Construction of a full-length cDNA library from the Aspergillus oryzae RIB40

Abstract:

[Objective] To construct a full-length expression cDNA library from Aspergillus oryzae RIB40 for screening and cloning genes related to the synthesized pathway of secondary metabolites in Aspergillus oryzae. [Methods] Total RNA was isolated from A. oryzae RIB40 using the method of RNAiso and mRNA was purified by PolyATract mRNA Isolation System III. Single-strand cDNA and double-strand cDNA were synthesized from about 5 μg mRNA using ZAP-cDNA Synthesis Kit and the cDNA were fractionated by the CHROMA SPIN-400 column, then ligated into Uni-ZAP express vector and packaged. [Results] A high quality full-length expression cDNA library from the A. oryzae RIB40 was constructed. The titer of the primary library was 2.96×106 CFU/mL with a recombination rate of 97.8% and the average length of inserted fragments was more than 1.5 kb. Furthermore, the titer of the amplified library achieved to 3.4×1010 CFU/mL. [Conclusion] The cDNA library which constructed in this study provides a basis for basic biological research and screening and cloning of the target gene of Aspergillus oryzae.

Key words:Aspergillus oryzae RIB40, Expression vector Uni-ZAP XR, Full-length cDNA library, Quality identification