农杆菌介导的红曲菌T-DNA插入突变库的构建及色素突变子的性质分析

以农杆菌EHA105为介导,将带有潮霉素抗性基因和gus基因的T-DNA片段转化到红色红曲菌(Monascusruber)中。通过转化条件的优化,成功构建了含有5132个转化子的T-DNA插入突变库。根据菌落颜色与色素分泌情况筛选到50株色素突变子,聚合酶链式反应(PCR)表明上述突变子均有T-DNA片段插入。经5代的继代培养后,94%的突变子具有稳定性(潮霉素抗性)。对突变子产色素和桔霉素能力的分析表明其分泌次生代谢产物的能力发生了较大变化。本方法的建立,为进一步深入研究红曲菌的代谢调节和基因功能奠定了基础。

Construction of T-DNA insertional library of Monascus mediated by Agrobacterium tumefaciens and characteristic analysis of the color mutants

T-DNA insertional library of Monascus ruber mediated by Agrobacterium tumefaciens was constructed, which consisted of 5132 transformants. 50 of color-producing mutants were screened from T-DNA insertional library based on differences of the colony color and pigment production between transformants and original strain M-7. All these mutants were identified by PCR amplification and the result showed that T-DNA was integrated into the genome of them. 94% of mutants screened was fully stable after five rounds successive culture and showed resistance to hygromycin B The abilities to produce pigment and citrinin were changed greatly by UV-VIS and HPLC analysis.The method of T-DNA transformation mediated by A. tumefaciens should be very useful for further studies on the metabolites of Monascus spp. and the function of gene of interest.