Expression Analysis of Two Chemical-Types of Artemisia Annua L. Terpenoids and Their Related Genes
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摘要:
目的 分析2种化学型黄花蒿萜类代谢物及其转录组数据信息, 探讨2种化学型黄花蒿差异代谢物及其可能形成原因。 方法 采集江苏盱眙县、河南中牟县两地黄花蒿种子于同一条件下种植, 通过顶空-气相色谱-三重四极杆串联质谱(HS-GC-QQQ-MS/MS)分析黄花蒿的挥发性成分, 超快速高效液相色谱-三重四级杆飞行时间串联质谱(UFLC-Triple TOF-MS/MS)非靶向分析2种化学型黄花蒿的代谢物, 并采用转录组测序分析挥发性成分相关萜类生物合成基因的表达。 结果 依据挥发性主成分类别分型, 江苏盱眙县种源黄花蒿为蒿酮型, 河南中牟县种源黄花蒿为樟脑型。UFLC-Triple TOF-MS/MS检测到的差异代谢物通过KEGG数据库分析可知蒿酮型和樟脑型黄花蒿分别在倍半萜、三萜生物合成和二萜生物合成路径显著上调。转录组数据中通向萜类骨架生物合成的甲基赤藓糖醇-4-磷酸(MEP)途径和甲羟戊酸(MVA)途径注释到11个关键酶的23个候选基因具有显著差异。单萜合成路径中, 1, 8-桉树脑合酶(TPS-Cin)共检测到10个候选基因。此外, 还发现2个冰片脱氢酶候选基因在樟脑型黄花蒿中高表达, 3个黄花蒿醇脱氢酶2(ADH2)候选基因在蒿酮型黄花蒿中高表达。 结论 该研究为揭示黄花蒿化学型形成分子机制提供科学数据。 Abstract:OBJECTIVE To investigate the differential metabolites of two chemical-types of Artemisia annua and their possible causes by analyzing the data of terpenoid metabolites and their transcriptome of two chemical-types of Artemisia annua. METHODS The seeds of Artemisia annua were collected from Xuyi County, Jiangsu Province, and Zhongmou County, Henan Province, in China, and grown under the same conditions. The volatile components of Artemisia annua were analyzed by headspace-gas chromatography-triple quadrupole mass spectrometry (HS-GC-QQQ-MS/MS), non-targeted analysis of the differential metabolites of 2 chemical-types of Artemisia annua were performed by ultra-fast liquid chromatography-triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS), and the expression of the biosynthetic genes of the volatile components was analyzed by transcriptome sequencing. RESULTS Based on the typing of volatile components, Artemisia annua from Xuyi County, Jiangsu Province was classified as artemisia ketone type, and Artemisia annua from Zhongmou County, Henan Province was classified as camphor type. The differential metabolites detected by UFLC-Triple TOF-MS/MS were significantly up-regulated in sesquiterpene, triterpene, and diterpene biosynthesis pathways by Kyoto encyclopedia of genes and genomes (KEGG) database analysis for artemisia ketone type and camphor types of Artemisia annua, respectively. The 2-methyl-D-erythritol-4-phosphate (MEP) pathway leading to terpene skeleton biosynthesis and the mevalonate (MVA) pathway annotated 23 candidate genes for 11 key enzymes that were significantly differential in the transcriptome data. In the monoterpene synthesis pathway, 10 candidate genes were detected for 1, 8-cineole synthase (TPS-Cin). In addition, two borneol dehydrogenase candidates were found to be highly expressed in camphor-type Artemisia annua, and three Artemisia annua alcohol dehydrogenase (ADH2) candidates were highly expressed in artemisia ketone-type Artemisia annua. CONCLUSION This study provides scientific data to reveal the molecular mechanism of Artemisia annua chemical-types formation. -
