Molecular Characteirzation Of The CDNAs Encoding Prophenoloxidases And Their Expression In Cabbage Butterfly Pieris Rapae

Phenoloxidase (EC.1.14.18.1, PO) is a key enzyme which is widelydistributed in-vertebrata and invertebrata animals, plants, bacteria and fungus.In insects, PO was uniquely associated with several different physiologicallyimportant biochemical processes, including the sclerotization and melanizationof the insects’ cuticle, defensive encapsulation for the foreign organisms, andwound healing. It is also a potential insecticide target in the future. In this study,the cDNAs encoding PPOs were cloned by means of reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE) and after that the bioinformation were analysed. In addition, expressionstudies were carried out in different life stages using Semi-quantitative RT-PCRand Real-time PCR. Then the Pieris rapae5th larvae were injected withBeauveria bassiana conidiophore to study the effects to the expression ofPrPPO1. Meanwhile, the PrPPO1expression in5th larvae injected withquercetin or kojic acid after4h,8h and12h were also characterized. Thecontents and results were summarized as follows: 1. The full-length cDNA of PrPPO1was3338bp. The deduced amino acidsequence in the ORF of the gene starts from the64th nucleotide and was2049bp long encoding a682amino acid long protein. The3’ untranslated region(UTR) extends from2102nd nucleotide and a predicted1226nucleotides longpolyadenylation signal was found. The MW weight of the deduced protein wasabout78.57kDa and the pI was6.37.2. The partial cDNA of PrPPO2was1679bp. The deduced amino acidsequence in the ORF of the gene starts from the1st nucleotide and was1173bplong encoding a391amino acid long protein. The3’ untranslated region (UTR)extends from1173th nucleotide and was506nucleotides long containing apredicted polyadenylation signal. The other predictions were not appliedbecause of the partial cDNA of PrPPO2.3. BLASTp search and neighbor-joining analysis showed that PrPPO1had ahigh identity to the published sequence of PPO1from other lepidopterousinsects, ranging from69.15%to73.15%, and PrPPO2had a high identity to thepublished sequence of PPO2from other lepidopterous insects, ranging from70.0%to72.0%. 4. Protein signature analysis revealed a putative thiolester site and twodistinct copper binding regions, which including6histidine residuessignificantly. There was no signal peptide in the N-terminal region of thepolypeptide chain. No possible transmembrane protein model was found.5. The secondary structure prediction showed that in PrPPO1alpha helixwas17.16%, random coil was63.20%which was the main structural element inPrPPO1, and extended strand was19.65%. There was no β-turn in the PrPPO1cDNA. The compatative modeling method was used to calculate the tertiarystructure of PrPPO, and the result showed that PrPPO1was the “roller” shape,which belongs to the α/β type.6. PrPPO1was highly abundant in eggs and the4th instar larvae withrelative quantifications of2.76-fold and5.60-fold compared with that of theadults stages. Relatively lowest expression was found in the5th instar larvae(0.21-fold) and pupae stages (0.26-fold). Nearly expression was found in1st,2nd,3rd instar larvae,2.16,1.82and1.52fold, separately. PrPPO2was highlyabundant in the4th instar larvae with relative quantifications of13.24fold, eggsand3rd instar larvae were followed compared with that of the adult stages. Relatively lowest expression in the5th instar larvae (0.96-fold) was found.Nearly expression was found in1st,2nd instar larvae,2.98, and2.65, separately.Consequently, the results of PrPPO1expression by real-time PCR were accordwith that of the Semi-quantitative RT-PCR.7. The Beauveria bassiana-injected and the PBS-injected both had an up-regulate to the expression of PrPPO1between6h and12h after treated,5.67and5.0fold in12h compared with that of6h, separately. But the down-regulate wasfound between12h and24h after treated, and no obvious differences was foundbetween the Beauveria bassiana-injected and the PBS-injected. The expressioneffect on PrPPO1for the Beauveria bassiana-injected to the Pieris rapae wasminor.8. There was no significantly decreased was found in PrPPO1transcriptlevels after quercetin or kojic acid treated.