Agrobacterium Tumefaciens-mediated Taxol-producing Bacteria Hqd <sub> 33 </ Sub> Genetic Transformation System

Discovery of taxol producing endophytic fungi isolated from T.brevifolia by Strovel in 1993, which made it possible to limitless produce taxol via microbial fermentation.The research group directed by Prof. Zhou also separated four strains of taxol producing fungus (Nodulisporium sylviforme) associated with T.cuspidate. Through a series of breeding, a high yield strain(448.52μg/L) was obtained. But it is not proven to be economically viable. In order to establish the foundation of the construction of higher yield engineering strain, we developed Agrobacterium-mzdiated transformation for endophytic fungi.According to the fact that HQD33 was sensitive to Hygromycin B, the Hygomycin B resistance gene(hph) was used as selectable marker gene for endophytic fungi HQD33 transformation. The Hygromycin B, concentration for transformants selection was tuned to be 30μg/mL. Using pBI121, a commonly used vector for plant transformation, as the backbone, the binary vector pBI121-43, in which the hph gen was under the promoter PtrpC and terminator TtrpC was constructed for HQD33 transformation. Mediated by A.tumefaciens strain LBA4404, T-DNA of pBI121-43 was successfully integrated into HQD33 genome with the frequency of 130 transformants/106 spores. After being perfected, transformation steps were described below. For A.tumefaciens preparation, a LBA4404 clony containing pBI121-43 was grown when culture absorbance value at 660nm was about 0.5, and dissolved in IM medium supplemented with 400μmol/L AS. After 48h co-culture, fungi and bacteria were transferred to the selection medium. 8-10 days later, hphresistance colonies were visualized.In the present studies, we have developed a simple, highly efficient and reliable A.tumefaciens-mediated transformation method for HQD33, established the foundation of the construction of higher yield engineering strain.