关键词: 米曲霉, 外源基因表达, 米赫根毛霉脂肪酶, 硝酸盐还原酶, 标记基因

Abstract:

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.

Key words: Aspergillus oryzae, heterologous gene expression, Rhizomucor miehei lipase, nitrate reductase, marker gene