【作者】 刘小宁；

【导师】 高希武；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2005， 博士

【摘要】 本论文以棉铃虫Heficoverpa armigera（Hubner）为对象,研究了槲皮素、2-十三烷酮、单宁酸等植物次生物质组合诱导对棉铃虫细胞色素P450s含量和活性的影响。明确了槲皮素和2-十三烷酮对棉铃虫P450s活性诱导的时间效应和剂量效应、槲皮素对棉铃虫不同组织总mRNA含量的影响以及植物次生物质对CYP686基因表达量的影响。对棉铃虫P450 CYP686基因进行了克隆和序列分析,并在原核细胞中进行了表达。 采用饲料添加法,用2-十三烷酮和槲皮素处理棉铃虫6龄幼虫48h后,两种植物次生物质对中肠P450s含量诱导具有累加效应,而对脂肪体P450s含量诱导表现为累加或拮抗作用。 利用毛细管气相色谱法灵敏度高,样品用量少,分辨率高的特点,建立了一个用于检测棉铃虫幼虫中肠和脂肪体细胞色素P450s O-脱甲基活性的小型反应体系（875μL）,其中含0.1 mol/L,pH 7.8的Tris-HCl缓冲溶液,0.5μmol NADPH,7.5μmol MgCl₂,0.085% BSA,0.157mmol对硝基苯甲醚及2.08mg/mL酶液。 槲皮素和2-十三烷酮对棉铃虫中肠和脂肪体P450s O-脱甲基活性具有明显的诱导作用。组合诱导也得到相似的结果。当两种植物次生物质浓度为0.5 mg/mL时,对中肠和脂肪体P450s O-脱甲基活性诱导倍数均达到最大,分别为2.63和3.88倍。虽然两种植物次生物质对活性的诱导不存在互作增效关系,但是随浓度增加其活性有增加的趋势。 诱导的时间和剂量效应表明,当槲皮紊浓度为0.1 mg/mL时,中肠利脂肪体P450s的诱导活性随时间是先降低后增强,到48h达到最大（分别为对照的2.03倍和2.4倍）,然后降低到正常水平;而槲皮素浓度为0.5 mg/mL时,诱导活性仅有一个高峰,发生在48 h,诱导倍数分别是2.8和2.79。2-十三烷酮对P450s诱导效应与槲皮素不同,2-十三烷酮对脂肪体P450s活性诱导与对照相比从4-48 h均为诱导增强,48 h后表现为诱导抑制。除2-十三烷酮浓度为0.1 mg/mL,处理时间为4 h和36 h时对中肠P450s活性为诱导增加之外,其余各处理时间点的活性和对照相比差异不显著。 研究了槲皮素对棉铃虫中肠利脂肪体总mRNA比含量影响的时间效应,结果表明在处理5 h、24 h和36 h时,槲皮素对中肠总mRNA含量具有明显的诱导增加作用。脂肪体总mRNA的量诱导组与对照相比没有显著差异。 用半定量RT-PCR检测植物次生物质对CYP686基因表达的影响,表明槲皮素对中肠和脂肪体的CYP686基因的表达量、单宁酸对脂肪体CYP686基因表达量没有明显的诱导作用。单宁酸对中肠CYP686基因的表达量具有明显的诱导作用。2-十三烷酮对中肠和脂肪体两个组织部位的CYP686基因表达量均具有明显的诱导作用。 依据已发表的棉铃虫CYP686基因设计2对引物,用RT-PCR的方法从棉铃虫中肠和脂肪体中分别克隆出P450的全长基因,生物信息学分析结果表明该基因为CYP686基因（注册号 AY950636和AY950637）。更多还原

【Abstract】 The content and activity of cytochrome P450s, the total mRNA, expression of CYP6B6 mRNA induced by 2-tridecanone, quercetin and tannic acid, molecular cloning and expression in Escherichia coli of CYP6B6 gene were studied in cotton bollworm, Helicoverpa armigera (Hiibner).1. The induction of the cytochrome P450s by 2-tridecanone and quercetin was investigated in sixth-instar larvae. Combinations of 2-tridecanone and quercetin showed an additive effect on the content of P450s in the midgut, but either an additive or antagonistic effect on the content of P450s in the fat body.2. An 875μl of reaction system, determining the activity of p-nitroanisole O-demethylation by cytochrome P450s, was developed based on the traits of the capillary gas chromatography (CGC) with higher sensitivity, less sample volume and higher resoling power than spectrophotometry. The total reaction system was 875 μl containing 0.1 mol/1, pH 7.8 Tris-HCl buffer, 0.5 μmol NADPH, 7.5 μmol MgCl2, 0.085% BSA, 0.157 mmol p-nitroanisole and 2.08 mg/ml enzyme solution. The p-nitrophenol, product ofp-nitroanisole O-demethylation by cytochrome P450 of the midgut and fat body in the cotton bollwarm larva, was detected by CGC.3. The O-demethylase activity was higher in the fat body than that in the midgut of the sixth-instar larvae fed on the artificial diets mixed with 2-tridecanone and quercetin. The similar results were obtained when induced by the combination of 2-tridecanone and quercetin at different concentrations. When the concentration of inducers, mixture of 2-tridecanone and quercetin, was 0.5 mg/ml in the diet, the maximum 0-demethyiase activities of P450s in the midgut and fat body were 2.6 and 3.88 times higher than those in the relevant control, respectively. There was no synergism between the effects of two allelochemicals, which both increased significantly the activities of P450s in midgut and fat body.4. The time-course and dosage-course of P450s O-demethylase activities were determined in the larvae treated by quercetin and 2-tridecanone. P450s activities induced by low dosage of quercetin, in midgut and fat body, dropped firstly and then increased. Up to 48 h, activity reached the maximum, which 2.03 and 2.4 times higher than those in the relevant control, respectively. Under the high dosage, activities at 48 h reached the maximum, which 2.8 and 2.79 times higher than those in the relevant control, respectively. P450s O-demethylase activities in fat body of the larvare induced by the high dosage of 2-tridecanone were higher than those by low dosage from 4 h to 48 h, 2-tridecanone obviously inhibited P450s O-demethylase activities at 60 h and 72 h. 2-tridecanone had no effect on P450s activities of midgut compared with the relevant control, except that P450s activities by 0.1 mg/ml 2-tridecanone increased significantly at 4 h and 36 h.5. The effect of quercetin on the total mRNA was investigated, the results showed that the total mRNA was obviously higher than the corresponding control at 5 h, 24 h and 36 h in the midgut, and it had no significant changes in the fat body compared with the control.6. A semi-quantitative RT-PCR method was used to determine the expression level of CYP6B6 mRNA in the midgut and fat body of the larvare induced by by 2-tridecanone, quercetin and tannic acid. The expression levels of CYP6B6 mRNA were obviously correlated with the concentrations of 2-tridecanone, but no correlation with the concentrations of quercetin. No induction of CYP6B6 mRNA expression by tannic acid in fat body was observed, but significant induction was found in midgut.7. Using two pairs of primers designed according to sequence of CYP6B6, we cloned full length of P450 from midgut and fat body tissue in cotton bollworm by RT-PCR. According to biological informatics analysis, the P450 genes (GeneBank accession number, AY950636 and AY950637) belong to CYP6B6.8. Expression vector pGEX-4T-I was used to express the CYP6B6 genes from the cotton bollworm in Echerichia coli; SDS-PAGE and CO difference spectrum analysis showed that t更多还原