| Protease inhibitors are present in living organisms including bacteria, plants and animals. According to the different active sites, protease inhibitors primarily can be grouped into four families:serine protease inhibitors, cysteine protease inhibitors, metalloproteinase inhibitors and aspartic protease inhibitors. These protease inhibitors are generally small, stable proteins. Protease inhibitors are capable of affecting insect survival and development by inhibiting protease activities. With the rapid development of transgenic technology, many protease inhibitor genes have been successfully expressed in transgenic plants for controlling insect pests. It is quite certain that protease inhibitors will be widely used in agricultural pest control in the future. Xbpi-1gene was cloned form Xenorhabdus bovienii strain BJFS526. The Xbpi-1recombinant protein was in vitro expressed and purified based on His-Tag by affinity chromatography. Xbpi-1has been demonstrated having effects on the survival, growth and reproduction of Myzus persicae. In this study, the biological activity of recombinant Xbpi-1against the pea aphid, Acyrthosiphon pisum Harris was evaluated; the effects on the activities of aphid proteases and content of amino acids were measured, and finally the tissue distribution of and damage by Xbpi-1were determined by using the fusion gene of Xbpi-1and green fluorescent protein (GFP). The main results were summarized as follows:1. The effects of Xbpi-1on the pea aphid’s mortalities and weights were measured by feeding of and dipping with the protease inhibitor protein, respectively. The pea aphids fed on artificial diet containing50,200,400,800μg/mL Xbpi-1showed calibrated mortality of2.1%,9.4%,19.7%,50.9%, and aphid weights were reduced12.3%,20.3%,25.7%,33.7%, respectively compared with those of the control. However, inhibiting effects were not significant when the aphids were treated with Xbpi-1by dipping method. The mortalities between treated and control aphids were not significantly different at24h and72h after the treatments with different Xbpi-1concentrations (50-800ug/mL). The results indicated that Xbpi-1would have a potential to be used as a bio-pesticide or as a gene resource for controlling of sucking insect pests.2. In order to determine the biochemical mechanisms underlying the insecticidal activity of Xbpi-1, the activities of insect proteases were measured and compared between the treated and control aphids. It was found that the total proteolytic activities of aphids fed on the artificial diet containing50,200,400,800μg/mL Xbpi-1were significantly reduced by13.1,13.6,23.2and29.6%, respectively, compared with control aphids at96h after treatments. Similarly, activities of trypsin, chymotrypsin and leucine aminopeptidase were significantly inhibited by26.1,23.5and23.8%respectively in aphids treated with800μg/mL Xbpi-1.3. The free amino acid contents were also measured to elucidate the insecticidal mechanisms of Xbpi-1. The result showed that the contents of total free amino acids were declined significantly by0.85,13.50,14.76and20.81%in aphdis fed with diet containing50,200,400,800μg/mL Xbpi-1respectively, and the contents of15free amino acids were reduced significantly in the aphids treated with800μg/mL Xbpi-1. It is concluded that the reduction in contents of free amino acids was resulted from the inhibition of insect protease activities by Xbpi-1.4. To further investigate the distribution and damage effects of Xbpi-1on tissues, the fusion gene of Xbpi-1and green fluorescent protein (GFP) was constructed and expressed in Escherichia coli. The purified Xbpi-GFP was mixed into artificial diet to feed the aphids, and then paraffin sections of pea aphid were made for the GFP observation using a focus fluorescence microscope. Green fluorescence was observed in both gut and fat body, indicating that Xbpi-1could enter into the blood cavity. It was also found that the midgut and the fatbody degradated gradually after the treatment with Xbpi-1. |
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