Study On Physiological And Biochemical Mechanism Of Cry8Ga1and Cry8Ea1Against Grubs

Holotrichia oblita Faldermann and Holotrichia palallela Motschulsky (Coleoptera: Melolonthidae) are the key underground insect pests seriously damaging crops such as wheat, peanut, soybean, corn, chinese sorghum and potato. HD8G and HD8E have larvicidal activity against H. Oblita and H. Palallela respectively. The relationship between enzymatic activity and toxicity of Bt was announced by assaying enzymatic activities of grubs which were fed with Bt toxin. By using electron microscope, we observed the positions of bonding Bt toxin in in midgut cells and the pathological changes of cells in midgut. The receptor proteins were separated from BBMV and identified by receptor pull-down platform. The main results are as follows:1.The larva of H. oblita was fed with two concentrations of Cry8Gal. Subsequently, the activities of6enzymes were assayed. It was found that when only applied with higher concentration of Cry8Ga1(50.767μg toxin/g soil), the activities of total protease, chymotrypsin-like, acetylcholinesterase (AChE), glutathione-S-transferase (GST) and aminopeptidase (APN) in larvae would significant rise in most time of the experiment. However, the differences were insignificant of the activities of AChE and APN in larvae between the lower concentration (5.0767μg toxin/g soil) group and control. The activities of trypsin-like did not show significant difference but individual processing time in present experiment.2. The influences of main proteases and detoxification enzymes in H. Palallela larvae treated by Cry8Ea1and Cry8Ga1respectively were compared. Results:Cry8Eal protein caused the activities of H. Palallela larvae total protease, chymotrypsin-like, APN and trypsin-like in certain periods to increase significantly; Cry8Gal only caused the rise of the APN activity, and total proteolytical activity had no obvious change, trypsin-like and chymotrypsin-like activities decreased significantly. The activity of AChE rised in H. Palallela larvae treated by Cry8Ea1, and the result of witch treated by Cry8Gal was quite the contrary. After being treated by Cry8Eal or Cry8Ga1, the GST activity of H. Palallela larvae showed a trend of drop after the first rise with toxin concentration increasing.3. Cry8Eal and Cry8Gal inhibited the feeding behavior of grubs. After being feed with Cry8Eal and Cry8Ga1, the organelles in midgut cells of H. Oblita and H. Palallela larvae occurred obvious pathological changes, such as the microvilli, longitudinal and circular muscles, the intercellular space, endoplasmic reticulum, mitochondria. Cry8Eal binded on muscle fibers of H. Palallela larva midgut. Cry8Ga1binded on membrane inner surface of intestinal epithelial cells in hindgut of H. Oblita and H. Palallela larvae.4. Cry8Eal was digested by trypsin, chymotrypsin and the intestinal juice in midgut of H. palallela larvae in thermostatic water bath with37℃. The results showed that the intestinal juice digested Cry8Ea1to55kDa and a small amount of60kDa protein. Digestion of Cry8Eal in H. Palallela larvae relies on the serine proteases mainly.5. Some proteins which were predicted reporters of Cry8Eal were APN, actin and V-ATPase subunit A which identified in Lepidoptera. The others were myotubularin, myosin, spectrin, transport protein, transmembrane protein,alcium pump, Na+/K+ATPase alpha subunit, H(+)-transporting ATPase, ADP/ATP translocase, ATP synthase beta subunit, trypsin-like enzyme precursor, serine carboxypeptidase, zinc finger protein.