| Cytochrome P450 monooxygenase which is the core enzyme of microsome mixed-function oxidase (MFO) is an important detoxifying enzymes in insects. It is involved in growing, developing and feeding, for example, it can catabolize exogenous material and secondary metabolites in plant body. Increased detoxification of insecticides mediated by CytP450 was proved to be an important mechanism of insecticide resistance. Besides, CytP450 plays an important role in synthesis and metabolism of some kinds of endogenous substances such as juvenile hormone, moulting hormone, fatty acid. Most foreign research on MFO in honey bee centered in the 1980s, and these researchers found MFO not only existed in honey bees but also involved in degradation of toxic substance. These years, the incidents of honeybee poisoning still happen occasionally, it is necessary to study CytP450 in honeybee, and the study can also lay foundations for the research on honeybee protection.CytP450 in Italian honeybee (Apis mellifera ligustica Spinola) was studied in the following aspects: assaying method, tissue distribution, the variation of the activity of ECOD of CytP450 in different ages and months, different species of honeybee, chemical inducing and so on. In addition, a new CytP450 gene was cloned by the means of Rapid Amplification cDNA Ends(RACE).Firstly, an optimal reaction system suitable for measuring the activity of 7-ethoxycoumarin O-deethoxylation (ECOD) of CytP450 in Apis mellifera L was established by orthogonal matrix method. The activity of ECOD was determined by fluorescence spectrophotometry, using 7-ethoxycoumarin as substrate. The optimal conditions of assaying the activity of ECOD were: pH 7.8, 40℃and 20 minutes. .Secondly, the activities of ECOD of CytP450 in different tissues, different ages, different months and different species were measured by using the eastablished method. For A. mellifera, the activity of ECOD of CytP450 in midgut was greater than that in head, and the thorax had few activity. The activity of ECOD rose gradually from the third day of larva, reached the maximum on the first day of sealed brood, then the decrease began till pupal stage, rose a bit at the stage of young bees and adult bees. The activity of ECOD of CytP450 on the first day of sealed brood was higher than that of any other ages notedly. The first day of sealed brood larva was collected from April to October in order to measure the activity of ECOD of CytP450. The results showed that the activity of ECOD of CytP450 from April to July was higher than that from August to October. The activity of ECOD of CytP450 was different in three species of honeybee. Apis mellifera ligustica and Apis mellifera carnica had higher activities of ECOD of CytP450 than Apis cerana cerana.Thirdly, the median lethal concentration (LC50) of tau-fluvalinate, amitraz, malathion and methomyl were used in bioassay with contact method, the activities of ECOD of CytP450 in honeybees treated by LC50 of four insecticides for 48 hours were measured, the result showed the enzyme activities both in midgut and head of treated honeybees were higher than that of control. The enzyme activities in midgut between treated honeybees and control group honeybees had significant difference except the honeybees treated by amitraz, and the enzyme activity in head between treated honeybees and control group honeybees had no significant difference.In addition, a CytP450 gene named as AmCYP6AQ1 by Nomenclature Committee of CytP450 was cloned by the means of Rapid Amplification cDNA Ends, its accession number in GENEBANK is HQ874630, the sequence was released on March 6th.The conclusions above can fill the gaps in the study of honeybees CytP450, lay foundations for further study on honeybees CytP450, provide theoretical basis for utilizing detoxification mechanism in bee protection.
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