898	1 452	1 669	4 303 999	68.54%	5 430 183	86.32%
S01	7 184 552	1 444	1 667	4 665 036	65.13%	6 003 589	83.56%
S02	5 715 955	1 403	1 612	4 045 698	70.84%	5 178 238	90.59%
S03	5 616 854	1 412	1 613	3 768 495	67.17%	4 855 092	86.44%

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表 3 新转录本注释

Table 3. New isoform annotation

注释数据库 Databases	被注释的新转录本数 Annotated new isoform number
COG	16 999
GO	36 663
KEGG	24 246
KOG	35 099
Pfam	31 029
Swiss-Prot	39 795
eggNOG	56 578
NR	58 139
All	58 398

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表 4 差异表达转录本注释

Table 4. Annotation of DETs

差异表达转录本 DET	差异表达转录本数目 DET Number	上调转录本数目 Up- regulated	下调转录本数目 Down- regulated	注释到的差异表达转录本数目 Annotated DET	注释到 COG的数目 COG	注释到 GO的数目 GO	注释到 KEGG的数目 KEGG	注释到 KOG的数目 KOG	注释到 NR的数目 NR	注释到 Pfam的数目 Pfam	注释到 Swiss-Prot的数目 Swiss-Prot	注释到 eggNOG的数目 eggNOG
L01_L02_L03_vs_R01_R02_R03	15 675	7 415	8 260	14 306	5 347	9 649	2 825	7 349	14 166	8 817	10 475	13 859
L01_L02_L03_vs_S01_S02_S03	9 228	4 224	5 004	8 497	3 120	5 865	2 911	4 075	8 423	5 277	6 238	8 243
S01_S02_S03_vs_R01_R02_R03	7 633	4 093	3 540	6 942	2 533	4 739	2 279	3 468	6 876	4 529	5 326	6 688

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表 5 部分叶与根差异转录本的KOG分类

Table 5. DETs KOG classification between leaf and root

KOG分类　　　　 KOG classification	KOG注释的转录本数 Number of annotated DETs
信号转导机制 Signal transduction mechanisms	650
能源生产与转换 Energy production and conversion	534
无机离子的转运和代谢 Inorganic ion transport and metabolism	398
防御机制 Defense mechanisms	168
细胞骨架 Cytoskeleton	136
辅酶的运输和代谢 Coenzyme transport and metabolism	108
RNA加工与修饰 RNA processing and modification	91
核苷酸运输与代谢 Nucleotide transport and metabolism	80
染色质结构和动力学 Chromatin structure and dynamics	30
真核细胞的细胞外结构 Extracellular structures	20
核结构 Nuclear structure	6
细胞运动 Cell motility	0

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