Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system

摘要

13?￠????Lipoxygenase (13?￠????LOX) and 13?￠????hydroperoxide lyases (13?￠????HPL) are the key enzymes for the production of the ?￠????green note?￠???? compounds hexanal, (3 Z )?￠???? and (2 E )?￠????hexenal in plant tissues. To produce high levels of 13?￠????LOX and 13?￠????HPL enzymatic activities for a biocatalytic process to generate C 6 ?￠????aldehydes on a large scale, soya bean 13?￠????LOX ( GmVLXC ) and watermelon 13?￠????HPL ( ClHPL ) genes were expressed in Nicotiana benthamiana using a viral vector system mediated by agroinfiltration. The N.????benthamiana leaves produced high activity of watermelon HPL, but not GmVLXC 13?￠????LOX. In addition, all leaves treated with bacterial suspension displayed a high activity of 9?￠????LOX, indicating that the internal tobacco 9?￠????LOX gene was highly induced through agroinfiltration because of wounding. GmVLXC and ClHPL transcripts could be detected in the corresponding transformed tobacco leaves by real?￠????time RT?￠????PCR analysis but the expression level of ClHPL was 24?￠????fold higher than that of GmVLXC . Western blot analysis showed that LOX was present in all tobacco leaves which were treated with bacterial suspensions, but not in the untreated wild?￠????type control. This result confirms that internal 9?￠????LOX was highly induced by agroinfiltration. The highest levels of ClHPL activity under optimal infiltration conditions were 80 times the HPL activity of wild?￠????type plants or plants transformed with control vector. A large amount of hexanal was formed when linoleic acid was incubated with extracts from N.????benthamiana leaves over?￠????expressing ClHPL in combination with GmVLXC ?￠????expressing yeast extracts. One gram of ClHPL ?￠????expressing N.????benthamiana leaves (fresh weight) could produce 17?￠???????±?￠????0.4?￠????mg hexanal from 50?￠????mg linoleic acid after 30?￠????min.