| Pardosa pseudoanulata is a big, ferocious, and predatory spider, which is dominant in the rice paddies, and has an important role in the regulation of pest populations. Now the mechanism of Bt insecticidal proteins in the transgenic rice on the target pests is reported largely. Among those, all the spider biosafety researches are mainly on the enrichment of Bt protein by the food chain in the spiders instead of the effects.Our study used the Pardosa pseudoannulata as the object, analyzed the enrichment of Cryl Ab protein and its effects on the activity of three detoxifying enzymes in this spider from the perspective of the food chain; furthermore, Cry1Ab protein could be detected in the spider oocyst, so we could investigate CrylAb protein transferred from parent to descendant, the chemical substance content and morphological characteristics in the embryonic development, and the activities of four protective enzymes in the eggs, and the features of the livetins. Therefore, it could provide a theoretical basis for the safety assessment of transgenic Cry1Ab protein rice.Drosophila and planthoppers were fed with transgenic CrylAb protein medium and rice respectively, to enrich Bt insecticidal protein, and then we feed the spiders with the drosophila and planthoppers. We used the enzyme-linked immunosorbent (ELISA) for Cry1Ab protein content in the Pardosa pseudoannulata body, and then detected changes of SOD, AchE, and GSH-Px enzyme activity with the kit; besides, took the birth oocysts as the embryonic development materials, determined CrylAb protein content by ELISA; assayed SOD, AchE, GSH-Px and CAT enzyme activities in the eggs with the kit; moreover, utilized the related methods to investigate differences of the chemical substance content during the embryonic development; observed changes in the embryonic morphological features by use of paraffin sections and transparent liquid paraffin technique; finally, exploited the electrophoresis to study the chemical properties of livetin.The results showed that:Cry1Ab protein enriched in the Pardosa pseudoannulata body (enrichment factor up to24.38), GSH-Px activity in experimental group increased significantly compared to the control (p<0.01), and reached the peak at7th day; AchE activity in experimental group reached its peak at3th day and was higher than the control group significantly (p<0.05) and got to the bottom at7th day with significance to the control (p<0.05); from the3th to1lth day, SOD activity in experimental group was markedly lower than the control group (p<0.01) and reached the bottom at7th day with highly significance to the control (p<0.01). During the embryonic development, Cryl Ab protein content in the eggs of experimental group was also higher than the control (p<0.05), and the SOD, AchE, GSH-Px and CAT enzyme activity detection showed that GSH-Px enzyme activity increased higher than the control (p<0.01) but that of other three all showed lower than the control group (p<0.01).As a contrast to the control, embryonic development duration in experimental group extended and embryonic development incompleted, observed by the liquid paraffin and paraffin sections. Through the determination of the chemical substances in the eggs, we found that the protein content in control group was significantly higher than the experimental (p <0.05), but no significant differences in sugar and fat content. Than, we found that livetin of the Pardosa pseudoannulata is a glycolipid-binding protein, by its extraction and electrophoresis. livetin molecular weight in the experimental group was279.64KD, and its subunit size was85KD and69KD the63.5KD and50K.D. respectively; moreover, that of the control was274.79KD, and its subunit size was85KD,72KD,65KD and51KD, separately. The amino acid composition analysis showed that the content of glutamic acid and arginine in the control group was higher than that of the experimental.LC-MS/MS the results showed that protein sequences is different between the experimental group and the control group. |
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