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CLC number: Q943.2

On-line Access: 2017-08-08

Received: 2016-05-17

Revision Accepted: 2016-09-10

Crosschecked: 2017-07-25

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Jing-ya Zhao

http://orcid.org/0000-0002-7387-1216

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Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.8 P.662-673

http://doi.org/10.1631/jzus.B1600223


Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua


Author(s):  Lu-yao Wang, Ying Zhang, Xue-qing Fu, Ting-ting Zhang, Jia-wei Ma, Li-da Zhang, Hong-mei Qian, Ke-xuan Tang, Shan Li, Jing-ya Zhao

Affiliation(s):  Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; more

Corresponding email(s):   zhaojy@sjtu.edu.cn

Key Words:  Salicylic acid, Artemisia annua L., Quantitative real-time polymerase chain reaction (qRT-PCR), Isochorismate synthase


Lu-yao Wang, Ying Zhang, Xue-qing Fu, Ting-ting Zhang, Jia-wei Ma, Li-da Zhang, Hong-mei Qian, Ke-xuan Tang, Shan Li, Jing-ya Zhao. Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua[J]. Journal of Zhejiang University Science B, 2017, 18(8): 662-673.

@article{title="Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua",
author="Lu-yao Wang, Ying Zhang, Xue-qing Fu, Ting-ting Zhang, Jia-wei Ma, Li-da Zhang, Hong-mei Qian, Ke-xuan Tang, Shan Li, Jing-ya Zhao",
journal="Journal of Zhejiang University Science B",
volume="18",
number="8",
pages="662-673",
year="2017",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1600223"
}

%0 Journal Article
%T Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua
%A Lu-yao Wang
%A Ying Zhang
%A Xue-qing Fu
%A Ting-ting Zhang
%A Jia-wei Ma
%A Li-da Zhang
%A Hong-mei Qian
%A Ke-xuan Tang
%A Shan Li
%A Jing-ya Zhao
%J Journal of Zhejiang University SCIENCE B
%V 18
%N 8
%P 662-673
%@ 1673-1581
%D 2017
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1600223

TY - JOUR
T1 - Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua
A1 - Lu-yao Wang
A1 - Ying Zhang
A1 - Xue-qing Fu
A1 - Ting-ting Zhang
A1 - Jia-wei Ma
A1 - Li-da Zhang
A1 - Hong-mei Qian
A1 - Ke-xuan Tang
A1 - Shan Li
A1 - Jing-ya Zhao
J0 - Journal of Zhejiang University Science B
VL - 18
IS - 8
SP - 662
EP - 673
%@ 1673-1581
Y1 - 2017
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1600223


Abstract: 
isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

青蒿异分支酸合酶基因的克隆及功能分析

目的:研究青蒿异分支酸合酶的表达模式,评价其对青蒿素含量的影响。
创新点:该研究首次克隆了青蒿异分支酸合酶基因(AaICS1),并发现AaICS1影响青蒿素的合成,为更有效地开发利用青蒿提供了新思路。
方法:根据青蒿转录组数据,利用聚合酶链式反应(PCR)克隆AaICS1基因和启动子,并进行多重序列分析和启动子作用元件预测。通过实时定量PCR(qRT-PCR)对AaICS1进行表达分析,用Southern杂交分析AaICS1的拷贝数。构建AaICS1过表达载体和干扰表达载体,转化青蒿获得转基因植株,用高效液相色谱法(HPLC)分析青蒿素含量。
结论:AaICS1含一个总长为1710 bp的完整阅读框,编码570个氨基酸,与其它植物的ICS基因具有较高的相似性。Southern杂交结果表明AaICS1为单拷贝(图4),qRT-PCR结果显示该基因能够响应伤害、干旱、盐胁迫和水杨酸的处理,处理后基因表达量提高(图6),和启动子作用元件预测相符。qRT-PCR结果显示过表达转基因青蒿中AaICS1表达量提高,干扰转基因青蒿中该基因表达量降低(图7)。HPLC显示过表达AaICS1转基因植株中青蒿素含量提升,最高可达对照的1.9倍(图8)。

关键词:水杨酸;青蒿;实时定量聚合酶链式反应;异分支酸合酶

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]Abdin, M.Z., Israr, M., Rehman, R.U., et al., 2003. Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production. Planta Med., 69(4):289-299.

