The Prokaryotic Expression And Expression Ievel Analysis Of Prophenoloxidases In Vivo

Phenoloxidase (PO, EC.1.14.18.1) is essential for insect physiological functions and initial immunity, such as melanization and encapsulation of invading pathogens, wound healing. Nowadays, there are many studies on the the activation pathway of proPO, but the whole activation process and its mechanism are not very clear. Some studies showed that the proPO is produced predominantly in oenocytoids in Lepidopteran insects, but there is no evidence to show the development and evolution. According to the reason above, this paper aims to study the proPO character and its function. The study showed that the whole CDS of PxPPO1and PxPPO2were cloned by means of reverse transcription polymerase chain reaction (RT-PCR) from Plutella xylostella. And recombined this gene and pET-30a prokaryotic expression vector, the recombinant proteins was expressed in Escherichia coli BL21(DE3), then induced by IPTG to produce fusion protein. In addition, the Pier is rapae4th larvae were infected with Beauveria bassiana conidiophore to study the effects to the expression of PrPPO1. Meanwhile, different life stages Pieris rapae were used to collect hemolymph and light microscopy were used to count the number of oenocytoids, in order to investigate the relation between oenocytoids density and expression level of PrPPO1at different life stages. The main content as follows:1. The ORF of PxPPO1was cloned and inserted into pET-30a prokaryotic expression vector, the recombinant plasmids pET-30a-PxPPO1was7451bp. pET-30a-PxPPO1was expressed in E. coli, and was confirmed with an83kDa (PxPPO178.56kDa, His-tag4.66kDa) visible, but insoluble protein. The fusion protein proportion was above50%of total bacterial protein.2. The ORF of PxPPO2was cloned and inserted into pET-30a prokaryotic expression vector, the recombinant plasmids pET-30a-PxPPO2was7480bp. pET-30a-PxPPO2was expressed in E. coli, and was confirmed with an84.5kDa (PxPPO279.86kDa, His-tag4.66kDa) visible, but insoluble protein. The fusion protein proportion was above50%of total bacterial protein.3. Optimization expression conditions showed that PxPPO1were induced with different concentration of IPTG, the fusion protein proportion was above60%of total bacterial protein, there was no significant difference in the protein expression volume between the different IPTG concentrations. IPTG inducting for different time, showed that the fusion protein proportion was about32%of total bacterial protein with3h inducing. And, the fusion protein proportion could reach62%with8h inducing. The results with different temperature inducting PxPPO1showed that the highest expression of fusion protein were induced at30℃, the fusion protein proportion was about84%of the total bacterial protein, this ratio reached73%when induced at15℃.4. PxPPO2was induced with different concentration of IPTG, the fusion protein proportion was above62%of total bacterial protein, there was no significant difference in the protein expression volume between the different IPTG concentrations. IPTG inducting for different time, showed that the fusion protein proportion was about27%of total bacterial protein with3h inducing. And, the fusion protein proportion could reach40%with6h inducing. This ratio reached58%when induced with8h. The results at15℃,20℃,25℃and30℃inducting PxPPO2showed that the fusion protein proportion was about60%,83.8%,86.6%and88.1%of the total bacterial protein, respectively.5. Western blot analysis confirmed that the fusion protein was the target protein. This paper established a basis for PxPPO1and PxPPO2polyclonal antibody preparation, and for the further study of the PxPPO function at protein level and PPO regulation of insect development and immune response.6. The expression of the PrPPO1in the4th-instar larvae was significantly down-regulated at6h and12h post-infection by B. bassiana. The PrPPO1transcripts were reduced to0.59-fold and0.18-fold at6h and12h. However, PrPPO1expression was up-regulated about9.00-fold at72h post-infection. There is no significant difference of PrPPO1expression was detected between post-infection at2h (1.12-fold),24h (0.33-fold), and48h (1.06-fold).7. The oenocytoids densities of hemolymph at P. rapae different life stages were calculated and compared. The oenocytoids density was highest in the4th-instar larvae as20.44×104cells/mL, is consistent with the highest expression of PrPPO1in the4th-instar larvae. And in the2nd and the5th larvae were13.78×104cells/mL and13.33×104cells/mL, respectively. Lower oenocytoids densities were found in the1st instar (10.22×104cell/mL) and3rd instar (8.89×104cell/mL) larvae, is consistent with the lower expression of PrPPO1in the1st instar and3rd instar larvae. This result showed that the PrPPO1expression levels were correlated with oenocytoids densities at different life stages.