Molecular Cloning, Function Analysis And Prokaryotic Expression Of The Tyrosine Hydroxylase Gene In Mealworm, Tenebrio Molitor (Insecta Coleoptera)

Cuticle tanning, pigmentation and melanization of insects play an important role in the process of their developmental metamorphism. It is well known that 3, 4-dihydroxyphenylalanine (Dopa) is essential for tanning of newly formed cuticle and the immune-associated melanization in insects. Dopa is produced by the hydroxylation of tyrosine, and it subsequently converts to oxidized catechols and their derivatives by a series of enzymes, which lead to tanning, melanization and immunity. 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).TH catalyzes the hydroxylation of tyrosine to Dopa in the presence of tetrahydrobiopterin (BH4) as cofactor. The reaction is the first and rate-limiting step in the production of catecholamines and their derivatives.THs are highly conserved enzymes found in both vertebrates and invertebrates. Studies of TH had demonstrated that TH was required for cuticle tanning and immune function in D. melanogaster and other insects. The prevenient researches on TH mainly focused on Lepidoptera and Diptera insects, but little on Coleoptera insect.In order to characterize tyrosine hydroxylase of Tenebrio molitor (TmTH) and to assess the preliminary function of TmTH in its cuticle tanning and immune defense. We cloned a full length cDNA clone encoding TmTH from the fat body of immunized T. molitor larvae by RACE and PCR methods. The cDNA consisted of 2103bp with a single open reading frame of 1605 nucleotides encoding 534 amino acid residues. The predicted molecular weight and pI of TmTH were 60.66 kDa and 5.70 respectively (GenBank No.GQ390366). Amino acid sequence of the deduced protein was compared and analyzed with other insect and vertebrate THs protein sequences based on a ClustalW alignment including 15THs protein sequences. The alignment results showed that its amino acid sequence was well conserved among insects. The identities of TmTH to Tribolium castaneum TH (GenBank No.EF592178) were 92.32%. The total sequence identities to lepidopteran THs were about 65.12%, and 43-47% to vertebrate THs. In the N-terminal conserved region of insect THs, there was a consensus site (RRXS) for phosphorylation by cAMP-dependent protein kinase at Ser 32, which corresponded to the Ser 40 in vertebrate THs. TmTH was not predicted to be a secreted protein as determined by SignalP. The encoded protein had no possible signal peptide. In addition, TmTH contains a biopterin_H domain, a conserved C-terminal catalytic (C) domain and an unrelated N-terminal regulatory (R) domain.To determine the expression characteristics and elementary immune biological functions of TmTH, we analyzed several expression profiles in different tissues of full-grown last instar larvale. TmTH mRNA was abundantly expressed in the brain, but much less in integument, fat body and hemolymph. But the gene expression was strongly induced in fat body and integument by UV-killed E.coli microbes. This indicated that the enzyme might be involved in neural activity in the larvae, and further investigation on this was needed. The gene transcripts reached the maximum levels at 6-12 h after the bacterial challenge, lasted for more than 36h at high levels before gradually attenuating after the infection, but weakly in the brain. In addition, the induction could be caused by Escherichia coli (G-), Bacillus subtilis (G+), Staphyloccocus aureus (G+) and Candida mycoderma (F).To understand whether the expression of TH was correlated with cuticle tanning during metamorphism, we used RT-PCR to determine the relative level of TH transcript in the three developmental stages (larvae, pupae and adults). The results showed that TmTH mRNA was detected in high levels in the fat body and integument in the initial stages of larvae, pupa and adults when the newly formed cuticle was just starting to tan and darken. Then the TmTH mRNA gradually decreased or disappeared until T. molitor was fully mature in each stage except pupa when cuticle tanning was towards the end. It demonstrated a correlation between the TH transcript abundance and the degree of tanning.To study the tyrosine hydroxylase of T. molitor in vitro, the TmTH was then cloned into pET28a(+) for prokaryotic expression in Escherichia coli BL21. The results of SDS-PAGE indicate that we got a fusion protein--62 kD protein with 6×His-tag in Escherichia coli BL21.This will lay the foundation for researches on enzymatic properties and regulation of TmTH in vitro. In the future our work will mainly face on the researches of TmTH in vitro.In our research, we characterized tyrosine hydroxylase of Tenebrio molitor (TmTH) of Coleoptera insects firstly. We cloned TmTH by RACE and PCR methods, analysed the expression pattern in vivo and prokaryotic expression in vitro. We assessed the preliminary function of TmTH in its neural activity, cuticle tanning and immune defense.This will not only lay the theoretical foundation for the study of epidermis formation mechanism of Coleoptera insects, but also provide practical reference to study the insect pest control.