丝状真菌米曲霉外源基因表达系统的构建

以米曲霉Aspergillus oryzae RIB40的基因组DNA为模板,PCR扩增得到启动子、终止子、筛选标记基因等表达元件，依次连接到载体pUC119上,构建了米曲霉的重组表达载体pNMA. 将米赫根毛霉脂肪酶基因(RML)连接于pNMA的启动子下,得到表达载体pNMA-RML,通过ApaI酶切线性化转化米曲霉宿主菌A.oryzae niaD300,得到整合型的阳性转化子A.oryzae ONL1. 其培养7天的培养液上清在以三丁酸甘油酯为底物的平板上形成清晰的水解透明圈,碱滴定法酶活测定表明培养液酶活可达2.5 U/mL,培养液上清的SDS-PAGE图谱在32.5 kDa处有RML的特征条带. 以上结果表明RML已经在米曲霉中成功表达,同时证明所构建的米曲霉外源基因表达系统是有效的.

Construction of Heterologous Gene Expression System for Filamentous Aspergillus oryzae

The expression elements (promoter, terminator, selection marker gene) were obtained by PCR using the genome DNA of A.oryzae RIB40 as template. To obtain the recombinant expression vector pNMA, these expression elements were cloned into the plasmid pUC119 one by one according to a designed orientation. Then Rhizomucor miehei lipase (RML) was cloned into pNMA vector by following its promoter, and the obtained pNMA-RML vector was identified by PCR, digestion and sequencing. For transformation of A.oryzae niaD300, the pNMA-RML vector was linearized by ApaI digestion. One integrative transformant was obtained through PCR and phenotype identification, designated as A.oryzae ONL1. The 7-day fermentation broth of A.oryzae ONL1 could hydrolyze tributyrin verified by forming transparent cleared zone on tributyrin plate, and the enzyme activity reached 2.5 U/mL. The SDS-PAGE electrophoretic map of the fermentation broth demonstrated that there was a typical RML band at 32.5 kDa. So the successful expression of RML in A.oryzae niaD300 suggested that the constructed heterologous gene expression system of A.oryzae was feasible.