农杆菌介导的紫色红曲霉遗传转化体系的建立和优化

Establishment and Optimization of Agrobacterium-mediated Transformation Systems for Monascus purpureus

通过优化各种转化因素,建立了根癌农杆菌(Agrobacterium tumefaciens)介导红曲霉(Monascus)的高效转化体系:红曲霉在PDA培养基培养21 d后收集孢子,制备红曲霉孢子悬浮液,浓度为106个/mL,根癌农杆菌浓度为OD600值0.5,诱导剂AS浓度为100μmol/L,农杆菌与红曲霉在25℃共培养3 d。采用此转化体系构建了含有530多个转化子的红曲霉T-DNA插入突变体库。随机选取50株转化子菌株进行分子验证和稳定性检测,证明T-DNA成功插入红曲霉基因组DNA中,并能稳定遗传。最后,通过形态观察筛选出8株变异较大的菌株,为以后的红曲霉基因功能研究奠定了一定的基础。

A high-effective Agrobacterium tumefaciens（At）-mediated Monascus transformation system was established through optimizing various transferring factors in this study.Spores of Monascus cultured on PDA were collected 21 d after cultivation and prepared for Monascus spore suspension at 106 /mL,and concentration of At was OD600 value at 0.5 with inducing agent concentration at 100 μmol/L.Then At and Monascus were cultured together for 3 d in 25 ℃.Adopted this transformation system an insert mutant library containing over 530 transformants of Monascus T-DNA was established.50 transformant strains were randomly selected to carry out their molecular verification and stability,the results showed that T-DNA had successfully inserted to genome DNA of Monascus purpureus and could be steadily inherited.Finally,eight strains of large variation were selected through morphological observation thus establish a certain foundation for further study on Monascus gene function.