Promotion of artemisinin biosynthesis in transgenic Artemisia annua by overexpressing ADS, CYP71AV1 and CPR genes

被引:27
作者
Lu, Xu [1 ,2 ]
Shen, Qian [1 ]
Zhang, Ling [1 ]
Zhang, Fangyuan [1 ]
Jiang, Weimin [1 ]
Lv, Zongyou [1 ]
Yan, Tingxiang [1 ]
Fu, Xueqing [1 ]
Wang, Guofeng [1 ]
Tang, Kexuan [1 ]
机构
[1] Shanghai Jiao Tong Univ, Sch Agr & Biol, Plant Biotechnol Res Ctr, Fudan SJTU Nottingham Plant Biotechnol R&D Ctr, Shanghai 200240, Peoples R China
[2] China Pharmaceut Univ, State Key Lab Nat Med, Nanjing 210009, Jiangsu, Peoples R China
关键词
Artemisinin biosynthesis pathway; Overexpression of ADS; CYP71AV1 and CPR; Southern blot; Real-time quantitative PCR; High-performance liquid chromatography; IN-VIVO TRANSFORMATIONS; AMORPHA-4,11-DIENE SYNTHASE; MOLECULAR-CLONING; DIHYDROARTEMISINIC ACID; EXPRESSION; PLANTS; L; REDUCTASE; CULTURES; PATHWAY;
D O I
10.1016/j.indcrop.2013.04.045
中图分类号
S2 [农业工程];
学科分类号
0828 ;
摘要
Artemisinin is an effective anti-malarial drug isolated from A. annua, which has an enormous commercialization demand all over the world. However, the low artemisinin content of A. annua greatly limits the commercialization of artemisinin. In this study, we report the results of our experiments, where for the first time we have achieved the overexpression of ADS, CYP71AV1 and CPR genes in A. annua. Eight transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR. Southern-blot analyses showed that some of the transgenic lines had low copies of the integration transgenes. The results of real time-qPCR showed that the expression levels of ADS, CYP71AV1 and CPR genes were significantly increased, too. The HPLC analyses showed that the artemisinin contents were significantly increased in these transgenic plants. One of the transgenic plants, ACR16, was found to contain 2.4-fold higher (15.1 mg/g DW) artemisinin than the control plants (pCAMBIA2300 transgenic plants). All above results showed that overexpression of ADS, CYP71AV1 and CPR genes in A. annua could promoted the metabolic flux flows toward biosynthesis of artemisinin and effectively increase the level of artemisinin content in transgenic A. annua plants. (C) 2013 Elsevier B.V. All rights reserved.
引用
收藏
页码:380 / 385
页数:6
相关论文
共 24 条
[1]   Over-expression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content [J].
Alam, Pravej ;
Abdin, M. Z. .
PLANT CELL REPORTS, 2011, 30 (10) :1919-1928
[2]   Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L. [J].
Banyai, Waleerat ;
Kirdmanee, Chalermpol ;
Mii, Masahiro ;
Supaibulwatana, Kanyaratt .
PLANT CELL TISSUE AND ORGAN CULTURE, 2010, 103 (02) :255-265
[3]   Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis [J].
Bouwmeester, HJ ;
Wallaart, TE ;
Janssen, MHA ;
van Loo, B ;
Jansen, BJM ;
Posthumus, MA ;
Schmidt, CO ;
De Kraker, JW ;
König, WA ;
Franssen, MCR .
PHYTOCHEMISTRY, 1999, 52 (05) :843-854
[4]   In vivo transformations of dihydroartemisinic acid in Artemisia annua plants [J].
Brown, GD ;
Sy, LK .
TETRAHEDRON, 2004, 60 (05) :1139-1159
[5]   In vivo transformations of artemisinic acid in Artemisia annua plants [J].
Brown, Geoffrey D. ;
Sy, Lai-King .
TETRAHEDRON, 2007, 63 (38) :9548-9566
[6]   A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3 [J].
Dietrich, Jeffrey A. ;
Yoshikuni, Yasuo ;
Fisher, Karl J. ;
Woolard, Frank X. ;
Ockey, Denise ;
McPhee, Derek J. ;
Renninger, Neil S. ;
Chang, Michelle C. Y. ;
Baker, David ;
Keasling, Jay D. .
ACS CHEMICAL BIOLOGY, 2009, 4 (04) :261-267
[7]   The Genetic Map of Artemisia annua L. Identifies Loci Affecting Yield of the Antimalarial Drug Artemisinin [J].
Graham, Ian A. ;
Besser, Katrin ;
Blumer, Susan ;
Branigan, Caroline A. ;
Czechowski, Tomasz ;
Elias, Luisa ;
Guterman, Inna ;
Harvey, David ;
Isaac, Peter G. ;
Khan, Awais M. ;
Larson, Tony R. ;
Li, Yi ;
Pawson, Tanya ;
Penfield, Teresa ;
Rae, Anne M. ;
Rathbone, Deborah A. ;
Reid, Sonja ;
Ross, Joe ;
Smallwood, Margaret F. ;
Segura, Vincent ;
Townsend, Theresa ;
Vyas, Darshna ;
Winzer, Thilo ;
Bowles, Dianna .
SCIENCE, 2010, 327 (5963) :328-331
[8]   Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L. [J].
Lei, Caiyan ;
Ma, Dongming ;
Pu, Gaobin ;
Qiu, Xiaofang ;
Du, Zhigao ;
Wang, Hong ;
Li, Guofeng ;
Ye, Hechun ;
Liu, Benye .
INDUSTRIAL CROPS AND PRODUCTS, 2011, 33 (01) :176-182
[9]   Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method [J].
Livak, KJ ;
Schmittgen, TD .
METHODS, 2001, 25 (04) :402-408
[10]   LEAF DISK TRANSFORMATION OF CULTIVATED TOMATO (LYCOPERSICON-ESCULENTUM) USING AGROBACTERIUM-TUMEFACIENS [J].
MCCORMICK, S ;
NIEDERMEYER, J ;
FRY, J ;
BARNASON, A ;
HORSCH, R ;
FRALEY, R .
PLANT CELL REPORTS, 1986, 5 (02) :81-84