Cloning And Function Analysis Of Prophenoloxidase Activating Proteinase Of Diamondback Moth, Plutella Xylostella

Prophenoloxidase activating proteinase (PAPs) is a key proteinase in the process of prophenoloxidase activation. The immune challenge can induce proteins in the upstream of prophenoloxidase activation cascade to activate the precursor of PAPs by proteolytic cleavage. Inactive prophenoloxidase can be activated to be active phenoloxidase by activated PAPs and ultimately produce toxic substances in order to kill foreign organisms. In this thesis, we cloned two PAP genes from the diamondback moth, Plutella xylostella and did a preliminary study on their fuction. The results are summarized as follows:1) The full cDNA sequences of two prophenoloxidase activating proteinase were obtained using Rapid Amplification of cDNA Ends (RACE). They were designated Px-PAPl and Px-PAP3b, the length of which were1595and1650bp, open reading frame involed in cDNA were1149and1323bp long respectively. The analyses of sequences characteristics predicted that Px-PAPl and Px-PAP3b both contained signal peptides and possessed integrity catalytic triad residues in carboxyl-terminal. There was only one clip domain in amino-terminal of Px-PAPl, whereas there were two clip domains in amino-terminal of Px-PAP3b. The results of multiple sequence alignment revealed that Px-PAPl and Px-PAP3b shared high similarity with PAPs of other lepidopteran species. Phylogenetic analyses reflected a close relationship between the two genes and other insect PAPs.2) E. coli challenge could induce the expression levels of Px-PAPl, Px-PAP3a and Px-PAP3b. Hemocytes of Plutella xylostella larvae were collected at0,2,4,8,12and24hour after E. coli injection and then transcripts levels were detected by rt-qPCR. The results indicated that E. coli injection could markedly up-regulated the transcript levels of Px-PAPl, Px-PAP3a and Px-PAP3b. After E. coli challenge, the transcript levels of Px-PAPl and Px-PAP3a increased gradually at0-4h, and arrived the peak levels at4h; at the following4-24h, the levels of these two genes went down. However, after E. coli challenge, the transcript levels of Px-PAP3b increased gradually at0-8h, and and then arrived its peak level at8h; at the following8-24h, the levels of this gene went down gradually. Compared to Px-PAPl and Px-PAP3a, the transcript levels of Px-PAP3b were significantly higher.3) CvBV could down-regulate the expression levels of Px-PAPl, Px-PAP3a and Px-PAP3b. Hemocytes of Plutella xylostella larvae were collected at4and8hour after E. coli+CvBV (0.05VREs) injection and then transcript levels were detected by rt-qPCR. The results indicated that E. coli+CvBV (0.05VREs) injection could markedly down-regulate the transcript levels of Px-PAPl, Px-PAP3a and Px-PAP3b as compared to the control group.4) RNA interference experiments were carried out to silence Px-PAPl, Px-PAP3a and Px-PAP3b. Plutella xylostella larvae were collected at6,12and24hour after E. coli+700ng dsPAP injection with the group of E. coli+700ng dsGFP injection as the control group, and then transcript levels were detected by rt-qPCR. The results indicated that the Px-PAP3a was unexpectedly silenced at12h after dsRNA injection when the Px-PAPl was targeted. Both Px-PAPl and Px-PAP3a were silenced synchronously at12h after dsRNA injection when the Px-PAP3a was targeted. When the Px-PAP3b was targeted, the transcript levels of Px-PAP3b were not affected at6h after dsRNA injection but significantly down-regulated at12and24h after injection and the interference effect declined gradually with the lapse of time while both Px-PAPl and Px-PAP3a were not influenced at the same time.