Molecular Cloning And Transgenic Research Of The Genes Encoding Taxadiene Synthase And Tumor Angiogenesis Inhibitor

Cancer is the second serious disease that threatens human health. It has immediate significance to develop new type anticancer drugs with high tumoricidal activity and free from side effects. The first objective of this dissertation was to develop new strategy to ease up the supply crisis of taxol, the most important anticancer chemotherapeutic drug found during the 20th century, by constructing recombinant Gibberella fujikuroi strains to product the precursor of taxol under the ingenious idea of using the tetracyclic diterpenoid compounds gibberellins biosynthesis pathway of Gibberella fujikuroi. On the other hand, blocking tumor angiogenesis has proven to be an important strategy for cancer therapy. Thus, the second aim of this doctorate thesis was to find an efficient, safe and cheaper expression system for the tumor angiogenesis inhibitor-Arresten which is an important anti-angiogenic vascular basement membrane-associated protein, isolated from human placenta, the 26kDa NC1 domain of the α1 chain of type IV collagen, and functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, and tubeformation.Ⅰ The cDNA sequence encoding taxadiene synthase was cloned by RT-PCR from the Taxus chinensis embryonic calli E3B. The cDNA was first cloned into the prokaryotic expression vector pET15b and induced by IPTG to express the targeted protein in E.coli strain BL21s. Then, the cDNA fragment that could be expressed in E. coli BL21s was sequenced and the resulting error-free cDNA fragment was subcloned into the BamHI site of the eukaryotic expression vector pAN52-1Not, just downstream the start codon ATG, plasmid pANts was obtained. Then, A XbaI fragment of hygromycin B resistance cassette from pCSN44 was cloned into the XbaI site in the flanking region next to the transcription unit of pANts, the taxadiene synthase cDNA-containing expression vector pANts-hyg was constructed.Ⅱ A 1743 fragment of the gene cps coding for copalyl diphosphate synthase which represents the first gene of the gibberellin biosynthesis pathway was amplified by PCR from the genomic DNA of the rice pathogen Gibberella fujikuroi and cloned into the HindIII site of the plasmid pCSN44, the recombinant vector was termed as pCSNcps which had been designed for disrupting the cps gene to block gibberellins biosynthesis pathway.Ⅲ The method for protoplast preparation from the hyphae of Gibberellafujikuroi was established by single element experiments. The results showed that the released protoplasts reached up to 106/mL after incubated in the 15% crude driselase solution for 4h, at 37°C. As for Lywallzyme, the protoplasts production was not more than 103/mL.IV A protoplast/PEG transformation system was established and used to transform the Gibberella fujikuroi strain CBS195.34 and some transformants were obtained. 1 and 4 transgenic Gibberella fujikuroi strains respectively transformed by plasmid pANts-hyg and pCSNcps were identified by PCR detection and southern blot analysis. However, the growth potential of the transgenic strains were not equal to parental strain CBS 195.34. As the generation times increased, the growth rate remarkably decreased. The results also showed that the cps gene disruption vector pCSNcps was not enough for constructing recombinant Gibberella fujikuroi strain with gibberellins biosynthesis pathway blocked via a single cross-over event, although this vector carried a 1743bp fragment of the cps gene as homologous sequence.V Three different transformation methods, i.e. protoplast/PEG, electroporation and biolistic process were employed to transform different Gibberella fujikuroi strains. The results showed that the transformation frequency of different strains was quite different. Electroporation method could not improve the transformation frequency and biolistic transformation did not produce a better result than PEG method.VI A 690bp cDNA fragment coding for the tumor angiogenesis inhibitor Arresten was cloned from human placenta by one-step RT-PCR. The error-free cDNA was recovered from T-vector by BspHI & BstEII digestion and ligated into the plant binary vector pCAMBIA1301 predigested by Ncol & BstEII, the plant expression vector pCAMBIAarr was constructed. Then the recombinant Agrobacterium tumefaciens LBA4404 (pCAMBIAarr) was obtained by freeze-thaw method.VII Two regenerated transgenic plants of Nicotiana tabacum Qinyan95 with hygromycin B resistance were obtained via Agrobacterium tumefaciens-mediated transformation and identified by PCR, southern blot analysis and RT-PCR.