Cloning Of Artemisinin Biosynthetic Genes, E. Coli Expression And Molecular Analysis

In order to break down the rate-limited steps in the artemisinin biosynthesis to improve the artemisinin production and realize the industrial production of artemisinin,related key genes in artemisinin biosynthesis must be cloned and the regulatory patterns of key genes should be studied. For this purpose molecular cloning of related key genes in artemisinin biosynthesis was performed in this thesis work.A novel 1886bp full-length sesquiterpene synthase (ASES) cDNA was cloned from a high-yield Artemisia annua strain 001 by RACE strategy. ASES is 59% identical to Artemisia cyclase cDNA clone cASCJ25,50% identical to epi-cedrol synthase from A. annua,48% identical to amorpha-4,11-diene synthase from A. Annua,39% identical to the 5-epi-aristolechene synthase from tabacco,38% identical to vetispiradiene synthase from H. Muticus,41% identical to the 8-cadinene synthase from cotton. The coding region of cDNA was cloned into procaryotic expression vector pET30a and overexpressed in E. coli BL21(DE3). The cyclase proteins extracted from bacterial culture were found largely in the insoluble protein fraction. The tissue specific expression patterns were analyzed by RT/PCR. The cDNA expresses in leaves,stems and flowers. The expression wasn't detected in roots.Amorpha-4,11-diene synthase cDNA (amsl) was cloned from a high-yield Artemisia annua strain 001 by reverse transcription/polymerase chain reaction. The cDNA was subcloned into prokaryotic expression vector pET3d and overexpressed in E. coli BL21(DE3). Southern blot analysis showed that AMS gene is at least three gene copies per genome. The AMS genomic DNA has complex organization including 7 exons and 6 introns. RT/PCR analysis showed that AMS gene expresses in leaves,stems and flowers. The expression wasn't detected in roots.For the first time a 1539bp squalene synthase (ASQS) cDNA was cloned from a high-yield Artemisia annua strain 001 by RACE strategy. ASQS is 70%,77%,44% and 39% identical to squalene synthases from arabidopsis thaliana,tobacco,humanand yeast,respectively. The ASQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or truncated cDNA was subcloned into prokaryotic expression vector pET30a and expression induced in E. coli BL21(DE3). No squalene synthase polypeptide of expected molecular mass was observed in E.coli containing the putative full-length squalene synthase cDNA,however,overexpression in E. coli was achieved by truncating 30 hydrophobic amino acids at the carboxy terminus.