橙色红曲菌Ku70基因缺失菌株的构建及功能分析

Construction and functional analysis of Ku70 gene deletion strain of Monascus aurantiacus

红曲菌作为传统的药食同源微生物,可产生多种有益的代谢产物,如:红曲色素、洛伐他汀、γ-氨基丁酸等。构建基因工程菌是研究红曲菌代谢途径的有效方法,同源重组(HR)和非同源末端连接(NHEJ)被认为是丝状真菌DNA双链断裂(DSB)修复的两种主要途径。有研究表明,抑制NHEJ中关键成分的活性通常会提高目的基因的同源重组效率。我们在橙色红曲菌(Monascus aurantiacus)AS3.4384中发现了参与NHEJ途径的Ku70蛋白。通过农杆菌介导的同源重组法成功敲除Ku70基因,发现不影响红曲菌的生长繁殖\\产孢能力以及色素的生物合成,因此可构建Ku70基因缺失菌株作为红曲菌高效基因敲除体系,为后续的基因敲除提供研究材料。

Monascus,as a traditional homologous microorganism of food and medicine,can produce a variety of beneficial metabolites,such as Monascus pigment,lovastatin,γ-aminobutyric acid and so on.The construction of genetically engineered fungi is an effective method to study the metabolic pathway of Monascus.Homologous recombination(HR)and non-homologous end joining(NHEJ)are considered to be the two main repair pathways of filamentous fungal double chain break(DSB).Studies have shown that inhibition of the activity of key components in NHEJ generally improves the efficiency of target gene knockout.We found Ku70 protein involved in the NHEJ pathway in Monascus aurantiacus AS3.4384.Ku70 gene was successfully knocked out by homologous recombination mediated by Agrobacterium tumefaciens,and it was found that the fall growth,reproduction,sporulation ability and biosynthesis of pigment of Monascus were not affected.Therefore,Ku70 gene deletion strain could be constructed as an efficient Monascus gene knockout system,providing material for subsequent gene knockout.