Key words:
- Artemisia annua L. /
- volatile components /
- chemical type /
- terpenoids /
- biosynthesis
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图 8 2种化学型黄花蒿挥发性成分相关萜类合成通路基因表达
注: AK-T.蒿酮型; C-T.樟脑型; DXS.1-脱氧-D-木酮糖-5-磷酸合成酶; HDR.4-羟基-2-甲基-2-E-丁烯基-4-焦磷酸还原酶; IPPI.异戊烯基焦磷酸异构酶; GPS.异戊烯基转移酶牻牛儿基焦磷酸合酶; GGPS.牻牛儿基牻牛儿基焦磷酸合成酶; AACT.乙酰辅酶A酰基转移酶; HMGR.3-羟基-3-甲基戊二酰辅酶A还原酶; MK.甲羟戊酸激酶; PMK.甲羟戊酸激酶; MPDC.甲羟戊酸焦磷酸脱羧酶; FDPS.法尼基焦磷酸合酶; CPS.内根-古巴焦磷酸合成酶; GA3.贝壳杉烯C19氧化酶; GA20OX.赤霉素双加氧酶; GA3OX.赤霉素3-β-双氧酶; TPS04.香叶基芳樟醇合酶; CYP82G1.三甲基十三烷四烯合酶; ADH2.黄花蒿醇脱氢酶2;TPS-Cin.1, 8-桉树脑合酶
Figure 8. Gene expression of terpenoid synthesis pathway related to volatile components of 2 chemical types of Artemisia annua
表 1 黄花蒿挥发性成分(n=4)
Table 1. List of volatile components of Artemisia annua samples(n=4)
编号 CAS 英文名称 中文名称 峰面积百分比/% JXAa HZAa 1 3208-16-0 2-Ethylfuran 2-乙基呋喃 1.87±1.2 0.7±0.32 2 66-25-1 Hexanal 正己醛 2.95±1.63 0.92±0.23 3 505-57-7 2-Hexenal 2-己烯醛 3.35±2.15 1.51±0.57 4 544-12-7 trans-3-Hexen-1-ol 反式-3-己烯-1-醇 0.91±0.46 0.55±0.12 5 13466-78-9 3-Carene 3-蒈烯 1.9±1.25 4.55±0.59 6 79-92-5 Camphene 莰烯 5.46±5.25 21.38±0.96 7 555-10-2 β-Phellandrene 3-异丙基-6-亚甲基-1-环己烯 - 1.13±0.4 8 99-84-3 β-Terpinene 对薄荷-1(7), 3-二烯 6.28±1.08 5.65±2.48 9 99-86-5 α-Terpinene 松油烯 - 0.37±0.05 10 527-84-4 o-Cymene 邻伞花烃 0.46±0.09 1.28±0.13 11 470-82-6 Eucalyptol 桉叶油醇 12.37±3.45 21.17±1.51 12 99-85-4 γ-Terpinene γ-松油烯 - 0.73±0.15 13 546-49-6 Artemisia ketone 蒿酮 51.28±8.31 - 14 17699-16-0 4-Thujanol 4-侧柏醇 - 1.13±0.57 15 15537-55-0 cis-(+/-)-4-Thujanol 顺-(+/-)-4-侧柏醇 - 0.43±0.12 16 76-22-2 Camphor 樟脑 6.8±6.14 30.7±4.44 17 30460-92-5 Pinocarvone 松香芹酮 - 0.61±0.09 18 564-94-3 Myrtenal 桃金娘烯醛 - 0.45±0.14 19 56423-40-6 Butanoic acid, 2-methyl-, phenylmethyl ester 2-甲基丁酸苯甲酯 0.52±0.23 - 20 87-44-5 β-Caryophyllene β-石竹烯 0.99±0.47 0.4±0.16 21 92692-39-2 Amorpha-4, 11-diene 青蒿二烯 0.53±0.17 - 22 54274-73-6 epi-Bicyclosesquiphellandrene epi-双环倍半水芹烯 1.57±1.21 0.7±0.41 注: -表示未检测出该成分。 表 2 樟脑型黄花蒿二萜通路上调差异代谢物
Table 2. Up-regulated differential metabolites in camphor type Artemisia annua diterpene pathway
序号 保留时间/min 分子式 质荷比m/z 化合物名称 加和离子 差异倍数 P值 VIP 1 8.565 C20H30O3 275.237 ent-7α-Hydroxykaur-16-en-19-oic acid [M-CO2+H]+ 0.056 3.896E-07 1.464 2 6.768 C20H32O 307.264 Kaur-16-en-18-ol [M+H2O+H]+ 0.072 9.921E-05 1.453 3 7.539 C20H30O 305.247 Kaur-16-en-18-al [M+H2O+H]+ 0.081 2.691E-03 1.401 4 7.953 C20H34O 291.269 香叶基芳樟醇[(E, E)-Geranyllinalool] [M+H]+ 0.289 5.714E-03 1.372 -
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