摘要
红曲霉中mok E基因是红曲霉次级代谢产物Monacolin K生物合成的一个相关基因,mok E基因表达量与Monacolin K产量呈正相关。以mok E基因为目的基因,构建红曲霉mok E基因过表达工程菌株,通过逆转录实时荧光定量聚合酶链式反应(reverse transcription-quantitative real-time polymerase chain reaction,RT-q PCR)确定mok E基因过表达转化子,对转化子Monacolin K产量进行测定,同时利用扫描电镜,对野生型红曲霉M1及转化子菌丝、孢子进行观察。结果表明:以潮霉素抗性基因为筛选标记,成功得到240个转化子,对其中9个转化子进行mok E基因表达量测定,有3株转化子mok E基因表达量增加,分别为T2、T8、T9转化子,确定其为mok E基因过表达转化子;T2、T8、T9转化子内酯型Monacolin K产量分别为2 159.7、4 177.6、3 365.7μg/g,与野生型红曲霉Monacolin K产量(1 447.8μg/g)相比,分别提高了49.2%、188.5%及132.5%。扫描电镜结果显示,mok E基因过表达,对红曲霉的菌丝体、孢子形态及生殖方式均有影响。
mok E gene belongs to the Monacolin K biosynthesis gene cluster,whose expression level is positively correlated with Monacolin K production.In this research,mok E gene was overexpressed in Monascus M1 and its expression level was measured by reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR).The Monacolin K production in transformants was measured by high performance liquid chromatography(HPLC).The morphological features of mycelia and spore of the wild-type strain and the transformants were observed by scanning electron microscope(SEM).The results showed that 240 transformants were obtained by hygromycin resistance selection.Nine of these transformants were selected for the measurement of mok E expression by RT-qPCR.The expression levels of mok E gene were enhanced in three transformants,T2,T8 and T9.The production rates of Monacolin K lactone in transformants T2,T8 and T9 were 2 159.7,4 177.6,and 3 365.7μg/g,respectively,which were increased by 49.2%,188.5%and 132.5%,respectively,compared with that in the wild-type strain(1 447.8μg/g).The results of SEM revealed that mok E overexpression contributed to the morphological changes of mycelia and spore.
作者
林琳
王昌禄
李贞景
陈勉华
武淑芬
任志远
LIN Lin;WANG Changlu;LI Zhenjing;CHEN Mianhua;WU Shufen;REN Zhiyuan(Key Laboratory of Food Nutrition and Safety,Ministry of Education,College of Food Engineering and Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2018年第8期45-49,共5页
Food Science
基金
国家自然科学基金面上项目(31330059)
关键词
红曲霉
基因过表达
MONACOLINK
逆转录实时荧光定量聚合酶链式反应
扫描电镜
孢子
Monascus spp.
overexpression
Monacolin K
reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)
scanning electron microscope(SEM)
spore
