[Isolation and characterization of promoter of ADS from Artemisia annua].

OBJECTIVE: To try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized. METHOD: 5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay. RESULT: The 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls. CONCLUSION: It was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.

Senescent leaves of Artemisia annua are one of the most active organs for overexpression of artemisinin biosynthesis responsible genes upon burst of singlet oxygen.

To dissect and penetrate complexicity regarding the tissue-specific and environment-induced expression modes of cytosolic and plastidial terpene biosynthetic genes in A. annua, corresponding mRNAs relevant to terpene biosynthesis were quantitatively compared among distinctive organs and during different growth stages. Although all examined mRNAs gradually elevate from June to August in tested organs, a putative artemisinin biosynthesis responsible DBR2 mRNA represents the most abundant transcript anyplace and anytime. Apart from others, senescent leaves endow global activation of artemisinin biosynthetic genes and ultimately lead to enhanced artemisinin production. Direct measurement of (1)O (2) burst from senescent leaves strongly supports an involvement of (1)O (2) in conversion from precursor(s) to artemisinin. In the context of environmental stresses, physical and chemical stress signals that include those invoking (1)O (2) burst were evaluated as if inducing artemisinin biosynthetic genes. The quantitative data have reiterated a common pattern of modulating artemisinin production in A. annua by triggering (1)O (2) burst during senescence and under chilling acclimatization. In conclusion, a missing link concatenating senescence-coupled (1)O (2) generation to (1)O (2)-induced upregulation of artemisinin biosynthetic genes has been re-established, which would provide a fertile base for future endeavors pursuing further enhancements of artemisinin production.

Quantitative transcript profiling reveals down-regulation of A sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants.

To investigate the dynamic fluctuation of terpenoid relevant transcriptomics in transgenic ARTEMISIA ANNUA plants that express the genomic integrated antisense squalene synthase gene ( ASSS), we have quantified the transcript levels of the sterol anabolic SS gene as well as artemisinin biogenetic amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 reductase (CPR) genes by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). The SS mRNA level in transgenic plants sharply droped to 7.4 % - 55.3 % (i. e., 44.7 - 92.6 % reduction as the wild-type control), strongly implying that the expression of endogenous SS gene is significantly suppressed by the exogenous ASSS gene. In a synchronous fashion, ADS, CYP71AV1 and CPR mRNA levels elevated with the decline of SS mRNA level in transgenic plants, and the maximal ADS, CYP71AV1 and CPR mRNA levels in transgenic plants were 3.0-, 4.4- and 2.5-fold, respectively, higher than those in the control. Without a lethal effect but with a distinguishable impact on the organogenesis and morphology of transgenic plants, the down-regulation of SS gene has also led to the coordinated overexpression of ADS, CYP71AV1 and CPR genes together with the overproduction of artemisinin although no fully perfect correlation among the available experimental data has been shown.