Plant Biotechnology Research Center, SJTU-Cornell Institute of Sustainable Agriculture and Biotechnology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
PLoS One. 2012;7(8):e43038. doi: 10.1371/journal.pone.0043038. Epub 2012 Aug 20.
In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of (1)H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.
为了提高长春花中抗癌二聚吲哚生物碱的产量，研究人员致力于培养细胞系和发根，并努力阐明单萜吲哚生物碱（MIA）生物合成的调控。在这项研究中，ORCA3（十八烷衍生物反应性长春花 AP2 结构域）基因单独或与 G10H（香叶醇 10-羟化酶）基因一起首次被引入长春花植物中。通过遗传修饰，获得了单独过表达 ORCA3 的转基因长春花植物（OR 系），或共过表达 G10H 和 ORCA3 的转基因长春花植物（GO 系）。ORCA3 的过表达诱导 AS、TDC、STR 和 D4H 转录本的增加，但不影响 CRMYC2 和 G10H 的转录。在 G10H 和 ORCA3 共过表达下，G10H 转录本显著增加。ORCA3 和 G10H 的过表达显著增加了斯特罗辛、文多灵、长春质碱和阿马碱的积累，但对脱水长春碱和长春碱水平的影响有限。基于 NMR 的代谢组学证实了 OR 和 GO 系中单体吲哚生物碱的积累更高。与对照相比，（1）H NMR 光谱的多变量数据分析显示 OR 和 GO 系中氨基酸、有机酸、糖和苯丙素水平的变化。结果表明，ORCA3 和 G10H 过表达增强 MIA 生物合成可能会影响长春花植物代谢中的其他代谢途径。
