cDNA cloning and partial characterization of tyrosine hydorxylase from the diamondback moth, Plutella xylostella = cDNA cloning and partial characterization of tyrosine hydorxylase from the diamondback moth, Plutella xylostella

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In inscts, 3,4-dihydroxyphenylalanine(DOPA) was required for cuticle tanning and the production of melanin during immune responses. DOPA was produced by the hydorxylation of tyrosine, and this reaction could be catalyzed by two types of enzyme; tyrosine hydroxylase(TH) and phenoloxidase(PO). Tyrosine hydorxylase(TH; tyrosine hydorxylase 3-monooxygenase, E.C.1.14.16.2)is enzyme catalyzing the first and rate limiting step in dopamine and other catecholamine biosynthesis. TH was required for cuticle tanning in Drosophila melanogater and for cuticle pigmentation in other insect species, but additional functions of TH had been uncertain. This study was carried out to confirm the existence of TH in Plutella xylostella(PxTH) and to chracterize PxTH. cDNA of 218 nucleotid was constructed using gradient PCR. RACE PCR was performed using the partial cDNA. The full length cDNA OF PxTH was 1686 nucleotides. The deduced polypeptide was composed of 562 amino acids. In the phylogenetic analysis, PxTH was clustered with the other lepiodpteran THs. the PxTH sequences such as Ser24, Ser31, Ser35, Ser53a and Ser65 has been highly predicted as a target for phosphorylation. Especially, Ser35 of PxTH is highly conserved in broad range of animals including rat and human. Western blot analysis using the PxTH-antibody verified the expression of PxTH in all stage larva, pupa and adult. To examine possible immune-related function of the PxTH in this species, the PxTH geen expression was investigated by RT-PCR and western blotting analysis when Plutella xylostella were challenged by microorganisms. The high expression of PxTH in accroding to inoculation of microorganisms was observed in 1h post infection and slightly induced during 2h to 5h post infection.