| Atropa belladonna L. is a valuable medicinal perennial herb, belonging to the family of Solanaceae, which produces pharmaceutical tropane alkaloids (TAs), simutanneously it is the commercial plant species that can be used to extract TAs in China. TAs have been widely used as anticholinergic agents affecting systemic nervous parasympatheticum with an enormous market demands. As the content of TAs is very low in plant, the production of TAs can not meet the need of market; in addition, as it is very hard and expensive to produce TAs by chemical synthesis, chemical synthesis can not go into mass production. Recently, with the development of the research in the biosynthetic pathwary of TAs, many relative genes in downstream pathway are isolated and identified, and this makes it workable to improve the content of TAs in plant by genetic engineering. However there are few studies in the upstream pathway, expecially the biosynthesis of putrescine (the precursor for TAs) is not completely clear. Using the technology of molecular biology and chemistry biology, this work researched the biosynthesis of putrescine and tried to figure out the molecular mechanism of enzymatic committed reaction step in upstream pathway and provide candidate gene for genetically modifying A. belladonna to improve the TAs content.We isolated two ODC genes in A. belladonna by the RACE technology, which were named to be AbODCl and AbODC2respectively. AbODC2gene was isolated for the first time and there was no report about its function at present. The results of bioinformatics analysis indicated that the full length cDNA sequence of AbODCl and AbODC2were respectively1527bp and1312bp including1293-bp and1194-bp coding sequences, which respectively encoded430and397amino acid residues. Compated with AbODC1, AbODC2had much lower similarity with reported ODC proteins of other plants in Solanaceae family. The prediction of signal peptide and subcellular location showed that both AbODC1and AbODC2belonged to cytoplasmic protein and located in cytoplasm. The secondary stuctural prediction indicated that AbODC1contained33.26%alpha helix,18.60%extended strand and48.14%random coil, and AbODC2contained34.76%alpha helica,14.86%extended strands and50.38%random coils.3-D structure prediction showed that both AbODC1and AbODC2had the similar3-D structure with Nicotiana glutinosa ODC of which function had been identified. AbODC1protein had the same substrate binding sites with Nicotiana glutinosa ODC, while AbODC2protein had a few amino acid residues at the same sites different from those of AbODCl. The upstream promoter sequences of both AbODCl and AbODC2were respectively isolated from the genome of A. belladonna. Promoter analysis suggested that AbODCl and AbODC2genes had the cis elements such as light-, anaerobic-elements in promoter region. In addition, AbODC1geae might also contain gibberellic acid-and MeJA-elements while AbODC2gene also had ABA-elements.The recombinant protein of AbODCl was purified from E. coli strain Rosetta using pET28as the expression vector. After induced by0.5mM IPTG at18℃for6h, AbODCl recombinant protein was produced and purified, and whose molecular weight was close to predicted one (46.5Kda). In the solvent of PLP, we detected that AbODCl recombinant protein could catalyze the L-ornithine to become putrescine, which indicated the recombinant AbODCl had the enzymatic activity of catalyzing L-ornithine to form putrescine.A. belladonna hairy root cultures were obtained by infecting sterile leaf discs by A. tumefaciens strain C58C1(pRiA4). The effects of100mM NaCl, UV-B and Chilling on expression levels of AbODCl and AbODC2, and the content of polyamine in hairy root of A.belladonna were detected. The results showed that the contents of both spermine and spermidine had a great improvement when the hairy root cultures were treated with NaCl/UV-B, which suggested stressed treatment improved polyamine biosynthesis and this would facilliate plant resistance. The content of putrescine did not have obvious changes at all time points in both NaCl and UV-B treatments; this might be caused by putrescine becoming spermine and spermidine. The results of Q-PCR showed that AbODCl gene expression levels was not improved but reduced at all time point in NaCl/UV-B/Chilling treatment.On the contrary, AbODC2gene expression levels had an obvious increase, especially at time point of four hour in the treatment of all the three treatments. AbODC2gene expression levels were about two times compared with that of control. Hence, it might be deduced that AbODC2had more closely relationship with plant resistance. In addition, we detected the relative gene expression of AbODCl and AbODC2in different tissues by Q-PCR. The results indicated that the highest expression levels of AbODC1gene were in secondary roots and main roots, following by flowers, and tender leaves were lowest. AbODC2gene showed tissue-specific expression, which was mainly expressed in secondary roots. Because the secondary roots were the main tissues for TAs biosynthesis and AbODC2gene was mainly expressed in secondary root, AbODC2gene might be a very important gene related to TAs biosynthesis.Then, we successfully constructed plant expression vectors harboring AbODC1or AbODC2based on pCAMBIA1305. The recombinant vectors were respectively named to be P1305-AbODC1and P1305-AbODC2, which could be used for genetic transformation (overexpression). In addition, we also successfully constructed plant expression vectors harboring AbODC1or AbODC2based on PBIN19. The recombinant vectors were respectively named to be PBIN19-AbODCl and PBIN19-AbODC2, which could be used for genetic transformation (RNAi). The four recombinant vectors were respectively introduced into A. tumefaciens strain C58C1(pRiA4) to generate engineered strains.In summary, molecular cloning and characterization of two ODC genes from A. belladonna will be helpful to understand biosynthesis of putrescine in planta and provide candidate gene for genetically modifying A. belladonna to improve the TAs content. |
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