苦碟子中对香豆酰转移酶基因克隆分析与功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2018-0340

生物技术通报 ›› 2018, Vol. 34 ›› Issue (11): 120-126.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0340

苦碟子中对香豆酰转移酶基因克隆分析与功能验证

李天真¹, 周威², 殷华², 张同存¹, 刘涛²

1. 天津科技大学,天津 300457;
2. 中国科学院天津工业生物技术研究所,天津 300308

收稿日期:2018-04-12 出版日期:2018-11-26 发布日期:2018-11-28
作者简介:李天真,女,硕士研究生,研究方向：天然产物合成生物学;E-mail：litianzhen@tib.cas.cn
基金资助:
国家自然科学基金资助项目（31770104）

Genetic Cloning and Functional Verification of Hydroxycinnamoyltransferase in Ixeris sonchifolia Hance

LI Tian-zhen¹, ZHOU Wei², YIN Hua², ZHANG Tong-cun¹, LIU Tao²

1. School of Biological Engineering,Tianjin University of Science & Technology,Tianjin 300457;
2. Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308

Received:2018-04-12 Published:2018-11-26 Online:2018-11-28

摘要/Abstract

摘要： 苦碟子含有绿原酸等多种酚酸类化合物,该类化合物具有抗氧化和清除自由基等生物活性。对香豆酰转移酶（Hydroxycinnamoyltransferase,HCT）是此类化合物生物合成途径中的关键酶。本研究以苦碟子叶片为实验材料,采用转录组测序、序列分析以及RT-PCR的方法,获得一条对香豆酰转移酶基因,命名为IsHCT（GenBanK：MH151086）。序列分析表明该基因长1 320 bp,编码439个氨基酸。通过氨基酸序列比对及生物信息学分析发现,IsHCT氨基酸序列含有两个保守结构域,属于BAHD家族中的HCT酶类。系统发育进化树结果表明,该序列与菊苣的CiHCT的同源性最高,序列相似性高达95%。通过在大肠杆菌中过表达IsHCT和来源于拟南芥的对香豆酰辅酶A连接酶（At4CL）,并在发酵培养基中饲喂底物咖啡酸和奎尼酸,在发酵液中检测到绿原酸的合成,表明IsHCT能够催化咖啡酰辅酶A与奎尼酸合成绿原酸,具有对香豆酰转移酶功能。

关键词: 苦碟子, 对香豆酰转移酶, 绿原酸, 大肠杆菌

Abstract: Phenolic acid compounds from Ixeris sonchifolia Hance,such as chlorogenic acid,etc.,are reported to have antioxidation and free radicals scavenging activities. Hydroxycinnamoyltransferase（HCT）is a key enzyme in the biosynthetic pathway of plant chlorogenic acid. A hydroxycinnamoyltransferase gene from I. sonchifolia Hance leaves was cloned using transcriptome sequencing,sequence analysis and RT-PCR strategies,and named as IsHCT（GenBank accession number：MH151086）. The open read frame of the gene was 1320 bp encoding a putative protein of 439 amino acids. Multiple sequence alignment and bioinformatics analysis results showed that IsHCT contained two conserved domains,and belonged to the HCT enzyme in the BAHD family. Phylogenetic tree analysis demonstrated that this gene had the highest homology with CiHCT from Cichorium intybus,and the similarity reached 95%.Finally,by overexpressing IsHCT and At4CL from Arabidopsis thaliana in Escherichia coli and feeding the substrates caffeic acid and quinic acid in the fermentation medium,and chlorogenic acid was detected in the fermentation liquid,indicating that the IsHCT catalyzed the chlorogenic acid synthesis from caffeyl coenzyme A and quinic acid,i.e.,it functioned as HCT

Key words: Ixeris sonchifolia Hance, HCT, chlorogenic acid, Escherichia coli

李天真, 周威, 殷华, 张同存, 刘涛. 苦碟子中对香豆酰转移酶基因克隆分析与功能验证[J]. 生物技术通报, 2018, 34(11): 120-126.

LI Tian-zhen, ZHOU Wei, YIN Hua, ZHANG Tong-cun, LIU Tao. Genetic Cloning and Functional Verification of Hydroxycinnamoyltransferase in Ixeris sonchifolia Hance[J]. Biotechnology Bulletin, 2018, 34(11): 120-126.

