Comparison Of Detoxification Enzyme Activity In Bombyx Mori And Bombyx Mandarina, And Cloning,Expression Of CYP9A20V1 Gene From B.mandarina

Glutathione S-transferases(GST),Esterases(EST) and Cytochrome P450 monooxygenases are three primary detoxifying enzymes of insects.By decomposing or combining with toxic and harmful substances,these eneymes make the toxic substances unable to reach the target sites.Bombyx mori and Bombyx mandarina both belong to Lepidoptera Bombycidae,evolved from the common ancestry.B.mori was under artificial breeding condition for long time while B.mandarina evolved by natural selection.So,the Insecticide resistance,genetic background and other aspects between them exist certain difference.Using enzyme activity kinetic methods,we measure the detoxifying enzymes activity in different tissues of B.mori and B.mandarina larvaes on day 3 of fifth instar. The results showed that the GST activity differed significantiy in different tissues (fatbody＞midgut).Among them,the GST activity of B.mandarina fatbody is 1.67-fold higher than B.mori fatbody(296±26.85,177.2±5.66 nmol/min/mg protein).There is a remarkable difference between them.The GST activity in midgut of B.mori and B.mandarina is almost the same.EST activity in midgut is highest in all tissues which indicate hydrolysis of ESTs mainly occurs in midgut.The EST activity of B.mandarina midgut is 1.98-fold higher than B.mori midgut(467±10.2,235.8±3.28 nmol/min/mg protein).But the EST activity in fatbody of B.mori and B.mandarina are both lower. P450s activity in fatbody is higher than midgut in B.mandarina while It is hard to detect the P450s activity in both fatbody and midgut of B.mori.The result that the activity of all three primary detoxifying enzymes in B.mandarina is higher than B.mori is in accordance with the conclution which shows that the insecticide resistance of B.mandarina is stronger than B.mori. There are two main insecticide resistance mediated by insect detoxifying enzymes. One is the change of gene function,the other is the gene over-expression.It has been studied that the increase of insecticide resistance in some insects is caused by the change of P450s.In order to study silkworm metabolic mechanism to insecticide,we compared the structure and function of cytochrome P450 from B.mori and B.mandarina.A member gene CYP9A20V1(GenBank accession number FJ378716) of P450 family was cloned from B.mandarina midgut using RT-PCR(reverse transcription and polymerase chain reaction).Sequence analysis revealed that this gene contains an ORF of 1 596 bp, encoding a protein of 531 amino acids with predicted molecular weight of 6.4 kD and isoelectric point of 8.1.Online Blast searches showed that B.mandarina CYP9A20V1 had the highest identity(98.7%) with B.mori CYP9A20,and its identity with Helicoverpa armigera CYP9A12 reached 57.1%as well.Comparing the six substrate recognition domains of P450s,B.mandarina CYP9A20V1 and B.mori CYP9A20 shared four identical domains namely SRS1,SRS2,SRS4 and SRS6,and two homologous domains namely SRS3(88.9%) and SRS5(94.1%).We deduced that the two genes have the same substrate recognition capability.B.mandarina CYP9A20V1 had four homologous domains with H.armigera CYP9A12,with sequence identity as 47.1% (SRS1),88.2%(SRS4),76.5%(SRS5) and 60%(SRS6) respectively,showing high homology in substrate recognition.We presume that cytochrome P450 CYP9A20V1 gene maybe associated with Pyrethroid resistance in the B.mandarina.To study the function of B.mandarina CYP9A20V1,we carry eukaryote expression experiment with Bac-to-Bac baculovirus expression system and examine products activity.A recombinant virus named Bac-CYP9A20V1 was constructed.Transfecting Sf9 cell with this recombinant,we finally get the expression product.SDS-PAGE and Western blotting showed that a specific band consistent with the expected size of protein was detected around 64 kD.we use PNA as reactant to measure the activity of expression product,The results showed that the expressed protein have higher O-demethylase activitie(0.692±0.075nmol/min/mg protein) while the nomal Sf9 cell and Sf9 cells transfected with Bacmid can't get the signal.It indicates that eukaryote expression was successful and is helpful to make further research into the structure and function of target gene.