Molecular Cloning And Expression Of Genes Encoding Key Enzymes In Terpenoid Indole Alkaloids Biosynthesis Pathway From Catharanthus Roseus

This thesis performed a large scale of cloning for genes encoding key enzymes in terpene indole alkaloids (TIAs) pathway from Catharanthus roseus (L.)G.Don.via Gateway technology. These genes' mRNA expression levels in C. roseus seedlings were also detected. The results are as follows:1. The full length sequences of dxs, dxr, g10h, sls, tdc, str and dat which encoded key enzymes involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway were obtained via two-step Gateway PCR method, with the transcripted cDNA from total RNA of Catharanthus roseus seedlings as templates.2. After PCR amplification, these attB-flanked DNA fragments were inserted into attPcontaining donor vector (pDONR201) by BP clonase to generate entry clones, respectively. Positive clones were screened first by colony cracking, PCR and restriction enzyme analysis, and then these inserted genes were confirmed by gene sequencing.3. In order to get prokaryotic expression systems, ORFs of dxr, g10h, sls and str have been transferred to their destination vectors: pETG10A, pETG20A and pETG30A, and been transformed into competent cells of E.cloi host strain BL21(DE3). SDS-PAGE analysis results indicated that those recombinant expression clones which encoded enzymes of DXR, SLS and STR were overexpressed after IPTG induction.4. Fusion proteins DXR-HIS, DXR-HIS-TrxA, DXR-HIS-GST, SLS-HIS and STR-HIS were purified by Ni-TED resin. According to the results of SDS-PAGE and Bradford, these purified proteins were qualified for antisera preparation.5. Northern blot results displayed that transcript levels of dxr, sls, str and tdc in root, stem, leave, flower and fruit from C roseus were detected, in general the transcript levels in root and young leave were higher than in other tissues. But there're no signals for Northern blot ofglOh, dxs and dat. For one reason maybe that there're no mRNA transcripts in the seedling stage we chosed for these genes, or maybe they're inclined to be under strict post-transcriptional regulation.