| Plant secondary metabolites are the largest group of natural products that are usually the products of interaction between plants and the environments. Plant secondary metabolites, such as anti-tumor camptothecin and anticholinergic tropane alkaloids, are widely used in the pharmaceutical industry.Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (1PP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. To investigate camptothecin biosynthesis at molecular level and provide a new target gene for metabolic engineering of camptothecin pathway, a new cDNA encoding IPI was isolated from Camptotheca acuminate by the RACE method. The full-length cDNA was 1143 bp in length, designated as CaIPI (GenBank(?) Accession Number: DQ839416), that contained a 930-bp open reading frame (ORF) encoding a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CalPI was homologous with other IPI gene and the deduced amino acid sequence of CalPI was highly similar to known plant IPIs. CalPI contained Cys-149 and Glu-212 active sites that were considered as the highly conservative sites of catalytic region of IPIs. The subcellular location analysis predicted that CalPI was located into plastid and demonstrated that CalPI was involved in MEP pathway. Phylogenic analysis indicated that all IPIs could be divided into five groups and CalPI belonged to plant IPIs' family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues including roots, stems, tender leaves, mature leaves and fruits. The results showed that CaIPI expression could be detected in roots, stems and tender leaves but could not be detected in mature leaves and fruits; and the expression levels were much higher in stems than that in roots and tender leaves. HPLC analysis was performed to detect the content of camptothecin in these tissues mentioned above. The results showed the camptothecin content was much higher in tender leaves than that in fruits and roots, and it was not consistent with the results of semi-quantitative RT-PCR. It was presumed that a transportable mechanism possibly existed in camptothecin storage that had been reported before. Finally, The ORF of CaIPI was isolated and substituted the Arabidopsis thaliana ipi gene of plasmid pTrcAtIPI to generate pTrcCaIPI. And then pTrcCaIPI was introduced into engineered Escherichia coli strain XLl-Blue in which the carotenoid pathway was reconstructed based on plasmid pAC-BETA. In engineered XLl-Blue, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orangeβ-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil an important step involved in the biosynthetic pathway of camptothecin precursor biosynthesis.Tropane alkaloids (TAs) including hyoscyamine and scopolamine are produced by Solanaceae plants. The two TAs especially scopolamine have strong anticholinergic activity, which are widely applied clinically as ataractic and narcotic agents. Several genes have been identified in TAs biosynthetic pathway, which were derived from polyamine biosynthetic pathway, making metabolic engineering of TAs pathway possible. The putrescine N-methyltransferase (PMT) and hyoscyamine 6β-hydroxylase (H6H) are important enzymes involved in catalyzing key steps of the biosynthesis of TAs. Now, the two enzymes were the targets for metabolic engineering of TAs pathway have been reported before. In the present study, Anisodus acutangulus, the primary-source plant of TAs, was used as plant material. Firstly, the bacteria-free plantlet culturing system was established. And the genetically transformation method of A. acutangulus was established, by infecting the leaves by Agrobacterium rhizogenes strain A4. The presence of rolB and rolC in A4-transformed hairy root lines were confirmed by genomic PCR analysis at the frequency of 100%. A plasmid pXI haboring tobacco pmt gene and Hyoscyamus niger h6h gene was introduced into disarmed Agrobacterium tumefaciens strain C58C1 (harboring pRiA4) to generate the engineered bacteria C58Cl-pXI. The leaves of A. acutangulus were transformed by C58C1-pXI, and the transformants were screened with 100 mg-L-1 kanamycin, to obtain the kanamycin-resistant hairy root lines. Genomic PCR analysis was carried out to detect the presence of rolB, rolC, pmt and h6h gene of C58C1-pXI-transformed hairy roots. The results showed that the presence of the four genes including rolB, rolC, pmt and h6h were simoutanously detected in kanamycin-resistant hairy root lines of A. acutangulus at the frequency of 32%. HPLC analysis was performed to analyze the content of scopolamine and hyoscyamine of transgenic hairy root lines, the A4-transformed hairy roots and the normal roots used as control, for confirmation of the effects of genetic modification in A. acutangulus. The best scopolamine-producing hairy root line of transgenic group produced 3.702±0.34l mg·g-1 dw scopolamine, which was nearly 6 folds of A4 group and nearly 25 folds of wildtype roots. The best hyoscamine-production line of transgenic group produced 8.794±0.563 mg·g-1 dw hyoscyamine, which was nearly 8 folds of A4 group and nearly 75 folds of wildtype. And total production of scopolamine and hyoscyamine of the best transgenic group was 11.804 mg·g-1 dw, which was nearly 7 folds of A4 group and nearly 44 folds of wildtype. Metabolic engineering of TAs pathway of A. acutangulus hairy roots was successfully carried out by using the strategy of multiple-gene co-transformation. The present study provided new transgenic materials for developing new resource of TAs. |
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