CytP450 Stegeman and Livingstone 1998 CytP450 CytP450 Scott 1999 CytP450 Schuler 1996 CytP ECOD CytP450 P450 О- PNOD PNP 412 nm Hansen

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1 檻檻檻檻檻檻檻檻檻檻檻檻檻檻檻檻檻檻 * P450 O- 1 2** 2 1*** Apis mellifera ligustica Spinol P450 О P450 О- P450 О- ph NADPH ph 3 P450 О- 3 > > ph 20 min 40 ph 7. 8 P450 О- 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-Wei 1 2** GAO Xi-Wu 2 DIAO Qing-Yun 1*** 1. Institute of Apicultural Research Chinese Academy of Agricultural Science Beijing 2. Department of Entomology China Agricultural University Beijing China China Abstract The 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 MFO 2001 P450 * 2006BAD06B04 ** txw @ 163. com *** dqyun1@ 126. com

2 CytP450 Stegeman and Livingstone 1998 CytP450 CytP450 Scott 1999 CytP450 Schuler 1996 CytP ECOD CytP450 P450 О- PNOD PNP 412 nm Hansen and Hodgson MROD 1. 1 Mayer ECOD PMSF Aitio 1978 Merrk 99% DTT AE 99% Ⅱ NADPH P450 99% Biomol Tris P450 Wolff et al. Promega BSA 1979 G25O 7-7-Hydroxycoumarin 7-7- Ethoxycoumarin Sigma Gly TCA EDTA P R Smirle et al Yu et al Eppendoff SPH-100B LS55 Smirle and Winston 1987 Kezic' et al Perkin Elmer P450s P Yao et al ml 0. 1 mol / L ph mmol / L EDTA 50 mmol / L PMSF 50

3 5 P450 O mmol / L DTT 10% g 10 min P450 NADPH Bradford 1976 excitation wavelength nm excitation slit - 5 nm emission wavelength nm emission slit - 5 nm 7 - P450 O μm / L H P450 pmol min -1 mgpro -1 = 2 Table 1 Experimental factors and levels for orthogonal project P450 O- ph Reaction Temperature Hung Sun Level A time min B C P ml 0. 1 mol / L Tris-HC NADPH A B C ph 220 Numbers and 4 represent 4 levels of factors letters TCA 15% A B C represent factors of ph reaction time temperature g 1 min μl 450 μl - Gly-NaOH ph μmol / L μmol / L μmol / L P450 O- 7 - pmol 0. 1 μm / L mg min 2. 2 P450 O ph 3 3 ph 3 SPSS L NADPH P450 O- NADPH > > ph 4 20 min 4 40 ph

4 Fig. 1 1 The effect of substrate concentration and NADPH concentration on the activity of O-deethylation of cytochrome P450 2 O- 3 L Table 2 Application of L orthogonal matrix in detecting the activity of O-deethylation of cytochrome P450 in Italian honeybee larva Number Combination of treatment Specific activity of ECOD pmol min - 1 mg Pro A 1 B 1 C ± A 1 B 2 C ± A 1 B 3 C ± A 1 C ± A 2 B 1 C ± A 2 B 2 C ± A 2 B 3 C ± A 2 C ± A 3 B 1 C ± A 3 B 2 C ± A 3 B 3 C ± A 3 C ± A 4 B 1 C ± A 4 B 2 C ± A 4 B 3 C ± A 4 C ± ± Every row of run number represents one experimental replicate every run was replicated three times and every replicate was measured three times. Values are mean ± SE. Table 3 The method of range analysis O- Specific Activity of ECOD pmol min - 1 mg Pro - 1 A B C K K K K k k k k R B > C > A A 3 B4 C4 A 3 C 4 K i i k i K i K i is the sum of the specific activity ECOD of the i level of different factors and k i represents the average of K i. 2. 4

5 5 P450 O P450 ph 2004 P450 O- 4 Table 4 Source of Variation Sum of square Degree of freedom Mean of square A B ** P < Two blank rows were consolidated into the error item. * and ** represent significant difference P < and extremely significant difference P < respectively. F B C P450 O- ECOD P450 O- ph P450 O- ECOD ph 3 ph ph ph P NADPH 2005 L ph Analysis of the results from orthogonal ph 7. 8 projects with ANVOA 30 10min 2010 ph References P4503A P4503A P450 O- C * 6 Consolidated error 6 * ** P < 6 P450 0 ~ 4

