畲药十二时辰基源植物重瓣铁线莲的全长转录组分析

何舒澜; 柯兰兰; 陈观水

畲药十二时辰基源植物重瓣铁线莲的全长转录组分析

1.
福建卫生职业技术学院，福建　福州　350101
2.
福建医科大学基础医学院，福建　福州　350108
3.
福建农林大学生命科学学院，福建　福州　350002

基金项目: 福建省职业院校教师教学创新团队立项建设项目；福建省大学生创新创业项目（S202110389044）；福建卫生职业技术学院教学创新团队专项（JG2021103）

详细信息

作者简介:
何舒澜（1980 —），男，硕士，副教授，主要从事中草药栽培与鉴定研究，E-mail: 181329113@qq.com

通讯作者:
陈观水（1979 —），男，博士，副教授，主要从事药用植物次生代谢研究，E-mail: gshchenfafu@163.com

中图分类号: S68
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出版历程
- 收稿日期: 2024-08-02
- 录用日期: 2024-10-16
- 修回日期: 2024-09-12
- 网络出版日期: 2024-11-11

Full-length Transcriptome of She Herbal Medicine Plant Clematis florida

1.
School of Pharmacy, Fujian Health College, Fuzhou, Fujian　350101, China
2.
School of Basic Medical Science, Fujian Medical University, Fuzhou, Fujian　350108, China
3.
College of Life Science, Fuian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的重瓣铁线莲是畲药十二时辰基源植物，以根入药，含有丰富的活性成分，本研究旨在了解重瓣铁线莲基因注释信息，次级代谢产物代谢通路及基因功能，丰富其转录组信息，为进一步筛选和鉴定其药用成分代谢通路相关基因奠定基础。方法利用-SMRT测序技术，对重瓣铁线莲根进行全长转录组测序，并借助生物信息学工具进行功能注释、结构分析以及萜类合成途径基因的挖掘。结果测序共获得高质量测序subreads数据量为62.21 Gb，通过数据处理分析，获到20540条高质量去冗余转录本。利用NR, GO, NT, Pfam, COG/KOG, SwissProt, KEGG等7个数据库对高质量去冗余序列进行基因功能注释，共有19909条转录本至少在1个数据库得到注释，有8888条转录本在NR、NT、COG/KOG、KEGG、GO等5个公共数据库中均有注释。GO注释结果显示，14911条转录本共富集在包含生物学过程、细胞组成和分子功能三大类的53个条目中。KEGG分析显示，19701条转录本序列被富集到6条主通路和44条子通路。COG/KOG注释发现，13204条转录本被注释，其中注释最多的功能类别为一般功能预测。对转录本结构分析发现，共预测到978个转录因子、224条LncRNA、7167个SSR。对萜类化合物生物合成途径进行挖掘，共鉴定到48个转录本（16个关键酶候选基因）参与萜类骨架生物合成。结论本研究成功对畲药十二时辰基源植物重瓣铁线莲的根进行了全长转录组测序，获得该物种的全长转录组相关基因功能信息，填补了重瓣铁线莲的基因信息的空白，为深入研究重瓣铁线莲的调控网络、生物学特征、相关代谢途径、信号通路及分子机制等提供参考。
- 重瓣铁线莲 /
- 全长转录组 /
- 转录因子 /
- 长非编码RNA
Abstract: Objective Transcription of herbal plant Clematis florida Thunb. var. plena D. Don was used to identify the key genes involved in the metabolic pathways of the active compounds in the famed “Shiershichen” of She medicine. Method PacBio single-molecule real-time (SMRT) was employed to sequence the full-length transcriptome of the roots of the herbal plant that contains the active compounds of the She medicine. Functional annotation, gene structure, and mining of the terpenoid biosynthetic pathway were conducted using bioinformatics tools to secure the transcript data. Result From the 62.21 G polymerase read bases generated, 20,540 non-redundant high-quality transcript sequences were identified after data treatment. At least one of the 7 major databases including NR, NT, Pfam, COG/KOG, SwissProt, GO, and KEGG applied annotated the gene functions of 19,909 transcripts, and 8,888 were in NR, NT, COG/KOG, KEGG, and GO. The GO annotation showed 14,911 transcripts enriched in 53 terms that included biological processes, cellular components, and molecular functions. The KEGG database annotated 19,701 transcripts and classified them into 6 major pathways and 44 sub-pathways with the largest number of transcripts enriched in metabolic pathways. The COG/KOG annotation identified 13,204 transcripts with the general function prediction being the most predominant. There were 978 transcription factors, 224 lncRNAs, and 7,167 SSRs predicted, and 48 transcripts with 16 key candidate genes involved in the terpenoid backbone biosynthesis identified. Conclusion The full-length transcriptome sequencing on the roots of C. florida was successfully obtained with the comprehensive gene function information. It provided a basis for further studies on the regulatory network, biological characteristics, related metabolic pathways, signaling pathways, and molecular mechanisms associated with the well-known She herbal medicine.
- Clematis florida /
- full-length transcriptome /
- transcription /
- LncRNA

