Abstract:

To utilize the high efficiency of glaA expression cassette of Aspergillus awamori and Agrobacterium-mediated transformation in the construction of A.awamori expression vector, a 2.1-kb glaA promoter fragment including signal peptidecoding sequence and a 1.1-kb glaA terminator fragment were amplified from the genomic DNA of A.awamori strain SG1 by PCR and then fused to form a foreign gene expression cassette. The Agrobacterium tumefaciens binary vector pCAMBIA1302 was used as backbone and its T-borders and necessary elements for E.coli and Agrobacterium-mediated transformation were retained whereas other regions in the vector were deleted. The foreign gene expression cassette described above and the filamentous fungal functional hygromycin B resistance marker from pAN7-1 were inserted into the region between T-borders. Finally a secretory integration Agrobacterium-mediated A.awamori expression vector pSUAA52am was constructed. Transformation experiment showed that transformation efficiency of pSUAA52am into A. awamori with protoplast method was 21 transformants/106 spores, while that with Agrobacterium-mediated method reached 1106 transformants/106 spores, 53 folds of the former. This indicated that the construction of this expression vector is successful.

Key words: Aspergillus awamori, glaA expression cassette, Agrobacterium-mediated transformation, expression vector