| Cytochrome P450 monooxygenases are important metabolic enzymes involved in catalyzing a number of endogenous and exogenous compounds.They have been found in all living organisms examined,including vertebrates,invertebrates(including insects), plants and microorganisms.As a major kind of Lepidopterous pest,Bombyx mandarina Moore shows a relatively close relationship to Bombyx Mori,and it seriously affects the growth of mulberry and the development of sericulture.To further address the structure and functions of P450,the induced expression of Cytochrome P450 CYP305BIV1 gene in different tissues of Bombyx mandarina Moore and promoter activity of the gene were investigated in the present study.The main results were shown as follows:After feeding Bombyx Mori and Bombyx mandarina with mulberry leaves soaked in different concentration gradient of NaF,Rutin,and Cypermethrin,the roles of different drugs on the main physiological characteristics of Bombyx Mori and Bombyx Mandarina were studied.After 48 hours,the mortality rate of Bombyx Mori and Bombyx Mandarina in each group are showed as follows:10-4 mol/L NaF(66.7%,13.3 %),5×10-3 ng/μL Rutin(60%,26.7%),0.1 ng/μL Cypermethrin(46.7%,11.3%), respectively.The mortality rate of Bombyx Mandarina is significantly higher than that of Bombyx Mori,suggesting that Bombyx Mandarina has higher tolerance for NaF, Rutin and Cyperrnethrin than Bombyx Mori within the tested concentration spectrum.Using the survival wild silkworms treated with four kinds of drugs as mentioned above,based on the mRNA sequence of Bombyx mandarina.P450 CYP305BIV1 gene accessed in GenBank,a pair of primers was designed for analyzing the tissue expression of Bombyx mandarina.CYP305B1V1 gene induced by NaF,rutin,cypermethrin and ecdysone with semi-quantitative RT-PCR.The homology analysis with amino acid sequence of this gene was carried out.The results showed that Cypermethrin,rutin and NaF had effects on the tissue expression of CYP305B1V1 gene in Bombyx mandarina. while ecdysone little effect.Homology analysis showed that this gene was the most similar to Bombyx mori CYP305B1 gene with 100%amino acid identity and highly similar to Tribolium casmneum probable CYP305A1,Apis mellifera probable CYP305A1,Drosophila melanogaster CYP305A1,Anopheles gambiae CYP305A2 and Culex pipiens quinquefasciatus CYP2L1.Two nucleotide sequences,located at 1000bp and 1500bp upstream of the transcriptional start site of P450 CYP305B1V1,were amplified from the genome of Bombyx mandarina by PCR.Luciferase Reporter vectors pGL-YTA and pGL-YTB were reconstructed with the 2 sequences,respectively.And then the vectors were cotransfected with internal control vector pRL-TK into Sf9 cells under the mediation of liposome.After in vitro inducted by NaF,Rutin,Ecdysone,and Cypermethrin,the transcriptional activity and the corresponding regulation factors of the promoter of P450 CYP305B1V1 gene were analyzed.Our results showed that the relative activity of pGL-YTA and pGL-YTB were 5.2 and 2.1-fold than that of pGL3-Basic,indicating that both of the 2 sequences contain promoter activity,and the sequence between 1000 and 1500bp may include some elements that related to transcription inhibition.The data of inducing by the 4 foreign inducers revealed that 10-2 mol/L NaF increased 1.8-fold relative activity of pGL-YTA promoter.Similarly,10-2 mol/L NaF and 0.05 ng/μl Cypermethrin increased the relative activity of pGL-YTB promoter by 2.25 and 5.39-fold,respectively.In summary,CYP305BIV1 gene might be involved in the metabolic process of ectogenic toxicant in Bombyx mandarina,and the precisely effect mechanisms needed further investigation.
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