丝状真菌米曲霉外源基因表达系统的构建

Construction of Heterologous Gene Expression System for Filamentous Aspergillus oryzae

以米曲霉(Aspergillus oryzae)R IB40的基因组DNA为模板,聚合酶链式反应(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.oryzaeniaD300,得到整合型的阳性转化子A.oryzaeONL1.其培养7天的培养液的上清液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2.5U/mL,培养液上清液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)图谱在相对分子质量32 500处有RML的特征条带.这说明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.

The expression elements such as promoter, terminator and selectable marker gene were obtained by means of polymerase chain reaction （PCR）, with the genome DNA of Aspergillus oryzae （A. oryzae） RIB40 as the template. The elements were then cloned one by one into the plasmid pUC119 to construct a recombinant expression vector pNMA. Moreover, Rhizomucor miehei lipase （RML） was cloned into pNMA vector by following its promoter, with a pNMA-RML vector being obtained. For the transformation of A. oryzae niaD300, the pNMA-RML vector was linearized by Apa Ⅰ digestion, and an integrative positive transformant A. oryzae ONL1 was obtained. The supernatant of 7-day fermentation broth of A. oryzae ONL1 could hydrolyze to form a transparent clear zone on the tributyrin plate, and the enzyme activity measured by the base titration method reached 2.5 U/mL. It is demonstrated by the SDS-PAGE electrophoretogram of the superuatant that there is a typical RML band at 32 500, which represents a successful expression of RML in A. oryzae niaD300 and verifies the feasibility of the constructed heterologous gene expression system of A. oryzae.