ABSTRACT
Claviceps pururea Cp-1 strain established in our lab is capable of producing variety of bioactive ergot alkaloids, and is broadly used by the pharmaceutical companies. To engineer the strain genetically for the production of specific ergot alkaloid, an effective transformation system must be set up first. However, the reported transformation system is not suitable for this strain due to different genetic backgrounds and the heterogeneity of Claviceps. Thus, in this paper, the hyphae of Cp-1 strain were used to prepare protoplasts by lywallzyme. The formation of protoplasts was investigated under different concentrations and incubation time of enzyme. The strain was tested for sensitivity to several antibiotics at different concentrations. Finally, the genetic transformation system of Cp-1 strain was established. The results suggest that protoplasts were formed efficiently by using 1% lywallzyme at 25℃ for 2 h. Transformants were obtained by PEG mediated protoplast transformation of Cp-1 strain with plasmid pAN7-1, using 1.5 mg·mL-1 hygromycin B as the selective marker. The exogenous gene in the plasmid pAN7-1 was integrated into the genome of Cp-1 strain transformant as demonstrated by PCR result. This study laid an important foundation for genetic manipulation of Cp-1 strain.
ABSTRACT
The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I (1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C (2), rhizoctonic acid (3), monomethylsulochrin (4), chaetominine (5), spirotryprostatin A (6), asperfumoid (7), and lumichrome (8). The structure of compound 1 was elucidated by various spectroscopic analyses (UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.