Prokaryotic Expression, Enzyme Assay And Function Of Melanization Of The Tyrosine Hydroxylase In Mealworm, Tenebrio Molitor (Insecta Coleoptera)

Melanization plays an important role in the process of cuticle tanning, pigmentation and immune defense of insects.It is that tyrosine can be hydroxyed to 3, 4-dihydroxyphenylalanine (DOPA), then DOPA is catalyzed to catecholamines and its derivatives, at last the research shows that the hydroxylation of tyrosine to DOPA can be generated by two types of enzymes: tyrosine hydroxylase (TH, EC 1.14.16.2) and phenoloxidase (PO, EC 1.14.18.1).The indepth studies of PO exist widely in each development stage of Lepidoptera, Coleoptera, Diptera, Blattariae and Orthoptera insect. But the prevenient researches on TH of insects mainly focused on Lepidoptera and Diptera insects, especially the D. melanogaster, but little on the TH gene and its function of Coleoptera insect.Therefore, this study made the Tenebrio molitor of Insecta Coleoptera as the research object. The prokaryotic expression vector with TmTH gene was constructed successfully,and expressed its active enzyme protein, and then determinated the the content of DOPA by high performance liquid chromatography (HPLC). On condition that inhibitors inhibited relevant enzymes, we made a primary study on melanization of TmTH and PO. This study not only lay a foundation for further comprehensive study of skin formation mechanism of Coleoptera and immune melanization, but also provide practical reference to study the insect pest control. The main content and the results as following:1 Vector construction and prokaryotic expression of TmTH The TmTH was cloned into the expression vector pET28a⁺, and induced the concentration expression and time expression by IPTG in Escherichia coli BL21. The results showed that protein expression could attain the largest amount when the Escherichia coli BL21 induced by 1mM IPTG and expressed 5h. And the results of SDS-PAGE and western-blotting indicated that we got an active protein--62 kD protein. This will lay the foundation for further researches on enzymatic properties and related founction of TmTH in vitro.2 HPLC method for determining the content of product DOPAThe active TmTH which expressed by prokaryotic and the samples of reactions compound between different organization from larva and inhibitors were added to catalysis 10 min in reaction system of TmTH. Then the high chlorine acid was used to inactivate enzyme, and determined L-DOPA content which was reaction product by HPLC. At the last, we used the formula to calculate the activity of enzymes. The results showed that the specific activity of TmTH was 10.028 U/mg which was gained from 3 ml prokaryotic expression bacteria, and both two inhibitors, 3IL and PTU, could inhibit the product L-DOPA, especially, 3IL, the inhibitor of TH, could obviously inhibit the enzyme activity.3 The effect of hemolymph melanization and coagulation of Tenebrio molitor larva when inhibit the activity of TmTHLarval hemolymph mixed reaction with different concentration of the TH inhibitor-3IL and the PO inhibitor -PTU, respectively, then recorded the melanization time at room temperature and photographed the hemolymph coagulation by inverted microscope. The results showed that 3IL and PTU had different degree of inhibition, and the more inhibitor concentration, the more obviously inhibit effect. And 3IL slightly extended the time of hemolymph melanization, and suppressed the aggregation of hemolymph coagulation.4 The effect of epidermis melanization of Tenebrio molitor larva when inhibit the activity of TmTH in vivoTenebrio molitor larva with different development body color were injected inhibitors of different concentration, then observed and recorded the melanization time of wound. We also injected the newly molting white larvae and recorded the time of developing to maturity body color. In order to deeply research the function of TmTH in epidermis melanization, white epidermis was dissected from larvae and was cultured 48h in different conditions, then observed the changes of cuticle color by inverted microscope. The result of the inhibition of 3IL and PTU showed both of them took part in the process of cuticle tanning.