【作者】 郑明奇；

【导师】 张文吉；

【作者基本信息】 中国农业大学 ， 农药学， 2003， 博士

【摘要】 尽管P450s酶系在昆虫中的重要性已经十分清楚，但由于多型P450共存于同一昆虫体中，要真正了解和调控P450的功能，需要剖析不同P450的底物特异性，首先必须分离纯化出单型P450，因此P450分离纯化在P450研究中具有重要的意义。 棉铃虫是一种在世界范围内普遍发生的重要害虫，能导致多种不同作物的严重损失。为深入了解棉铃虫P450的结构与功能，我们就棉铃虫P450的诱导、分离纯化及性质进行了研究，主要研究结果如下。 比较萘、五甲基苯、槲皮素、芸香苷和单宁酸对棉铃虫微粒体P450含量的影响发现：萘和五甲基苯能明显诱导微粒体P450，中肠微粒体P450的含量分别提高了2.60和2.34倍，脂肪体微粒体P450的含量分别提高了2.51和3.15倍。槲皮素、芸香苷和单宁酸对脂肪体微粒体P450未表现出诱导作用，对中肠微粒体P450有一定的抑制作用，并导致棉铃虫对溴氰菊酯的敏感性增加。 对CHAPS、Lubrol PX、Emulgen 911和胆酸钠四种去垢剂的比较研究发现：CHAPS对中肠和脂肪体微粒体P450的增溶效率最高，Lubrol PX、Emulgen 911和胆酸钠较差。CHAPS增溶中肠和脂肪体微粒体P450的最适浓度分别为0.5％和0.5％或0.8％。在0.5％浓度下，四种去垢剂均未使中肠和脂肪体微粒体P450变性。 PEG沉淀法被用于P450的初步纯化。当PEG的浓度为2％时，约30％的微粒体蛋白和10％的P450被沉淀；PEG为5％时，约55％的微粒体蛋白和20％的P450被沉淀；PEG为8％时，约78％的微粒体蛋白和30％的P450被沉淀；PEG为11％时，约85％的微粒体蛋白和70％的P450被沉淀。其中，8％CHAPS适合于微粒体P450的初步纯化。经PEG沉淀后，中肠微粒体P450纯度提高了2.1倍，回收率为72％；被沉淀出的蛋白主要集中在14.4-40kDa和66-97kDa范围内。 本文建立了HPHIC（Phenyl-5PW）分离P450的优化条件：①检测波长为405nm；②流速0.5ml／min；③50min线性梯度洗脱，从100％HICA₂至100％HICB。在该条件下，中肠微粒体经HPHIC分离后，P450主要存在于馏分7（tR=39′0″-44′0″）和馏分8（t_R=44′01″-49′0″）中；与微粒体相比，P450纯度提高3.04倍，回收率为55.3％。 本文还建立了HPIEC（DEAE-5PW）分离P450的优化条件：①检测波长为405nm；②流速0.5ml／min；③40min线性梯度洗脱，从100％IECA₂至100％IECB₂。在该条件下，馏分7和馏分8中的P450均在t_R=5-7min范围内洗脱出来；与微粒体比，P450纯度提高6.3倍，回收率为17.3％。 SDS-PAGE（银染）显示：经HPIEC分离后，馏分7得到分子量为59±1.0kDa和48±1.9kDa的两条蛋白带；馏分8得到分子量为55±0.2kDa和46±1.9kDa的两条蛋白带。更多还原

【Abstract】 Although many evidences show P450 play important roles in insects, knowledge regarding the structure and function of individual P450 in insects is limiting. Isolation and purification of individual P450 have been vastly improving our understanding of several important aspects of P450 in insect such as P450 diversity, insecticide resistance, tolerance to plant toxins and metabolism of endogenous compounds, which have been the focus and nodus of P450 research .Helicoverpa armigera is an economically important pest around the world. It has caused damage to many different crops. In order to know more about the structures and functions of individual P450 in H. armigera, attempts were given to develop purification methods suitable for P450s in this pest. The main results are as followed.The effects of naphathalene (NA) , pentamethylbenzene (PMB) , quercetin, rutin and tannic acid on P450 content of microsomes in H.armigera were compared. NA and PMB had significant induction effects on microsomal P450s, and increased the total P450 of midgut microsome up to 2.60 and 2.34 fold, respectively, and that of fatbody microsome up to 2.51 and 3.15 fold, respectively. Quercetin, rutin and tannic acid had no induction effects on P450 of fatbody but decreased the total P450 of midgut, and increased the susceptibility of the cotton bollworm to deltamethrin.CHAPS efficiently solubilized the P450 of microsomes in midgut and fatbody, while lubrol PX , emulgen 911 and sodium cholate were less efficient. 0.5% and 0.5% or 0.8% CHAPS were suitable for solubilization P450 of microsomes in midgut and fatbody, respectively. Four detergents above mentioned didn’t denature P450 of microsomes in midgut and fatbody at concentration of 0.5%.PEG precipitation was used in prefractionation of microsomes P450. At concentration of 2% PEG8000, 30% microsomal protein containing 10% of total P450 was precipitated. At concentration of 5% PEG8000, 55% microsomal protein containing 20% of total P450 was precipitated. At concentration of 8% PEG8000, 78% microsomal protein containing 30% of total P450 was precipitated. At concentration of 11% PEG8000, 85% microsomal protein containing 70% of total P450 was precipitated. 8%PEG was found to be suitable for prefractionation of microsomes P450. After precipitation by PEG, purity of P450 from midgut microsome was increased up to 2.1 fold, and recovery was 72%. Most proteins with molecular weights of 14.4-40kDa and 66-97kDa were precipitated by PEG8000.The optimal conditions of HPHIC for purifying P450 of microsomes were established, i.e.(1)detecting wavelength is 405 nm,(2)flow rate is 0.5ml per min,(3)50min linear gradient from 100% HICA2 to 100% HIC B. Separated by HPHIC, P450s of microsomes were eluted during 39’0’’-44’0’’(fraction 7) and 44’01’’-49’0’’ (fraction 8). Compared with microsomes, purity of P450 increased upto 3.04 fold, recovery of P450 was 55.3%.The optimal conditions of HPIEC for purifying P450 of microsomes were established, i.e. (1)detecting wavelength is 405 nm,(2)flow rate is 0.5ml per min,(3)40min linear gradient from 100%IEC A2 to 100%IEC B2. Separated by HPIEC, P450s in fraction 7 and fraction 8 are all eluted during 5’0’’-7’0’’.Compared with microsomes, purity of P450 increased up to 6.3 fold, recovery of P450 was 17.3%.SDS-PAGE (stained with silver) showed that,separated by HPIEC, fraction 7 and 8 both have two protein brands with molecular weights of 59±1.0, 48±1.9kDa and 55±0.2, 46±1.9kDa, respectively.更多还原