(1中国农业科学院蜜蜂研究所北京100093； 2中国农业大学昆虫学系北京100193)摘要荧光分光光度法检测意大利蜜蜂Apis mellifera ligustica Spinol细胞色素P450 О脱乙基活性方便快捷、较灵敏，以7乙氧基香豆素为底物，通过测定其产物7羟基香豆素的荧光变化来确定细胞色素P450 О脱乙基的活性。在利用荧光分光光度法检测意大利蜜蜂幼虫细胞色素P450 О脱乙基活性过程中，反应体系pH值、反应时间、反应温度对实验结果影响较大，本实验在确定最佳底物终浓度和辅酶NADPH终浓度的前提下用正交法研究了pH值、反应时间、反应温度3个因素对意大利蜜蜂细胞色素P450 О脱乙基活性的影响。极差分析和方差分析表明,3个因素对实验结果影响大小顺序为：时间>温度> pH值，并且得到最佳组合条件为：反应时间20 min、温度40℃ 及pH值78。
关键词意大利蜜蜂， 细胞色素P450 О脱乙基活性， 正交试验， 荧光分光光度法， 最佳条件

The optimum conditions for assaying 7ethoxycoumarin
Odeethoxylation of cytochrome P450 from the Italian
honeybee(Apis mellifera ligustica Spinol)
by orthogonal matrix method TANG XiaoWei1,2**GAO XiWu2DIAO QingYun1***
(1Institute of Apicultural Research，Chinese Academy of Agricultural Science, Beijing100093, China;
2Department of Entomology，China Agricultural University，Beijing100193, China)

AbstractThe 7ethoxycoumarin Odeethoxylation（ECOD) activity of cytochrome P450（CytP450) was determined using fluorescence spectrophotometry with 7ethoxycoumarin as a substrate. This method was sensitive and convenient. The optimal conditions for assaying the ECOD activity of cytochrome P450（CytP450) in Apis mellifera ligustica Spinol were determined using the orthogonal matrix method. The results show that reaction time was the most important factor determining the ECOD activity of CytP450 in honeybees. The relative importance of time, temperature and pH on the ECOD activity of CytP450 were, in descending order, time > temperature>pH. The optimal conditions were pH of 78, reaction temperature of 40℃ and reaction time of 20 minutes.
Key words Apis mellifera ligustica, Odeethoxylation, cytochrome P450 monooxygenase,  orthogonal experiment, fluorescence spectrophotometry, optimal conditions