Preliminary Study On Expression Of Helicoverpa Armigera ALP Gene And Its Function Associated With Action Of Cry1Ac In Cultured Insect Cells

Bacillus thuringiensis (Bt) is one of the most widely used biological pesticides, which is a Gram-positive bacteria, and blongs to the Bacillus cereus group. The transgenic plants with cry genes are highly effective against target insect pests.The interactions between the receptors of insect intestinal epithelial cells and the Bt toxin play important roles in insecticidal activity, and the main object of the experiment is to investigate the role of ALP in Cry1Ac-mediating toxicity on insect cells. At the present, there are four kinds of Bt toxin receptors identified on the insect midgut cells. They are alkaline phosphatase (ALP), aminopeptidase N (APN), cadherin (Cadherin) and glycolipid substances. Cadherin and APN have been well documented. The mutations of them are associated with Bt resistance in insect. The role of ALP is under investigation.In the present experiments, the Cry 1 Ac receptors such as cadherin, ALP2 and APN1 from H. armigera were expressed using plasmids for transient expression in Spodoptera litura S1-HP cells and their influences on the toxicity of activated Cry1Ac toxin against the cells were investigated. When EGFP was inserted just before the GPI anchor site of HaALP2, the location of the fusion protein on cell membrane was the most obvious. Immunofluorescence showed that HaALP2 without label was localized on cell membrane in S1-HP cells transfected via plasmid for transient expression, which was similar with HaALP with EGFP label before the anchor site. An enzymatic assay revealed that the expressed HaALP 1 labeled EGFP had the activity of enzyme. For HaAPN1,EGFP was also inserted before the anchor site and the location of HaAPN1 with EGFP label on cell membrane was demonstrated too. For Hacadherin, EGFP was linked to the carboxyl terminus of the protein, and the cadherin-EGFP fusion protein was localized on the cell membrane. When the three receptors with EGFP or without were respectively expressed in the cells, the trypsin-activated Cry1 Ac specifically binded to the cell membrane of S1-HP cells expressing only hacadherin and hacadherin-EGFP, and the cells showed aberrant cell morphology. HaALP2 or HaAPN1 with EGFP or without on cell membrane did not enhance the toxicity of the activated Cry1Ac against the cells expressing Hacadherin or Hacadherin-EGFP. Lactate dehydrogenase (LDH) activity analysis showed that only cadherin-expressing cells released significant LDH after the treatment of Cry 1 Ac, which was consistent with the result described above.In a word, we have studied the function of the putative Cry1 Ac receptors using the in vitro cell culture, and provided some clues and new ideas to the Bt action and the mechanisms of resistance.