产黄曲霉毒素解毒酶真菌(E-20)cDNA文库的构建

Construction of a cDNA library of the Fungi(E-20)producing aflatoxin-detoxifizyme

目的 :构建产黄曲霉毒素解毒酶 (ADTZ)真菌 (E - 2 0 )的cDNA文库 ,为进一步筛选和克隆 (E - 2 0 )菌的特异表达基因做准备。方法 :用SMARTTMcDNA文库构建试剂盒构建 (E - 2 0 )菌的cDNA文库 ,检测未扩增文库滴度和重组率后 ,进行文库的扩增 ,并检测扩增文库的质量。随机挑取 1 0个阳性克隆进行测序。结果 :未扩增文库滴度达 1 .0× 1 0 6pfu/mL ,重组率约为 98.9% ,扩增后滴度为 3× 1 0 8pfu/mL ,容量约为 4× 1 0 1 0 。测序结果得到 9条新的表达序列标签 (EST) ,1个 (E - 2 0 )菌的NADH -辅酶Q氧化还原酶 (NuoC)基因。结论 :E - 2 0表达型λ噬菌体cDNA文库的成功建立 ,对于该菌种中有明确功能的特异基因如ADTZ基因的克隆 。

Object: To construct a cDNA library of Fungi(E 20) that produce aflatoxin detoxifizyme for screening the genes of the fungi(E 2O). Methods: The cDNA library of Fungi(E 20) were constructed using the SMARTTM cDNA library construction kit. After having constructed the cDNA library, the titer and the recombination rate of the unamplified library were detected and then amplified. Then, ten clones were selected randomly and sequenced. Results: The titer and the recombination rate of the unamplified library were about 1.0×10 6 pfu/ml and 98.9%, and the titer and the capacity of the amplified library were about 3×10 8 pfu/ml and 4×10 10 .Ten expressed sequence taqs (ESTs) were gained from ten clones being sequenced. Blasting in the GenBank, one sample shows high homology with the data of NADH ubiquinone oxidoreductase (NuoC) gene. Accomplishment of this paper is an initial key work for building the resource information databank of the fungus(E 20).