Study On Citrinin Related Genes In Monascus Spp.

Red fermented rice (RFR), which has been used for more than one thousand years in China and exported to European counties, USA and Japan etc, is a fermentation product of rice and other starch materials by Monascus spp.. However, in 1995 professor Blanc found that there was a mycotoxin citrinin in RFR, which triggered a controversy about the safety of RFR in the world. So how to control or reduce citrinin content in RFR became one of the hot research areas of RFR. In this research, a transformants library of Monascus ruber, which contains more than 5,000 transformants, was established by Agrobacterium-mediate DNA transfer. Then, inhibition zone method and high performance liquid chromatography (HPLC) were used to screen citrinin mutants from the transformants library, and thermal asymmetric interlaced PCR (TAIL-PCR) was performed to obtaine the gene sequences adjoined to the transfer DNA (T-DNA). These results provide research bases for the acquirement of full-length citrinin related genes. The research contents were followed:1. The transformants library of Monascus ruber M-7, which contains more than 5,000 transformants, was established by Agrobacterium-mediate DNA transfer. The optimal transformation conditions were as follows: The original strain M-7 was cultivated on PDA medium at 30℃ for 20 days, then the Monascus spores were collected and diluted to 10⁶/mL with Agrobacterium tumefaciens suspension, whose optical density at 600nm was 0.5. The mixture was co-cultured at 30℃ for 3 days with 0.8mmol/L acetosyringone (AS).2. 40 transformants, whose colonial morphology was markedly different from that of the original strain, were screened from the 5,000 transformants which had been incubated for 7 days on PDA medium at 30℃. The color, morphology, growth speed, aerial hypha and the spore production of the mutant colonies were altered compared to these of the original strain.3. HPLC technique was established for the determination of citrinin in this research. The optimal chromatographical conditions were as follows: column (chromasil C₁₈, 5μm, 250×4.6mm); Mobile phase (acetonitrile-water=50:50, pH 2.5); flow rate (1.0mL/min); column temperature (room temperature); injection volume (10μL); fluorescence detector (λex=325nm, λem=512nm). It can detect the citrinin concentration ranged from 0.05～...