Studies On The Biochemical Characterization And Function Of Phenoloxidase In Bombyx Mandarina And So On

Phenol oxidase (PO, and was known as tyrosinase in mammalian), which is widely present in organism, is one of very important enzymes in insects. The enzyme have an important function in the development metamorphisis and immunity system of insects. Cuticle sclerotization catalyzed by PO is a vital process occurring during each stage of insect development, which can hard and stabilize the newly secreted exoskeleton. The theory of phenol oxidase inhibitors provides important clues to the control of insect pests,ultimately. Bombyx mandarina and Phthonandria atrineata (Butler) are the chief pests in mulberry garden, endangering mulberry all the year round. There are the problems of drug resistance and environmental pollution by chemical control. Hence, there is the extensive practical prospect in the exploiture of the special inhibitors and friendly to environment, In the paper, purification and characterization of phenol oxidase from Bombyx mandarina and Phthonandria atrineata (Butler) , the cloning,RNAi and function of PPO genes in Bombyx mandarina, and the comparison of PPO gene expression in Bombyx mandarina and Bombyx mori were studied. The main results present as follows:1. PO from Bombyx mandarina was purified by 0-40% saturated (NH₄)₂SO₄, and the result showed that 2.33-fold purification was achieved from the enzyme;Then, after Sephadex G-100 gel filtration, the activity of PO was 57.14-fold higher than crude enzyme.2. PO from Phthonandria atrineata (Butler) was purified by 0-80% saturated (NHO₂SO₄, and the result showed that 1.55-fold purification was achieved from the enzyme;Then, after Sephadex G-100 gel filtration, the activity of PO was 6.28-fold higher than crude enzyme.3. The optimum pH was 7.0, the activity has almost no change from 33℃to 43℃, and the best temperature was 37℃or so for the tested PO from Bombyx mandarina. Bombyx mandarina PO of the remainder activity would be 28% after incubation at the temperature of 70℃for 15 min.4. The optimum pH was 7.0, and the best temperature was 37℃for the tested PO from Phthonandria atrineata (Butler). Phthonandria atrineata (Butler) PO of the remainder activity would be 12% after incubation at the temperature of 70℃for 15 min.5. The affinities of PO in Bombyx mandarina with substrates from high to low were catechol, L-dopamine (L-DOPA) and pyrogallol, and the K_m with the three substrates were 2.06 mmol/L, 3.17mmol/L and 3.39mmol/L,respectively. The affinities of PO in Phthonandria atrineata (Butler) with substrates from high to low were L-dopamine (L-DOPA), catechol and pyrogallol, and the K_m with the three substrates were 4.30 mmol/L, 6.82mmol/Land 9.64mmol/L,respectively. The relations between reaction time and the rate of reaction, the relations between concentrations of substrate (catechol) and the rate of reaction, and that between concentrations of enzyme and the rate of reaction were also studied, respectively.6. The effect of some metal ions and EDTA-2Na on the PO activity were studied. The results showed that Mg²⁺,Ca²⁺å’ŒZn²⁺ activitied the enzyme activity from Bombyx mandarina, EDTA-2Na and Cu²⁺ inhibited strongly the activity of the PO. However, Mg²⁺and Ca²⁺ activitied strongly the enzyme activity from Phthonandria atrineata (Butler), EDTA-2Na,Zn²⁺and Cu²⁺ inhibited strongly the activity of the PO.7. The effect of some organic solvents and many kinds of enzyme inhibitors on the activity of PO from Bombyx mandarina and Phthonandria atrineata (Butler) was researched. The results showed that methanol, ethanol, isopropylalcohol, glycerol and many kinds of enzyme inhibitors had inhibitory effect on the enzyme activity in different degree, and the dependence of concentrations.8. Inactivation kinetics of PO from Bombyx mandarina and Phthonandria atrineata (Butler) during inhibition by quercetin were studied. The results indicated that quercetin could inhibit PO activity through competitive inhibition with L-DOPA as substrate, and the inhibitory constants (K_i) were determined to be 44.61μmol/L and 20.5μmol/L, respectively.9. Inactivation kinetics of PO from Bombyx mandarina and Phthonandria atrineata (Butler) during inhibition by thiourea were studied. The results indicated that thiourea could inhibit PO activity through non-competitive inhibition with catechol as substrate, and the inhibitory constants (K_i) were determined to be 0.07μmol/L and 0.18mmol/L, respectively.10. The PPO genes (PPO1 and PPO2) in Bombyx mandarina were cloned by RT-PCR and the accession number of them in GenBank are EU569724 and EU047703. The cDNA length of them are 2086bp and 2134bp, respectively, with an ORF of 2058 bp and 2082 bp coding 685 and 693 amino acid residues, which contain a possible cleavage site for proteolytic activation of the enzyme, two putative highly conserved copper-binding sites, six conserved histidine residues, five possible N-linked glycosylation sites, no signal peptide sequence. Molecular weight and pI of the proteins coded by PPO1 and PPO2 from prediction online are 78.703KDa, 80.075KDa and 6.28, 5.62. Multiple sequences alignment of homologs indicate that the two gene sequences, including the coded amino acid sequences from Bombyx mandarina and Bombyx mori have high identity, there are only difference in 14 bp, 5 amino acid from PPO1, and 21bp, 3 amino acid from PPO2 between Bombyx mandarina and Bombyx mori. The results of BLASTN search of the two genes in silkworm genome shown that they both have only one copy in genome. The comparison between cDNA and genome indicates that 13 exons and 12 introns are in PPO1, and 11 exons and 10 introns are in PPO2, and all of the boundaries of exon/intron is GT-AG. Compared with other 17 species, and constructed phylogenesis tree, the result indicated that wild silkworm was most closely related to silkworm ,but far from organism and mammal. This also showed furtherly that silkworm had origined from wild silkworm.11. RT-PCR experiment indicated that the two genes (PPO1 and PPO2) were expressed much differenty in all tested different tissues and different developmental stages between wild silkworm and silkworm, which imply its different role in wild silkworm and silkworm development.12. The coding partial sequences of PPO2 were synthesized corresponding dsRNA in vitro. The development of pupa cuticle was affected after injecting the PPO2 dsRNA into wild silkworm of 1 day before cocooning. Pupa integument was soft and light-colored on the sixth day after pupation. We could detect obvious reducibility of PPO2 mRNA of injection dsRNA into wild silkworm individuals comparing with control by RT-PCR, northern blot could detect nothing in the disposal individuals. It is proved that PPO2 was specifically interfered in pupa developmental stages. The results of RNAi reveal that PPO2 is related to pupa cuticle sclerotization.