RESUMEN
Methyl jasmonate (MeJA) is one of the most effective inducers of taxol biosynthetic genes, particularly the tasy gene. However, the mechanism underlying the regulation of tasy by MeJA is still unknown. In this study, a 550-bp 5'-flanking sequence was obtained and confirmed as the promoter of the tasy gene. Deletion analysis revealed that the fragment containing a GCC-box from -150 to -131 was the crucial jasmonate (JA)-responsive element, designated as JRE. Using JRE as bait, two binding proteins, namely TcERF12 and TcERF15, were discovered. Sequence alignment and phylogenetic analysis showed that TcERF12 was related to the repressor AtERF3, while TcERF15 was more related to the activator ORA59; these are typical GCC-box-binding ethylene-responsive factors. Both could significantly respond to MeJA for 10 and 4.5 times, respectively, in 0.5 h. When the two TcERFs were overexpressed in Taxus cells, tasy gene expression decreased by 2.1 times in TcERF12-overexpressing cells, but increased by 2.5 times in TcERF15-overexpressing cells. Results indicated that TcERF12 and TcERF15 were negative and positive regulators, respectively, in the JA signal transduction to the tasy gene by binding the GCC-box in the JRE of the tasy promoter. Our results promote further research on regulatory mechanisms of taxol biosynthesis.
Asunto(s)
Ciclopentanos/metabolismo , Genes de Plantas , Oxilipinas/metabolismo , Paclitaxel/biosíntesis , Taxus/genética , Taxus/metabolismo , Región de Flanqueo 5' , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Taxol is a secondary metabolite with prominent anti-tumor activity, but the yield cannot meet the growing clinical demand due to lower content in yew. Now, most enzyme genes involved in taxol biosynthesis have been cloned and identified, so that obtaining this drug by using synthetic biology method has become a hotspot in recent years. However, most hydroxylases involved in taxol biosynthetic pathway have not been explored. Here, we reviewed the progress on the biosynthesis pathway of taxol, especially concerning hydroxylase. The future research areas of taxol biosynthesis through synthetic biology were also discussed to provide basis for the discovery of uncharacterized hydroxylase genes and the mass taxol production by synthetic biology technology.