Isolation and expression characteristic of cyp71av1 promoter from Artemisia annua / 中草药

ABSTRACT

Objective: Trying to find the ways to enhance the expression of cyp71av1 gene encoding cytochrome P450 mono-oxygenase which is a key enzyme in artemisinin biosynthesis pathway accelerating the artemisinin synthesis, the promoter of cyp71av1 was isolated and characterized. Methods: 5′ untranslated regions of cyp71av1 were isolated from Artemisia annua with thermal asymmetric interlaced PCR. For functional characterization, the isolated fragments were fused with β-glucuronidase GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5′ untranslated regions of cyp71av1 in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay. Results: Two DNA fragments upstream of cyp71av1 coding sequence, a long fragment and a truncated fragment, were isolated from A. annua and introduced into N. tabacum respectively. Histochemical staining showed that two isolated fragments confered stable GUS expression in transgenic plants, and no significant difference was found between the two fragments on the GUS activity. The quantitative results also showed that the GUS activity in transgenic tobacco plants treated by dehydration, low-temperature (4 °C), and ultraviolet irradiation were 1.4 to 2.7 folds higher than that in the controls. Conclusion: It suggests that the isolated fragments has promoter activity and may be responsive to adverse environmental stresses.