RESUMO
Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.
RESUMO
The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.
RESUMO
Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.