[2]Aftab, T., Masroor, M., Khan, A., et al., 2010. Salicylic acid acts as potent enhancer of growth, photosynthesis and artemisinin production in Artemisia annua L. J. Crop Sci. Biotechnol., 13(3):183-188.

[3]Aquil, S., Husaini, A.M., Abdin, M.Z., et al., 2009. Overexpression of the HMG-CoA reductase gene leads to enhanced artemisinin biosynthesis in transgenic Artemisia annua plants. Planta Med., 75(13):1453-1458.

[4]Baldi, A., Dixit, V.K., 2008. Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua. Bioresour. Technol., 99(11):4609-4614.

[5]Banyai, W., Kirdmanee, C., Mii, M., et al., 2010. Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L. Plant Cell Tiss. Organ Cult., 103(2):255-265.

[6]Delabays, N., Simonnet, X., Gaudin, M., 2001. The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars. Curr. Med. Chem., 8(15):1795-1801.

[7]Duke, M.V., Paul, R.N., Elsohly, H.N., et al., 1994. Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L. Int. J. Plant Sci., 155(3):365-372.

[8]Eulgem, T., Rushton, P.J., Schmelzer, E., et al., 1999. Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J., 18(17):4689-4699.

[9]Garcion, C., Lohmann, A., Lamodière, E., et al., 2008. Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis. Plant Physiol., 147(3):1279-1287.

[10]Graham, I.A., Besser, K., Blumer, S., et al., 2010. The genetic map of Artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin. Science, 327(5963):328-331.

[11]Guo, X.X., Yang, X.Q., Yang, R.Y., et al., 2010. Salicylic acid and methyl jasmonate but not Rose Bengal enhance artemisinin production through invoking burst of endogenous singlet oxygen. Plant Sci., 178(4):390-397.

[12]Haas, B.J., Delcher, A.L., Wortman, J.R., et al., 2004. DAGchainer: a tool for mining segmental genome duplications and synteny. Bioinformatics, 20(18):3643-3646.

[13]Han, J.L., Liu, B.Y., Ye, H.C., et al., 2006. Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L. J. Integr. Plant Biol., 48(4):482-487.

[14]Hunter, L.J.R., Westwood, J.H., Heath, G., et al., 2013. Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis. PLoS ONE, 8(6):e66530.

[15]Loake, G., Grant, M., 2007. Salicylic acid in plant defence— the players and protagonists. Curr. Opin. Plant Biol., 10(5):466-472.

[16]McCormick, S., Niedermeyer, J., Fry, J., et al., 1986. Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep., 5(2):81-84.

[17]Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15(3):473-497.

[18]Nafis, T., Akmal, M., Ram, M., et al., 2011. Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L. Plant Biotechnol. Rep., 5(1):53-60.

[19]Patra, N., Srivastava, A.K., 2014. Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor. Appl. Biochem. Biotech., 174(6):2209-2222.

[20]Pu, G.B., Ma, D.M., Chen, J.L., et al., 2009. Salicylic acid activates artemisinin biosynthesis in Artemisia annua L. Plant Cell Rep., 28(7):1127-1135.

[21]Sadeghi, M., Dehghan, S., Fischer, R., et al., 2013. Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius. Plant Signal. Behav., 8(11):e27335.

[22]Shen, Q., Chen, Y.F., Wang, T., et al., 2012. Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae). Genet. Mol. Res., 11(3):3298-3309.

[23]van Tegelen, L.J., Moreno, P.R., Croes, A.F., et al., 1999. Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus. Plant Physiol., 119(2):705-712.

[24]Wildermuth, M.C., Dewdney, J., Wu, G., et al., 2001. Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 414(6863):562-565.

[25]Yang, R.Y., Feng, L.L., Yang, X.Q., et al., 2008. Quantitative transcript profiling reveals down-regulation of a sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants. Planta Med., 74(12):1510-1516.

[26]Yin, H., Kjaer, A., Fretté, X.C., et al., 2012. Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L. Process Biochem., 47(11):1559-1562.

[27]Zhang, F., Lu, X., Lv, Z., et al., 2013. Overexpression of the Artemisia orthologue of ABA receptor, AaPYL9, enhances ABA sensitivity and improves artemisinin content in Artemisia annua L. PLoS ONE, 8(2):e56697.

[28]Zhang, L., Ding, R., Chai, Y., et al., 2004. Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc. Natl. Acad. Sci. USA, 101(17):6786-6791.

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