HTML全文

图 1 重瓣铁线莲转录本在七大数据库注释结果

Figure 1. Transcripts annotated in 7 databases

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图 2 5个常用数据库功能注释维恩图

Figure 2. Venn diagram of isoform annotations according to 5 databases

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图 3 NR数据库中比对到前10种物种名称及转录本数目

Figure 3. Top 10 species distribution annotated by NR database

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图 4 重瓣铁线莲全长转录本的GO功能注释

Figure 4. Gene ontology classification in isoforms of C. florida

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图 5 重瓣铁线莲全长转录本的COG/KOG功能注释及分类

Figure 5. COG/KOG function annotation and classification in isoforms of C. florida

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图 6 重瓣铁线莲全长转录组SSR分布图

Figure 6. Distribution of SSR motifs in isoforms of C. florida

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图 7 SMRT 预测的前10个转录因子的数量和家族

Figure 7. Number and family of top 10 transcription factors predicted by SMRT

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图 8 重瓣铁线莲长非编码RNA分析结果

Figure 8. Long non-coding RNAs (lncRNAs) in isoforms of C. florida

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图 9 重瓣铁线莲LncRNA与mRNA长度分布图

Figure 9. Distribution of lengths of LncRNA and mRNA in C. florida

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表 1 单分子实时测序数据统计

Table 1. Statistics of sequencing data by SMRT

类别 Type	总数 Total number	最小长度 Min length/bp	最大长度 Max length/bp	平均长度 Mean length/bp	N50长度 N50 length/bp
原始序列中的单个子序列 Subreads	27863322	—	—	2233	2469
环形一致性序列 Circular consensus sequence (CCS)	595827	54	14994	2488	2659
全长非嵌序列 Full-Length non-chimericRead （FLNC）	494732	57	14860	2333	2531
优化一致性序列 Polished consensus sequence	41768	87	6251	2272	2468
去冗余高质量序列 Isoform	20540	87	6251	2248	2457

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表 2 转录本KEGG注释结果

Table 2. KEGG annotated transcripts

KEGG主通路 Main pathway	KEGG子通路 Sub-pathway of KEGG	转录本数量 Number of transcripts	KEGG主通路 Main pathway	KEGG子通路 Sub-pathway of KEGG	转录本数量 Number of transcripts
细胞过程 Cellular Process	运输与催化 Transport and catabolism	595	代谢 Metabolism	异种生物降解和代谢 Xenobiotics biodegradation and metabolism	85
	细胞群落-原核生物 Cellular community-prokaryotes	93		核酸代谢 Nucleotide metabolism	198
	细胞群落-真核生物 Cellular community-eukaryotes	124		萜类和聚酮类代谢 Metabolism of terpenoids and polyketides	203
	细胞运动 Cell motility	39		其他氨基酸代谢 Metabolism of other amino acids	333
	细胞生和与死亡 Cell growth and death	276		辅助因子和维生素代谢 Metabolism of cofactors and vitamins	345
环境信息处理 Environmental information Processing	信号转导 Signal transduction	984		脂类代谢 Lipid metabolism	437
环境信息处理 Environmental information Processing	膜运输 Membrane transport	51		聚糖生物合成和代谢 Glycan biosynthesis and metabolism	171
遗传信息处理 Genetic information Processing	翻译 Translation	806		全局和总览图 Global and overview maps	718
	转录 Trancription	480		能量代谢 Eenergy metabolism	540
	复制与修复 replication and repair	176		碳水化合物代谢 Carbohydrate metabolism	1010
	折叠，分类和降解 Folding, sorting and degradation	657		其他生长代谢物合成 Biosynthesis of other secondary metabolites	243
人类疾病 Human Diseases	药物依赖 Substance dependence	44		氨基酸代谢 Amino acid metabolism	561
	神经退行性疾病 Neurodegenerative diseases	165	组织系统 Organismal Systems	感官系统 Sensory system	15
	感染疾病-病毒 Infectious diseases:Viral	496		神经系统 Nervours system	294
	感染疾病-寄生生物 Infectious diseases:Parasitic	217		免疫系统 Immune system	359
	感染疾病-细菌 Infectious diseases:Bacterial	309		分泌系统 Excretory system	51
	免疫疾病 Immune diseases	35		环境适应 Environmental adaptation	362
	内分泌与代谢疾病 Endocrine and metabolic diseases	141		内分泌系统 Endocrine system	404
	耐药性：抗肿瘤药 Drug resistance:Antineoplastic	94		消化系统 Digestive system	77
	心血管疾病 Cardiovascular diseases	88		发育 Development	17
	癌症：特殊类型 Cancers:Specific types	86		循环系统 Circulatory system	57
	癌症：总览 Cancers:Overview	384		老龄化 Aging	148

