Studing The Sporulation Conditions And Optimizing The Formation And Regenaration Of Protoplasts Of Monascus Aurantlacus AS3.4384

Monascus, a typical filamentous fungus, is an important microorganism resource of China, which is originated from China a thousand years ago, has been recorded in ancient articles. Recently, some metabolic products, which are produced by Monascus, have been found with important physiological activity. Citrinin, however, a mycotoxin that does harm to humans and animals, was isolated from most cultures of Monascus strains recently. To prevent the pollution of citrinin by modifying ferment technology and screening Monascus strains without excreting citrinin in traditional way, but most of research results show it is very hard to completely suppress the excretion of citrinin by these way. Analysing the process that Monascus aurantiacus (AS3.4384) produces citrinin by molecular biological technology and constructing Monascus strains without excreting citrinin by genic engineering technology is the effective way to suppress the excretion of citrinin.Protoplast is an important tool to fuse and transfer of filamentous fungus, which is the foundation of molecular genetics. In this paper, sporulating, formation and regenaration of protoplast condition were optimized, to make the foundation of genic knocking. The main research results are as follows:1 The sporulating condition were optimized, inoculating 1.2 mL submerged mycelium of Monascus aurantiacus in MES agar medium with 6 °Be and pH 7, 28℃, cultivating for 25 day with light, spores were collected using sterile distilled water containing 0.05 % tween 80. The number of spores were counted by a hemacytometer, the highest concentration is 1.91×10⁹/mL.2 The formation of protoplast condition were optimized. The spores suspension up to a final concentration of 1-4x10⁷/mL, which were froze in -20℃, are inoculated in the liquid medium with MPPY, cultured at 30℃, 200 rmp for 35 h. Mycelial masses were resuspended in suitable enzyme solution (1 % snaliase + 0.3 % lysing enzyme + 4 % cellulase, lytic enzyme dissolved in 1 mol / L MgSO₄, adjusting pH to 6), and incubated at 30℃ with orbital shaking (60 rmp). The number of...