The Study Of Polyketide Synthase Gene In Dothiorella Sp. HTF3

In this study, Cytosporone B polyketide synthese gene of Dothiorella sp. HTF3 was investigated. Cytosporone B, one of a series of polyketides generated with HTF3 proliferation, had great antifungal and cytotoxin activities,and maybe had potential pharmic value. Cytosporone B PKS gene was studied to reveal its biosynthses pathway and facilitate its modification and application in future. On the other hand, a feasible methodology would be established in study progress, so that more research about fungi secondary metabolites will be done.Research contents in this study included: HTF3 genomic library construction and search clone containing PKS gene by homologous probe; Searching the whole PKS gene in clone by ORF analysis; Study the relationship between PKS gene and Cytosporone B by gene knockout.Two PKS gene fragments 0601 and 1101 were amplified from HTF3 genome by degeneracy PCR. Alignment results showed that, 0601 was a NR-PKS gene, and 1101 was a PR-PKS. 0601 may be refered to Cytosporone B biosynthesis according to the deduction of biosynthesis pathway and was chosen as the homologous probe.The genomic library was constructed on cosmid SuperCos 1 and clone efficiency was 5×10~3clones/μgDNA.One positive clone was screening from genomic library with 0601 probe.The analysis of two ends of insert sequence showed that, one PKS gene maybe contained in positive clone, and was gained by gene walking on positive clone, assigned as pks0601. pks0601, with a size of 5340bp, was a typical NR-PKS encoding a putative PKS with five conserved domains: KS,AT,2 PP-binding domain and TE, and without any domain catalyzingβ-keto reduction. And this PKS had high identities with Colletotrichum lagenarium PKS1, which synthesize precentor of melanin --THN,.Two fragments in upstream and downstream of pks0601 were intercepted from positive clone, and was linked by hygromycin resistance gene hph to constructed pGK55hyg. pGK55hyg would knockout pks0601 from HTF3 genome by homologous recombination, to study the effect of gene deficiency on Cytosporone B synthesis. pGK55hyg was transformed into HTF3 potoplast by potoplast-PEG method, and the condition of protoplast preparation and regeneration were optimized for high transformation efficiency. The cell wall degrading enzymes mixture was 1.5% prolywallzyme, 1.5% cellulase and 1.0% snailase, and regeneration medium was PDA(50% sea water) with 1mol/L NaCl.After transformation, 6 transformers were screened out by hygromycin B medium. Results of PCR test showed that, hph had transformed into HTF3 genome, but did not knockout pks0601. these transformers'broth were tested by TLC and show positive signal of Cytosporone B. Sum up these results, we can not reveal any relative between pks0601 and Cytosporone B, and study go on.