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表 3 重瓣铁线莲转录组中与萜类化合物合成相关的转录本

Table 3. Isoforms encoding key enzymes involved in terpenoid biosynthesis in C. florida

合成途径 Pathway	关键酶名称 Key enzyme name	酶的系统编号 Enzyme No	转录本数 No. of Isoform
MVA途径 MVA Pathway	羟甲基戊二酰辅酶A合酶 hydroxymethylglutaryl-CoA synthase (HMGS)	EC:2.3.3.10	1
	羟甲基戊二酰辅酶A还原酶 hydroxymethylglutaryl-CoA reductase(HMGR)	EC:1.1.1.34	2
	甲羟戊酸激酶 mevalonate kinase(MK)	EC:2.7.1.36	1
	磷酸甲羟戊酸激酶 phosphomevalonate kinase(PMK)	EC:2.7.4.2	1
	二磷酸甲羟戊酸脱羧酶 diphosphomevalonate decarboxylase (MVD)	EC:4.1.1.33	2
MEP途径 MEP Pathway	1-脱氧-D-木酮糖5-磷酸合酶 1-deoxy-D-xylulose-5-phosphate synthase(DXS)	EC:2.2.1.7	11
	1-脱氧-D-木酮糖5-磷酸还原异构酶 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)	EC:1.1.1.267	5
	2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase(MCT)	EC:2.7.7.60	1
	4-二磷酸二胺-2-C-甲基-D-赤藓糖醇激酶 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase(CMK)	EC:2.7.1.148	1
	4-羟基-3-甲丁-2-烯基二磷酸合酶 4-hydroxy-3-methylbut-2-enyl-diphosphate synthase(HDS)	EC:1.17.7.1	9
	4-羟基-3-甲丁-2-烯基二磷酸还原酶 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR)	EC:1.17.7.4	2
分支点 Branch points	异戊烯二磷酸Delta-异构酶 Isopentenyl-diphosphate delta-isomerase(IPPI)	EC:5.3.3.2	2
	牻牛儿基牻牛儿基焦磷酸合酶 geranylgeranyl diphosphate synthase(GGPS)	EC: 2.5.1.29	6
	法呢基焦磷酸合酶 farnesyl diphosphate synthase(FPPS)	EC:2.5.1.10	1
	蛋白香叶烯基转移酶 protein farnesyltransferase subunit (FNTB)	EC:2.5.1.58	1
	内肽酶endopeptidase(STE24)	EC:3.4.24.84	2

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参考文献(19)

