黑曲霉pepD基因阻断突变菌株的构建及功能分析

运用同源重组技术破坏了黑曲霉基因组中的pepD基因,该基因编码一种类subtilisin的胞外蛋白酶PEPD。实验以黑曲霉GICC2773基因组DNA为模板,PCR扩增pepD基因,并在此基因中间插入潮霉素抗性基因(hph)表达单元,由此产生了3.7kb的pepD阻断基因片段。将此阻断基因片段与载体pBS连接,构建成pepD基因阻断质粒pBSDH。采用原生质体-CaCl2/PEG法将酶切阻断质粒得到的含pepD基因和hph表达单元的3.7kb线性片段转化AspergillusnigerGICC2773菌株,在含潮霉素的平板上筛选潮霉素抗性转化子,从这些抗性转化子中经PCR检测分离到到1个pepD基因阻断突变菌株?pepD66。外源漆酶分泌活性分析显示,黑曲霉pepD基因的破坏使其外源漆酶的分泌表达有所提高。

CONSTRUCTION OF PEPD GENE DISRUPTION MUTANT IN ASPERGILLUS NIGER AND ITS FUNCTIONAL ANALYSIS

The Aspergillus niger pepD gene encoding PEPD,a kind of subtilisin-like extracellular protease, was disrupted using homologous recombination technology. The experiment was performed as follows. A pepD gene fragment was amplified by PCR with GICC2773 genomic DNA as template and cloned into vector pBS. Then the selection marker, a hygromycin resistance (hph) expression cassette, was inserted into the pepD gene resulting in the pepD disrupted plasmid pBSDH. The plasmid was completely digested with Stu I and the resultant 3.7kb linear fragment, in which the hygromycin resistance expression cassette was flanked with a 1067bp 5’ fragment of the pepD gene and a 1180bp 3’ fragment of the pepD was then used as transforming DNA for GICC2773 strain. The recipient GICC2773 contains an integrated laccase expression cassette. Total 84 transformants were obtained by hygromycin resistance selection, and then were screened by PCR using a 5’ primer annealing to the hph gene and a 3’ primer annealing to sequences outside the 3’ homologous fragment. Transformant pepD66 was identified to be a pepD gene disruption strain. The effect of pepD disruption in ?pepD66 on laccase expression was analysed. The result indicated that the disruption of pepD improved heterologous protein laccase production to some extent without any negative effect on total protein production. The disruption of pepD gene did not have any negative effect on growth neither. Thus the disruption of pepD in a recipient strain would be helpful for protecting the heterologous proteins from degradation.