参考文献

[1] 袁晓娇, 任越, 张丽鹏, 等. 苦碟子研究现状[J]. 人参研究, 2014, 26(2):59-63.
[2] 高晟, 周静. 苦碟子注射液临床应用研究进展[J]. 现代药物与临床, 2012, 27(2):180-184.
[3] Feng XZ, Xu SX, Dong M.Two novel flavonoids from Ixeris sonchifolia[J]. J Chin Pharm Sci, 2000, 9:134.
[4] Ma JY, Wang ZT, Xu LS, et al.Sesquiterpene lactones from Ixeris sonchifolia[J]. Phytochemistry, 1998, 48:201.
[5] 刘颖, 卢建秋, 张加余, 等. 苦碟子药材及其注射液中化学成分的HPLC-ESI-MSⁿ分析[J]. 中国中药杂志, 2013, 38(16):2675-2681.
[6] 刘睿, 孙璐, 赵雯, 等. HPLC同时测定苦碟子注射液中单咖啡酰基酒石酸、咖啡酸、绿原酸、阿魏酸、菊苣酸的含量[J]. 中国中药杂志, 2013, 38(19):3287-3290.
[7] Yin R, Deng XX, Han F, et al.Determination of five components in Ixeris sonchifolia by high performance liquid chromatography[J]. J Pharm Biomed Anal, 2007, 43(4):1364-1369.
[8] Frankel EN, Meyer AS.The problem of using one dimensional methods to evaluate multifunctional food and biological antioxidants[J]. Sci Food Agric, 2000, 80:1925-1941.
[9] Cuvelier ME, Richard H, Berset C.Comparison of the antioxidative activity of some acid phenols:structure activity relationship[J]. Biosci Biotechnol Biochem, 1992, 26:322-325.
[10] Licandro-Seraut H, Roussel C, et al.Sensitivity to vinyl phenol der-ivatives produced by phenolic acid decarboxylase activity in Esche-richia coli and several food-borne Gram-negative species[J]. Appl Microbiol Biotechnol, 2013, 97(17):7853-7864.
[11] Didry N, Seidel V, Dubreuil L, et al.Isolation and antibacterial activity of phenylpropanoid derivatives from Ballota nigra[J]. Journal of Ethnopharmacology, 1999, 67(2):197-202.
[12] Silva FA, Borges F, Guimaraes C, et al.Phenolic acids and derivatives studies on the relationship among structure, radical scavenging activity, and physicochemical parameers[J]. Agric Food Chem, 2000, 48:2122-2126.
[13] Cuvelier ME, Richard H, Berset C.Comparison of the antioxidative activity of some acid-phenols:structure activity relationship[J]. Biosci Bio technol Biochem, 1992, 56(2), 324-325.
[14] Lallemand LA, McCarthy JG, McSweeney S, et al. Purification, crystallization and preliminary X-ray diffraction analysis of a hyd-roxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransfer-ase(HCT)from Coffea canephora involved in chlorogenic acid biosynthesis[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2012, 68(Pt7):824-828.
[15] Legrand G, Delporte M, Khelifi C, et al.Identification and characterization of five BAHD acyltransferases involved in hydroxycinnamoyl ester metabolism in Chicory[J]. Front Plant Sci, 2016, 7, 741.
[16] He L, Xu X, Li Y, et al.Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb[J]. PLoS One, 2013, 8(4):e62922.
[17] Sonnante G, D’Amore R, Blanco E, Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid[J]. Plant Physiol, 2010, 153(3):1224-1238.
[18] Vogt T.Phenylpropanoid biosynthesis[J]. Mol Plant, 2010, 3(1):2-20.
[19] Li X, Bonawitz ND, Weng JK, et al.The growth reduction associated with repressed lignin biosynthesis in Arabidopsis thalianais indepe-ndent of flavonoids[J]. Plant Cell, 2010, 22(5):1620-1632.
[20] D’Auria JC. Acyltransferases in plants:a good time to be BAHD[J]. Curr Opin Plant Biol, 2006, 9(3):331-340.

苦碟子中对香豆酰转移酶基因克隆分析与功能验证

Genetic Cloning and Functional Verification of Hydroxycinnamoyltransferase in Ixeris sonchifolia Hance

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