| Atropa belladonna L. is an important medicinal perennial herb, belonging to the family of Solanaceae, which produces the secondary metabolites pharmaceutical tropane alkaloids(TAs), Hyoscyamine and scopolamine are the two most valuable TAs, which are widely used as anti-cholinergic drugs and are urgently demanded in word market. So far, TAs are mainly extracted from the plants of Solanaceae family such as Atropa belladonna、Datura stramonium and Hyoscyamus niger. Atropa belladonna is the main medicinal herbal resource of TAs for commercial values, and is one of the medical plants according to pharmacopeia. However, the contents of both hyoscyamine and scopolamine are very low in wild type A. belladonna, which making it difficult to meet the demand of the word market. What is more, TAs can not be produced in mass duing to it’s too expensive and difficult to produce by chemical synthesis. Recent years, with the quick development of metabolic engineering, it is possible to improve the content of TAs by gene engineering technology. The arginine decarboxylase is one of the key enzyme in the upstream pathway of the biosynthesis of TAs.We isolated two ADC genes in A. belladonna by the RACE technology, which were named as Ab ADC1 and Ab ADC2 respectively. Bioinformatics analysis indicated that the full length c DNA sequence of Ab ADC1 was 2817 bp including 2139 bp coding sequences, 568 bp of 5’untranslated region and 275 bp of 3’ untranslated region, encoding 712 amino acid residues.The full length c DNA sequence of Ab ADC2 was 2992 bp, including 2148 bp coding sequences, 492 bp of 5’untranslated region and 186 bp of 3’ untranslated region, encoding 715 amino acid residues.The amino acid sequence analysis showed that the amino acid sequence of Ab ADC1 had the highest identity as 89% with ADC from Solanum tuberosum, while the amino acid sequence of Ab ADC2 had the highest identity as 90% with ADC from Datura stramonium. The secondary stuctural prediction indicated that Ab ADC1 contains 36.52 % alpha helix, 16.15% extended strand and 47.33% random coil, and Ab ADC2 contains 35.38% alpha helica, 17.34% extended strands and 47.27% random coils. The prediction of signal peptide and subcellular location showed that both Ab ODC1 and Ab ODC2 belonged to cytoplasmic protein. Ab ADC1 was located in cytoplasm, Ab ADC2 was located in the cytoplasmic membrane. Both Ab ADC1 and Ab ADC2 had much similarity with reported ADC of Datura Stramonium and Nicotiana tobacum respectively according to the phylogenetic tree. The expression level of Ab ADC1 was highest in secondary roots, following by main roots and young stem. Ab ADC2 was mainly expressed in main roots, follwing by secondary roots.A.belladonna hairy roots were obtained by infecting sterile leaf discs by A. tumefaciens strain C58C1.The hairy roots were cultured in triangular flask, and treated with 100 m M Na Cl and Chilling. The expression levels of Ab ADC1 and Ab ADC2, and the content of TAs in hairy root of A.belladonna were detected in four different time points after one month. The result showed that the content of hyoscyamine had no change and the content of scopolamine reduced obviously in Na Cl treatment. In chilling treatment, the hyoscyamine content only had a reduce after treated in 4 hours, the scopolamine content had no obvious change. The expression levels of both Ab ADC1 and Ab ADC2 were reduced under the two treatments, and it was contrary with the expression of Ab ODC2, which had an obvious increase after treated in 4 hours. We speculated that the abiotic stress may increase the putrescine content by suppressing the expression of arginine decarboxylase and increasing the expression of ornithine decarboxylase, but had no influence on the synthesis of TAs.What’s more, we constructed plant expression vectors harboring Ab ADC1 or Ab ADC2 based on PBIN19, the recombinant vectors were named to be PBIN19-Ab ADC1 and PBIN19- Ab ADC2, which can be used for genetic transformation(RNAi).The vectors and the plasmid PBIN19 were transformed into Agrobacterium tumefaciens to form the genetically modified bacteria, C58C1-PBIN19- Ab ADC1、C58C1- PBIN19- Ab ADC2 and C58C1- PBIN19. Transgenic hairy roots were obtained by infecting sterile leaf discs by the engineering bacteria. In order to analysis the relationship of arginine decarboxylase and the TAs, both the expression of Ab ADC and the content of TAs were detected in transgenic lines.In summary, we got two arginine decarboxylase genes from A.belladonna and characterized the genes at the bioinformatics and expression levels, which provided new candidate genes for engineering biosynthetic pathway of tropane alkaloids. |
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