Construction Of Transformation Library Of Monascus Ruber Mediated By Agrobacterium Tumefaciens And Study On Transformants' Characters

As a kind of microbial resource in China, Monascus ruber can produce many bio-active substrates, such as pigments, monacolins, GABA, various enzymes etc. Many rsearches have been done about metabolic fermentation conditions of M. spp, but only a few studies were related to Monascus genetics. In this research, a transformation system of M. ruber mediated by Agrobacterium tumefaciens had been established and some transformants characters had been studied. The research contents were showed as follows:1 Some factors, which could affect transformation rate, were optimized. The factors contained medium and culture time of M. ruber, AS concentration, combination of D-galactose and AS, A. tumefaciens concentration, co-culture temperature and time of A. tumefaciens and spores of M. ruber. The results indicated that the optimal transformation conditions were as follows: cultivation of M. ruber for 20 days on PDA medium, 80mmol/L AS, coculture of 10~6/ml M. ruber spores with A. tumefaciens, whose optical density value at 600 nm was 0.5, and co-culture for 3 days at 30℃. Until now, about 5,100 transformants were obtained according to those conditons.2 To identify the transformants, genomic DNA of transformants MZ1~10 were. identified by PCR with primers designed according to Gus gene sequences of T-DNA. The result showed that there was T-DNA insertions in transformants genome.3 To examine the genetic stability of transformants, 14 randomly selected transformants and two transformants' spores were cultivated on PDA and PDA containing 50mg/L hygromcin B for 6 generations and 4 generations, respectively. The results revealed that all tested transformants were genetically stable.4 The transformants whose colonial and microscopical morphology diversified markedly from the parental strain were screened from the 5,100 transformants, whichcultured for 14-30 days on PDA medium at 30°C. The results showed that 32 transformants' colony color, aerial hypha, colony apophysis and growth rate had aberrances from the parental strain. 16 transformants of them had different microscopical morphology from the parental strain. Their sporangium wall, conidiophore shape and granules on hypha surface had some differences.5 Pigments from aberrant transformants was analyzed. 7 randomly selected transformants were growed on rice medium for 14 days at 30°C, and then the color values were measured and pigments solutions were UV-scanned. The results showed that, compared with the parental strain, all of those transformants color value decreased; three transformants' water-soluble and ethanol-soluble pigments both had one more peak; one transformant' ethanol-soluble pigment had one more peak.