多拷贝脂肪酶基因在黑曲霉中表达研究  

Expression of multicopy lipase gene in Aspergillus niger

作　　者：李杰[1] 张贺 王欣[1] 黄超群 徐玥 张会[1] 刘天奇 LI Jie;ZHANG He;WANG Xin;HUANG Chaoqun;XU Yue;ZHANG Hui;LIU Tianqi(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2018年第4期49-58,共10页Journal of Northeast Agricultural University

基　　金：黑龙江省应用技术研究与开发计划重大项目(GA15B203)

摘　　要：文章在使用含6个gla A box强启动子Pgla A6R和高效分泌信号肽Sgla A调控黑曲霉脂肪酶基因Anlip A表达基础上,用pyr G双向筛选标记,应用多拷贝黑曲霉脂肪酶基因策略,构建含1个拷贝Anlip A基因表达载体p SZHA6R-Anlip A和含2个拷贝Anlip A基因表达载体p SZHA6R-2Anlip A,通过农杆菌介导法转化黑曲霉CICC2462(Δpyr GΔas AA::pyr G),获得as AA基因位点整合纯合同源重组转化子CICC2462(p SZHA6R-Anlip A)和CICC2462(p SZHA6R-2Anlip A)。重组菌株经摇瓶发酵后,作荧光定量PCR、SDS-PAGE和脂肪酶活力检测。结果表明,重组菌株可分泌表达约37.0 ku目的蛋白条带,CICC2462(p SZHA6R-2Anlip A)中脂肪酶表达在m RNA水平和酶活水平皆高于CICC2462(p SZHA6R-Anlip A)。发酵第5天CICC2462(p SZHA6R-2Anlip A)中Anlip A基因转录水平为CICC2462(p SZHA6R-Anlip A)1.26倍。二者酶活力在发酵第8天达峰值,此时CICC2462(p SZHA6R-2Anlip A)是CICC2462(p SZHA6R-Anlip A)1.32倍,酶活力达1 480 U·m L-1。对重组菌株CICC2462(p SZHA6R-2Anlip A)第8天发酵液作酶学性质分析,结果表明,最适温度为35℃,最适p H为6.0。获得1株高效分泌表达脂肪酶食品级黑曲霉工程菌。The strong promoter PglaA6R containing six copies of the glaA box and efficient secretion of signal peptide SglaA were used to regulate the expression of AnlipA.Constructed expression vectors pSZHA6R-AnlipA containing one copy of AnlipA and pSZHA6R-2AnlipA containing two copies of AnlipA using pyrG bidirectional selection marker and applying the multicopy Aspergillus niger lipase gene strategy.Two expression vectors were transferred into Aspergillus niger CICC2462(ΔpyrGΔasAA::pyrG)by Agrobacterium-mediated method,then the homologous recombination homozygotes CICC2462(pSZHA6RAnlipA)and CICC2462(pSZHA6R-2AnlipA)at the asAA integration site were obtained.After fermentation in shake flask,detections of fluorescence quantitative PCR,SDS-PAGE and lipase activity indicated the pure homologous recombination strains had about 37.0 ku protein band.The mRNA and enzyme activity levels of lipase in CICC2462(pSZHA6R-2AnlipA)were both higher than those in CICC2462(pSZHA6R-AnlipA).The transcription level of CICC2462(pSZHA6R-2AnlipA)was 1.26 times greater than CICC2462(pSZHA6RAnlipA)on the 5th day.The enzyme activity of the two homologous recombination strains peaked on the 8th day of fermentation.At this time,CICC2462(pSZHA6R-2AnlipA)was 1.32 times greater than CICC2462(pSZHA6R-AnlipA)and the enzyme activity reached 1 480 U·mL-1.The enzymatic properties of the 8th day fermentation broth of the recombinant strain CICC2462(pSZHA6R-2AnlipA)was analyzed.The results showed that the optimum temperature was 35°C and the optimum pH was 6.0.In this way,food grade Aspergillus niger engineering strain that secreted lipase efficiently was obtained.

关 键 词：脂肪酶 多拷贝 分泌表达 黑曲霉 

分 类 号：Q815[生物学—生物工程]