The Construction Of Genetic Engineering Strains Of Cladosporium Cladosporioides MD2

Taxol is a kind of high efficient, low toxic and broad-spectrum antitumor drug. Producing taxol by microorganism fermentation is one of the effective ways to solve the problem of shortage of medicine source. However, the taxol yield of the separation of taxol-producing endophytic fungi are so low that still cannot meet the requirements of large-scale industrial production. In this study, we hoped to build high yield engineering strains by genetic transformation of taxol-producing fungi with genetic engineering technology, which can be benift for its industrialization application.On the basis of the establishment of the genetic transformation system of Cladosporium cladosporioides MD2 by Agrobacetrium tumefaciens-mediated transformation, the study was described as follows:(1) The key enzyme gene, 10-deacetyibaccatin III-10β-O- acetyltransferase gene (dbat) in taxol biosynthesis pathway from Taxus media cell was coloned;(2) The dbat gene was cloned into the pBI121 plasmid by replacing the site of gus gene to create the CaMV35S-dbat-Nos expression box, then the 35S-dbat-Nos expression box was cloned into multiple cloning site of pCAMBIA1303 plasmid to construct the expression vector p1303-SdbatN for genetic transformation, and the vector p1303-SdbatN was introduced into Agrobacterium tumefaciens LBA4404 by electroporation;(3) The spores of C. cladosporioides MD2 were used for receptors in transformation, and the vector p1303-SdbatN was transformed into C. cladosporioides MD2 by Agrobacetrium tumefaciens-mediated transformation. Then, the positive transformants were obtained by screening on selective medium containing 30μg/mL hygromycin and 100μg/mL cefotaxime;(4) 6 positive transformants were selected randomly to determine the T-DNA integration condition through the PCR analysis of hygromycin resistance gene (hph). The results showed that the hph gene band was amplified from all 6 positive transformants, which demonstrated T-DNA had inserted into the genome of C. cladosporioides MD2. And T-DNA integration pattern was analysed further by southern blot. The results showed that the transformants of C. cladosporioides MD2 obtained by ATMT almost had a single-copy T-DNA insertion at different sites in the genome;(5) Western blot and fluorescence detection were used to analyse the expression of report gene gfp. The results proved that the report gene gfp could be expressed in C. cladosporioides MD2 under the control of the 35S promoter;(6) In order to analyse genetic stability of transformants, C. cladosporioides MD2 positive transformants were transformed to potato dextrose agar (PDA) selective medium after growing successively five generations on PDA medium without hygromycin B, we found positive transformants still kept hygromycin resistence under no selection pressure conditions, which proved that stability of T-DNA insertion and reliability of the transformation method;(7) One engineering strain with taxol production significantly improved was obtained by HPLC analysis, and its taxol production was the 3 times of wild strain. Thus, the research work laid a foundation for the taxol industrialization production by microorganism fermentation in the future.