根癌农杆菌介导的定点敲除技术在红色红曲菌中的应用

The Targeted-deletion Technology in the Monascus ruber Mediated via Agrobacterium tumefaciens

红曲菌(Monascus spp.)是一种重要的丝状真菌,广泛应用于食品和医药领域中。目前,由于对红曲菌遗传背景了解的很少,因而对其重要功能基因的研究报道很少。本研究采用根癌农杆菌介导的转化技术对红色红曲菌(Monascus ruber)中推断的G-蛋白信号调节子mrfA基因的RGS功能域进行定点缺失研究,探讨基于同源重组为基础的基因定点缺失技术在红曲菌基因功能鉴定中的可行性。构建的敲除载体pC805S左右同源臂长度分别为958bp和824bp,将其转入受体菌M.ruber中,得到的138株转化子中,有26株转化子发生了同源重组,重组率达到18.8%。实验结果表明该技术作为红曲菌基因功能鉴定的方法是可行的。

Monascus spp., a kind of filamentous fungi, produce abundant of important metabolites which were widely used in the fields of food and medicine. Until now, there are few reports on the important func- tional genes of the Monascus spp. due to little genetic information. In this paper, the feasibility of gene dele- tion mediated via Agrobacterium tumefaciens on the basis of homologous recombination was analyzed by studying on the deletion of the RGS domain of putative G-protein signaling regulator gene mrfA in Monas- cus ruber. The length of homologous arms of deletion vector pC805S were 958 bp and 824 bp, respectively. There were 26 transformants in which homologous recombination occurred in 138 transformants and the re- combination rate was 18.8%. The result showed it was feasible to identify the function of unknown gene in M. ruber with the targeted-deletion technology mediated via A. tumefaciens.