Isolation And Genetic Transformation Of Taxane Endophytic Fungi From Taxus Chinensis Var. Mairei

Taxol is a kind of diterpenoid alkloid Which had been first isolated from the bark of Taxus brevifolia Nutt.. Taxol has been proved that had great power against a wide range of cancers including ovarian cancer. Because the purification procedures are complicated and the yields are low, the price of the drug is very high which limits its more application. As the demand of Taxol increases, the needs are conflicting with supply which encourage people to make efforts to expand the resources of Taxol. Since the first Taxol-producing fungus Taxomyces andreanae has been isolated from the bark of Taxus species by Stierle in1993, more and more Taxol-producing fungus have been isolated which provide a possibility to produce Taxol with large scale by microorganism fermentation.In this study, a endophytic fungus which could produce BacctinⅢ (important precursor of Taxol) had been isolated from the bark of a wild Taxus chinensis var. mairei of one hundred years old. The fungus had been classified as Pestalotiopsis sp. according to its morphological characteristics and molecular proofs and named as TL9. The most suitable carbon and nitrogen sources had been optimized by single factor test and L9(34) orthogonal tests.Using the genomic DNA of fungus TL9as template, designing primers based on dbat gene of Taxus chinensis var. mairei, a fragment of706bp was gained by PCR amplification which had been confirmed as fungal dbat gene fragment by sequence alignment on BLAST. Reverse PCR technique had been applied to amplify the neighboring region of the fragment and another fragment of753bp was gained. By analyzing and editing the two fragments, a fragment of1281bp had been gained which had great similarity of97%with plant dbat gene.The growth curve of TL9was drawn to find the most appropriate time (about4days) for generating protoplasts. By choosing proper digestion enzymes, adjusting enzymatic temperature, digestion time, the most proper protoplast-producing procedure were established. Hygromycin B was used as selection agent to select positive transformants, and150ug/L was set as selection concentration. Plasmid pCAMBIA1304and fungal expression vectors pAN7-1were used to construct hph gene-harboring fungal expression vectors. TL9protoplasts were used as recipients, Agrobacterium tumefaciens mediated transformation procedure were established, many factors including different culturing supporting maretial, Agrobacterium tumefaciens strains, different addition period of AS, AS concentration, protoplast concentration, Agrobacterium tumefaciens concentration, co-cultivation conditions were in consideration. The highest transformation efficiency reached55positive transformants per107protoplasts and the Hygromycin B resistance inherited stably. Three taxol synthetic pathway key genes (ts, dbat, bapt) of Taxus chinensis var. mairei were gained by RT-RCR and incorporated into pCAMBIA1304’-pAN7-1to construct taxol synthetic pathway key genes-harboring fungal expression vectors (pTS, pDBAT, pBAPT). The three plant genes were introduced into TL9genomic DNA by ATMT method which had been established. HPLC-MS results showed that dbat gene expressing in TL9could increase BacctinⅢ yield. RT-PCR results showed that dbat gene RNA expression levels had obvious correlation with BacctinⅢ yields.This study lay foundation for large scale production of taxanes by using transgenic endophytic fungi fermentation and provide some clues for understanding fungal taxol synthetic pathway at molecular level.