正调控元件拷贝数对黑曲霉PglaA启动子的影响  

Effect of positive regulatory element copy numbers on Aspergillus niger PglaA promoter

作　　者：李杰[1] 丁纯洁 鄢健楠 安欣 刘天奇 张会[1] LI Jie;DING Chunjie;YAN Jiannan;AN Xin;LIU Tianqi;ZHANG Hui(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

出　　处：《东北农业大学学报》2019年第6期28-35,共8页Journal of Northeast Agricultural University

基　　金：黑龙江省自然科学基金联合引导项目(JJ2019LH1309)

摘　　要：为获得转录水平更高的强启动子,提高黑曲霉中重组蛋白表达量,研究在黑曲霉葡萄糖淀粉酶基因启动子PglaA基础上,构建分别含2、4、6个拷贝的正调控元件片段PglaA2R、PglaA4R、PglaA6R启动子。以黑曲霉木聚糖酶基因xynB为目的基因,研究正调控元件拷贝数对PglaA启动子的影响。构建黑曲霉表达载体pSZHG2R-xynB、pSZHG4R-xynB、pSZHG6R-xynB,采用农杆菌介导法转化黑曲霉CICC2462。获得目的基因表达框整合在葡萄糖淀粉酶基因位点的纯合黑曲霉重组菌株PglaA2R-xynB、PglaA4R-xynB、PglaA6R-xynB。经SDS-PAGE检测观察到重组菌株分泌约24.0ku木聚糖酶蛋白条带。经酶活检测表明,木聚糖酶活力在第8天达最高,重组菌株PglaA4R-xynB(7705.36U mL^-1)、PglaA6R-xynB(8466.32U mL^-1)比PglaA2R-xynB(5890.77U mL^-1)分别提高1.31倍和1.44倍。荧光定量PCR结果表明,重组菌株PglaA4R-xynB、PglaA6R-xynB的xynB基因mRNA比PglaA2R-xynB分别提高2.2倍和5.3倍。可知,PglaA启动子正调控元件多拷贝显著提高木聚糖酶表达。In order to obtain a strong promoter with higher transcription level and increase the expression of recombinant protein in Aspergillus niger,the PglaA2R,PglaA4R,PglaA6R promoters of containing 2,4 and 6 copies positive control element fragment was constructed based on the PglaA promoter of Aspergillus niger glucoamylase gene.Using the Aspergillus niger xylanase gene xynB as the target gene,the effect of positive regulatory elements copy numbers on the PglaA promoter was investigated.The Aspergillus niger expression vectors pSZHG2R-xynB,pSZHG4R-xynB and pSZHG6 RxynB were constructed and transformed into Aspergillus niger CICC2462 by Agrobacterium-mediated transformation.The homozygous Aspergillus niger recombinant strains PglaA2R-xynB,PglaA4R-xynB and PglaA6R-xynB integrated into the glucoamylase gene locus were obtained.The recombinant strains secreted a xylanase protein band of about 24.0 ku by SDS-PAGE.Enzyme activity assay showed that xylanase activity reached the highest on the 8 th day,and the recombinant strains PglaA4 RxynB(7 705.36 U·mL^-1)and PglaA6R-xynB(8 466.32 U·mL^-1)were 1.31 and 1.44 times higher than that of PglaA2R-xynB(5 890.77 U·mL^-1),respectively.The results of real-time PCR showed that the xynB gene m RNA of the recombinant strains PglaA4R-xynB and PglaA6R-xynB were 2.2 and 5.3 times higher than that of PglaA2R-xynB,respectively.Multiple copies of the positive control elements of the PglaA promoter significantly increased in the xylanase expression.

关 键 词：黑曲霉 启动子 多拷贝 木聚糖酶 

分 类 号：Q936[生物学—微生物学]