Study Of Aspergillus Niger Acid Protease Gene Expression In Aspergillus

Filamentous aspergilli are excellent potential hosts for protein expression and important industrial strains, widely used in fundamental researches and industrial applications. The gene engineering of aspergilli has become increasingly active, and their genetic transformation systems turned to be essential. In this paper, the transformation system of aspergillus has been investigated. Aspergillus awamori CBS115.52 was successfully transformed with Aspergillus niger acid protease gene PepA using agrobacterium-mediated transformation method (AMT), and the obtained A. awamori transformants could express secretory A.niger PepA. We also transformed Aspergillus oryzae with PepA using protoplast PEG-CaCl2 transformation and AMT method respectively.In this study, the hygromycin B resistance gene were used as the selection marker in A.awamori CBS115.52 transformation, which were determined through the inhibition experiments of hygromycin B on A.awamori CBS115.52. The expression vector of PepA (pHGW-aPA) was constructed using entry clones of expression elements and the destination vector pHGW via Gateway technology. pHGW-aPA was transformated into A. awamori CBS115.52 by AMT method, and PCR and sequencing confirmed that the A.niger PepA was tranformated into A.awamori CBS115.52. Real-time quantitative PCR (qPCR) and Western blotting experiments illustrated that positive A. awamori transformants could express and secrete PepA at transcriptional level and translation level.The transformation of A. oryzae was studied in this paper on the basis of A.awamori transformation. The susceptibility of A. oryzae to hygromycin B was enhanced by adding chlopromazine to the culture medium. 200 mg/ml hygromycin B plus 0.1 mmol/L chlorpromazine could perfectly inhibit the growth of A. oryzae in CD medium, so the hygromycin B resistance gene could be used as the selection marker in A. oryzae AS3.951 transformation via AMT method. Subsequently, pHGW-aPA was transformed into A. oryzae AS3.951 and positive transformants were identified by PCR amplification. The inhibition of pyrithiamine (PT) to A. oryzae AS3.951 was also studied, The result showed that the growth of A. oryzae AS3.951 was totally inhibited by 0.1μg/mL PT in CD medium, so the pyrithiamine resistance gene ptrA could be used as the selection marker for A. oryzae AS3.951 transformation. With ptrA as selection marker, A. oryzae AS3.951 was transformed with the expression vector of PepA, by protoplast transformation method in three ways (co-transformation, circle plasmid vector transformation and linear plasmid vector transformation).