6 P450 O Aitio A A simple and sensitive assay of 7- ethoxycoumarin deethylation. Anal. Biochem Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem / Hansen LG Hodgson E Biochemical characteristics of insect microsomes. Biochem. Pharm Hung CF Sun CN Microsomal monooxygenases in diamondback moth larvae resitant to fenvalerate and piperonyl butoxide. Pestic. Biochem. Physiol P Kezic' N Lucic' D Sulimanovic' D Induction of mixed function oxidase activity in honey bee as a bioassay for detection of environmental xenobiotics. Apidologie Wolff T Deml E Wander H Aldrin epoxidation a P450 highly sensitive indicator specific for cytochrome P dependent mono-oxygenase activities. Drug Metab Dispos P450 O. Yao MC Hung CF Sun CN Fenvalerate resistance and aldrin epoxidation in larvae of the diamondback moth Mayer RT Jermyn JW Burke MD Prough RA Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal O-dealkylases. Pestic. Biochem. Physiol P4503A Schuler M The role of cytochrome P450 monooxygenases in plant-insect interactions. Plant Physiol Scott JG Cytochromes P450 and insecticide resistance. Insect Biochem. Molec. Biol Smirle MJ Winston ML Intercolony variation in pesticide detoxification by the honey bee Hymenoptera Apidae. J. Econ. Entomol Smirle MJ Winston ML Woodward KL Development of a sensitive bioassay for evaluating sublethal pesticide effects on the honey bee Hymenoptera Apidae. J. Econ. Entomol Stegeman JJ Livingstone DR Forms and functions of cytochrome P450. Comp. Biochem. Physiol /3 Pestic. Biochem. Physiol P450 CO Yu SJ Robinson FA Nation JL Detoxication capacity in the honey bee Apis mellifera L. Pestic. Biochem. Physiol

7 檻檻檻檻檻檻檻檻檻檻檻檻檻檻 * ** electrical penetration graph EPG EPG EPG EPG EPG EPG Some key points in applications of electrical penetration graph technique TANG Qing-Bo ZHANG Da-Shan JI Kun DING Shi-Bo YAN Feng-Ming ** Institute of Chemical Ecology Henan Agricultural University Zhengzhou China Abstract The electrical penetration graph EPG has been extensively used in research on plant-insect interactions virus transmission mechanisms and plant resistance mechanisms. However some misunderstandings in the application of EPG have resulted in mistakes in experimental design or misinterpretation of results. This paper provides solutions to some particular problems in EPG applications including EPG application fields how to deal with plant and insect materials equipment manipulations interpretations of EPG waveforms and their biological correlations selection of variables statistics as well as EPG recordings on artificial diets. Key words application scopes selection of materials adjusting of equipments biological correlation of waveforms artificial diet electrical penetration graph EPG Woodford et al Nisbet et al EPG Rahbeet al Mauck et al DC-EPG DC-EPG EPG 1 EPG EPG EPG EPG EPG EPG EPG EPG * ZX B ** fmyan@ henau. edu. cn

8 EPG EPG Tjallingii and Mayoral 1992 Tjallingii 1988 EPG 3 EPG 2. 2 EPG 24 h EPG EPG Walker and Janssen 2000 EPG Eenink et al Will et al Yin et al EPG CO 2 CO EPG 2 EPG 20 ~ 30 s EPG Martin et al DC-EPG

168 健等木醋对几种小浆果扦插繁殖的影响第1期 the view of the comprehensive rooting quality, spraying wood vinegar can change rooting situation, and the optimal concent

168 健等木醋对几种小浆果扦插繁殖的影响第1期 the view of the comprehensive rooting quality, spraying wood vinegar can change rooting situation, and the optimal concent 第 31 卷第 1 期 2013 年 3 月经济林研究 Nonwood Forest Research Vol. 31 No.1 Mar. 2013 木醋对几种小浆果扦插繁殖的影响健 1,2 杨国亭 1 刘德江 2 (1. 东北林业大学生态研究中心黑龙江哈尔滨 150040 2. 佳木斯大学生命科学学院黑龙江佳木斯 154007) 摘要为了解决小浆果扦插繁殖中生根率及成活率低等问题