[1]	黄泽豪, 张月玲. 畲药十二时辰的本草考证 [J]. 中国民族民间医药, 2014, 23(1):2−3,5. HUANG Z H, ZHANG Y L. Textual research on she medicine’s twelve o’clock materia Medica [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2014, 23(1): 2−3,5. (in Chinese)
[2]	何舒澜, 杨谨, 黄娴, 等. 重瓣铁线莲鲨烯合酶SQS基因的克隆及表达分析 [J]. 福建农业学报, 2022, 37(11):1430−1437. HE S L, YANG J, HUANG X, et al. Cloning and expression of squalene synthase gene from Clematis florida Thunb. var. plena D. don [J]. Fujian Journal of Agricultural Sciences, 2022, 37(11): 1430−1437. (in Chinese)
[3]	沈廷明, 黄春情, 吴军军, 等. GC-MS法分析畲药十二时辰花中挥发油成分 [J]. 中草药, 2020, 51(24):6362−6366. SHEN T M, HUANG C Q, WU J J, et al. GC-MS analysis of volatile oil from traditional She medicine Clematis florida var. plena flower [J]. Chinese Traditional and Herbal Drugs, 2020, 51(24): 6362−6366. (in Chinese)
[4]	YANG N N, ZHANG Y F, ZHANG H T, et al. The in vitro and in vivo anti-inflammatory activities of triterpene saponins from Clematis florida [J]. Natural Product Research, 2021, 35(24): 6180−6183. doi: 10.1080/14786419.2020.1833203
[5]	SUN K H, MA X H, ZENG X M, et al. A new indole-type alkaloid from the roots of Clematis florida var. plena [J]. Natural Product Research, 2019, 33(20): 2925−2931. doi: 10.1080/14786419.2018.1510396
[6]	WU Y, ZHANG Y F, ZHANG H T, et al. A new cyclopeptide alkaloid from Clematis Florida [J]. Natural Product Research, 2022, 36(7): 1693−1699. doi: 10.1080/14786419.2020.1809399
[7]	黄丽容, 郭晓云, 胡营, 等. 香附子全长转录组测序及生物信息学分析 [J]. 中国现代中药, 2023, 25(7):1428−1440. HUANG L R, GUO X Y, HU Y, et al. Full-length transcriptome sequencing and bioinformatics analysis of Cyperus rotundus L [J]. Modern Chinese Medicine, 2023, 25(7): 1428−1440. (in Chinese)
[8]	米琪, 赵艳丽, 徐萍, 等. 滇黄精全长转录组测序及生物信息学分析 [J]. 药学学报, 2024, 59(6):1864−1872. MI Q, ZHAO Y L, XU P, et al. Full-length transcriptome sequencing and bioinformatics analysis of Polygonatum kingianum [J]. Acta Pharmaceutica Sinica, 2024, 59(6): 1864−1872. (in Chinese)
[9]	HE B, SHAN T Y, XU J Y, et al. Full-length transcriptome profiling of Acanthopanax gracilistylus provides new insight into the kaurenoic acid biosynthesis pathway [J]. Physiology and Molecular Biology of Plants, 2024, 30(3): 383−399. doi: 10.1007/s12298-024-01436-7
[10]	BUCHFINK B, XIE C, HUSON D H. Fast and sensitive protein alignment using DIAMOND [J]. Nature Methods, 2015, 12(1): 59−60. doi: 10.1038/nmeth.3176
[11]	ZHENG Y, JIAO C, SUN H H, et al. iTAK: A program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases [J]. Molecular Plant, 2016, 9(12): 1667−1670. doi: 10.1016/j.molp.2016.09.014
[12]	THIEL T, MICHALEK W, VARSHNEY R K, et al. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L. ) [J]. TAG Theoretical and Applied Genetics Theoretische und Angewandte Genetik, 2003, 106(3): 411−422. doi: 10.1007/s00122-002-1031-0
[13]	SUN L, LUO H T, BU D C, et al. Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts [J]. Nucleic Acids Research, 2013, 41(17): e166. doi: 10.1093/nar/gkt646
[14]	KANG Y J, YANG D C, KONG L, et al. CPC2: A fast and accurate coding potential calculator based on sequence intrinsic features [J]. Nucleic Acids Research, 2017, 45(W1): W12−W16. doi: 10.1093/nar/gkx428
[15]	FINN R D, COGGILL P, EBERHARDT R Y, et al. The pfam protein families database: Towards a more sustainable future [J]. Nucleic Acids Research, 2016, 44(D1): D279−D285. doi: 10.1093/nar/gkv1344
[16]	LI A M, ZHANG J Y, ZHOU Z Y. PLEK: A tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme [J]. BMC Bioinformatics, 2014, 15(1): 311. doi: 10.1186/1471-2105-15-311
[17]	沈廷明, 陈懿冲, 黄春情, 等. 畲药十二时辰的质量标准研究 [J]. 海峡药学, 2023, 35(8):36−41. SHEN T M, CHEN Y C, HUANG C Q, et al. Quality standard of she medicine Clematis florida var. plena [J]. Strait Pharmaceutical Journal, 2023, 35(8): 36−41. (in Chinese)
[18]	陈懿冲, 沈廷明, 陈艳柠, 等. 基于网络药理学探讨畲药十二时辰镇痛作用机制 [J]. 海峡药学, 2024, 36(1):1−6. CHEN Y C, SHEN T M, CHEN Y N, et al. Based on the network pharmacology to explore the mechanism of analgesic action of she medicine Clematis florida var. flore-pleno [J]. Strait Pharmaceutical Journal, 2024, 36(1): 1−6. (in Chinese)
[19]	JIANG C H, BI Y K, MO J B, et al. Proteome and transcriptome reveal the involvement of heat shock proteins and antioxidant system in thermotolerance of Clematis florida [J]. Scientific Reports, 2020, 10(1): 8883. doi: 10.1038/s41598-020-65699-2