根癌农杆菌介导酸性蛋白酶在泡盛曲霉中表达的研究  

Transformation and expression of pepA in Aspergillus awamori mediated by Agrobacterium tumefaciens

作　　者：潘力[1] 李俊星[1] 苗小康[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《食品工业科技》2011年第5期178-182,共5页Science and Technology of Food Industry

基　　金：国家科技支撑计划(2007BAK36B03);广东省科技攻关项目(2008A010900002)

摘　　要：利用Gateway克隆技术快速构建丝状真菌表达载体,以米曲霉α-淀粉酶A启动子(amyB)、α-糖苷酶终止子(agdA)及黑曲霉酸性蛋白酶基因(pepA)分别构建入门载体,以带有潮霉素抗性标记(hygB)的农杆菌双元载体pHGW为目的载体,通过LR连接反应,构建根癌农杆菌介导丝状真菌重组表达载体pHGW-apA。转化泡盛曲霉宿主菌,筛选的转化子经传代培养确定其遗传稳定性,PCR及测序分析表明,hygB和目的基因pepA整合到了泡盛曲霉基因组。转录水平上的RealTime-PCR定量分析、表达水平上的Western Blotting分析以及培养液酶活分析表明酸性蛋白酶基因pepA在泡盛曲霉中得到正确表达。为丝状真菌表达载体快速构建与外源基因在丝状真菌中快速表达提供了可行的方法。The entry clones which include Aspergillus oryzae amyB promoter,agdA terminator fragment and Aspergillus niger pepA fragment were constructed by using the Gateway technology.And the Agrobacterium tumefaciens binary expression vector pHGW-apA containing mark gene and expression gene was successfully constructed through the LR reaction.Conidiospores were incubated with induced Agrobacterium,after a cocultivation and selection period,hygromycin-resistant transformants were screened with an efficiency of 25 transformants per 1×106 conidiospores.The integration of the hygB gene and pepA gene into Aspergillus awamori genome was determined by PCR and sequencing.RT-PCR,Western Blotting analysis and determination of acidic protease enzyme activity showed that pepA gene was expressed correctly in A.awamori.The system of expression in filamentous fungi was successful.

关 键 词：根癌农杆菌 PEPA 双元载体 遗传转化 

分 类 号：TS201.3[轻工技术与工程—食品科学]