米曲霉(Aspergillus oryzae) RIB40全长cDNA文库的构建

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

摘要:

【目的】构建米曲霉RIB40的全长cDNA表达文库，为米曲霉功能基因的开发以及次生代谢产物合成途径相关基因的筛选与克隆奠定基础。【方法】采用RNAiso法从米曲霉RIB40菌体中提取总RNA。选用PolyATract mRNA Isolation System III试剂盒分离纯化mRNA。以5 μg mRNA为模板，按照ZAP-cDNA Synthesis Kit试剂盒说明书要求合成单、双链cDNA，使用CHROMA SPIN-400柱离心层析纯化后连接于Uni-ZAP XR表达载体上，体外包装后转染Escherichia coli XL1-Blue宿主菌。【结果】构建了米曲霉RIB40的全长cDNA文库，初级文库滴度约为2.96×106 CFU/mL，重组率约为97.8%，插入片段平均长度大于1.5 kb，达到一个高质量cDNA文库的要求。文库扩增后，滴度达到3.4×1010 CFU/mL。【结论】米曲霉RIB40全长cDNA表达文库的成功构建，将会对米曲霉基础生物学研究及相关基因的筛选与克隆奠定 基础。

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.