Cloning And Expression Analysis Of Prophenoloxidase Activating Proteinase From Larvae Of The Asian Corn Borer,Ostrinia Furnacalis Guenee (Lepidoptera:Pyralidae)

In order to investigate the regulation mechanism of prophenoloxidase activating proteinase (PAP) in insect immunity, PAP gene was cloned from the Ostrinia furnacalis (Guenee) larvae with RACE, The fusion protein of PAP was expressed in the Escherichia coli RIL and further purified with Ni2+chelated affinity chromatography. Bacterial infection was applied to elucidate the defense mechanism of the larvae, and the expression of PAP in transcription level in various tissues of the larvae was analyzed by the Real time PCR. The main results are as follows:(1) In this research, RT-PCR and RACE (Rapid amplification of cDNA ends) methods were used for the cloning of full length cDNA encoding PAP from the5th instar larvae of0. furnacalis. The full length cDNA of PAP was1455bp, containing an ORF of1200bp, encoding400amino acid residues, with a5’UTR of66bp and a3’UTR of189bp. The PAP has a predicted molecular weight of44.8kDa and pI value of6.66. Bioinformatics analysis showed that a conserved catalytic triad (H, D, S) in the serine protease like domain was found, and two clip domains was existed (23-70amino acids and153-394amino acids), and the153-394amino acid is also called serine protease domain (serine proteinase, SP). There was an signal peptide in the N terminal of the peptide, and the cutting site is between18amino acids and19amino acids. No possible transmembrane protein model was found. There was no O-glycosylation site in PAP predicted with Dictyo Glyc. Nineteen phosphorylation sites were predicted in the whole peptide by NetPhos2.0Server. BlastP analysis showed that the amino acid sequence of the cloned cDNA from O. furnacalis had the high identity to those of Manduca sexta PAP3precursor、Manduca sexta PAP3、Manduca sexta PAP2、Bombyx mori PPAE、Samia cynthia ricini PAP and Spodoptera litura PPAE3, the consistency of the amino acid is more than37%. Phylogenetic analysis showed that the genetic relationship of O. furnacalis, M. sexta PAP3and S. litura PPAE3was closer, while that of O. furnacalis, Fenneropenaeus indicus PPAF and Penaeus monodon PPAE protein was more distant.(2) In this study, the second Clip domain (153-394amino acid) was sub-cloned from the5th instar larvae of O. furnacalis, and three different Clip domains were obtained:PAP-SP1(144-400amino acid), PAP-SP2(154-400amino acids) and PAP-SP3(44-400amino acids).The pET expression system was used to express PAP protein and SP domain, after gradient IPTG inducement the PAP protein was expressed in BL21, and the size of the recombinant protein was same as the predicted, about46.64kDa. After0.2mmol/L and0.1mmol/L IPTG induced in37℃temperature the PET-28b-PAP-SP1recombinant protein was significantly expressed, while that of the PET-28b-PAP-SP1recombinant protein was significantly expressed after0.1mmol/L IPTG inducement in28℃temperature. PAP-SP3was expressed with the recombinant express vector pET-28a-HMsbT-PAP-SP3after0.1mmol/L IPTG inducement in25℃temperature. To verify the PAP-SP3expression, the Western blot was used with the anti-FLAG tag, and the results was same as SDS-PAGE. After the purification with the Ni2+affinity chromatography, we obtained the HMsbT-PAP-SP3recombinant protein in elution1. We used acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (IEARpNa) as the substrate to determinate the enzymatic activity of PAP. And0.1mmol/L IPTG can induce the PAP、PAP-SP1、PAP-SP2to express with some activities about139.2U/mg、1853.1U/mg、1743.3U/mg, respectively. While they also had the activity, about25.0U/mg、1573.6U/mg、568.7U/mg without IPTG inducement, respectively. There was no significant difference after the IPTG induced.These results showed that we had successfully expressed the protein of PAP and SP domain, and the purified HMsbT-PAP-SP3had a high enzymatic activity.(3) To investigate the influence of bacteria injection on the expression levels of PAP mRNA in larvae of O. furnacalis Guenee, the Real time PCR was used to detect changes of gene expression after the physiological saline, gram-negative Escherichia coli and gram-positive Bacillus subtilis injection, and at different times (0h,4h,8h,12h,24h,36h) after bacteria injection, in differenttissues such as, haemocytes, fat body, midgut tissues, integument. The results showed that gene expression of PAP had an obvious change in haemocytes injected by Escherichia coli only, a sharp increase reached115.3±23.2fold at4h, and then decrease sharply, while the others had no significant change (P>0.05). The expression of PAP mRNA in fat body was not obvious (P>0.05). The expression of PAP mRNA in midgut tissues in the larvae injected by Bacillus subtilis had obvious differences with the control group, and changes were rising with time. The PAP mRNA had the highest level of expression at36h, reached107.6±7.2fold, and other groups had no significant change (P>0.05). The expression of PAP mRNA in the integument treated with different injection all had some degree increase compared with the control. The larvae treated with B. subtilis was the most obvious, PAP mRNA expression reached105.2±1.6fold at12h, the larvae treated with physiological saline reached36.4±0.5fold at8h, and the larvae treated with Escherichia coli reached21.3±0.3fold at36h.