中文摘要：

目的构建烟曲霉KpsF基因缺陷株,初步了解KpsF基因在烟曲霉生长及与钙调磷酸酶相互作用关系。方法PCR扩增烟曲霉KpsF基因及其上、下游各约1.0kb的DNA片段,以pyrG为筛选标记,原生质体法构建KpsF基因缺陷株ΔKpsF;观察ΔKpsF一般生长状况;实时荧光定量PCR方法检测Ku80（对照组）和ΔKpsF（实验组）在含有不同浓度CaCl2的液体GMM培养基中钙调磷酸酶催化亚基（CnaA）相对表达水平。结果烟曲霉ΔKpsF和Ku80径向生长差异无统计学意义（P〉0.05）;表型未见明显差别;ΔKpsF在10mmol/L CaCl2GMM液体培养基的CnaA相对表达量显著高于对照组Ku80（P〈0.01）,而100mmol/L CaCl2的CnaA相对表达量却没有明显变化（P〉0.05）。结论 KpsF基因缺陷对烟曲霉的径向和表型生长没有明显影响,对CnaA表达量的影响与Ca2＋浓度有关。在低浓度Ca2＋条件下,KpsF可能参与对钙调磷酸酶的负反馈调节作用,而在高浓度的Ca2＋条件下,这种负反馈调节作用受到抑制。

英文摘要：

Objective To construct the KpsF gene defect strainsΔKpsF and to explore the interaction between KpsF gene and calcineurin.Methods Amplificated the upstream and downstream of KpsF about 1.0kb DNA fragments,the pyrG of plasmid pJW 24 was used as a biomarker when constructting the knockout plasmid.Recombinant plasmid was transformed into A.fumigatus Ku80 pyrGand gotΔKpsF.To observer the radial and phenotypic growth of Ku80andΔKpsF,Real-time PCR was used to determine the relative expression of calcineurin catalytic subunit（CnaA）of Ku80（control group）andΔKpsF（experimental group）in GMM liquid medium containing different concentration of CaCl2.Results Compared with Ku80,the radial growth ofΔKpsF had no obvious change（P〈0.05）,and the phenotypic growth had no obvious difference.In GMM Liquid medium containing 10mmol/L CaCl2,the relative expression of CnaA ofΔKpsF strain was significantly higher than Ku80（P〈0.01）.However,the relative expression of CnaA did not obviously changed（P〈0.05）in GMM Liquid medium containing 100mmol/L CaCl2.Conclusions The KpsF had no effect in radial and phenotypic growth of A.fumigatus.Under the condition of different concentrations of Ca2＋,the relative expression levels of CnaA gene were different betweenΔKpsF and Ku80,suggesting that the KpsF might be involved in the feedback regulation of calcineurin under the condition of low concentration of CaCl2.