The Seperation And Identification Of The Cry1A Binding Proteins In Larval Midgut Of Helicoverpa Armigera And Their Alterations In Cry1Ac-resistant H.armigera Larvae

The wide spread plant of Bt cotton has played an important role in cotton pest control, pests are selected under the pressure of high-dose Bt toxin produced by Bt cotton for a long time, therefore the resistance evolution of pest to Bt cotton should not be ignored. The resistance mechaniam of Bt toxin to insects is complex and variable, but the change of the binding proteins in the midgut is the main one. The known receptors in cotton bollworm (Helicoverpa armigera (Hübner), CBW) are Aminoopeptidase N (APN) and Cadherin-like proteins. According to the test results of predecessors, no disruption and inactivation of cadherin gene but only a few change of amino acids had been found in APN and Cadherin-like proteins of the Cry1Ac-resistant CBW strain preserving in this lab, probably its Cry1Ac-resistant relates to the change of expressional level of receptor protein or there may have some other related receptors proteins.Firstly we compared two systems of two-dimension electrophresis (2-DE) and three prepared methods of BBMV, then build the proteomic analysis approach for CBW midgut proteins. Secondly we separated and identified Cry1Ac binding protein in CBW midgut by using proteomic approach. Thirdly the binding proteins in susceptible and resistant strain of CBW were compared, so as to find the possible resistant mechanism of Bt toxin. And finally the binding proteins of different Bt in susceptible and resistant CBW strains were also compared. The results were as follows:1. Methods: The ISO-DALT and IPG-DALT 2-DE system were compared, results showed that these two system could be used to separate the Cry1Ac binding proteins, though the separation results of ISO-DALT were better than IPG-DALT, especially for the larger protein separation, because of the limit protein loading quantity, this system was adverse for the mass spectrum. The IPG-DALT was more expensive, but it was good for the following mass spectrum work, so we chose IPG-DALT for 2-DE. The three different BBMV preparation protocols (ER, ARE and W) did not affect the number or types of CBW Cry1Ac binding proteins detected, However, different methods yielded different protein quantities. We consolidated a BBMV isolation protocol according to advantage of these reported protocols, this protocol could get high protein quantities and also suitable for 2-DE.2. Bacillus thuringiensis Cry1Ac binding proteins in CBW BBMV were investigated using proteomic analysis, we identified four other Cry1Ac binding proteins in CBW BBMV for the first time: vacuolar ATP synthase subunit B, actin, a heat shock cognate protein (HSP) and a novel protein. Actin and vacuolar ATP synthase subunit A had been reported as Cry1Ac binding proteins in Heliothis virescens. Heat shock cognate and this novel protein was the first report as Cy1Ac binding protein in insect.3. The types and expression quantities of Cry1Ac binding proteins of susceptible- and resistant-CBW were compared, it showed that the types of binding proteins were the same between susceptible- and resistant-strain, but the novel protein in the resistant-strain were 6.49-fold lower expression than the susceptible-strain. Studies before said that less binding of Bt in the midgut or less number of receptors were the main resistant mechanism for this resistant strain. Combined studies before and this paper, much lower expression of the novel protein might be one of the resistant reasons.4. Binding proteins of Cry1Ah, Cry1Ab and Cry1Ac in susceptible- and resistant-CBW were compared, the binding of Cry1Ab to binding proteins was weak than the other two toxins whether in susceptible- or resistant-strain. In susceptible-strain, Cry1Ab bound to novel protein and HSP, did not bind to actin and V-ATPase, but Cry1Ac and Cry1Ah bound to these four binding proteins, maybe novel protein and HSP are essential for the toxin action, actin and V-ATPase may be related to toxicity. Predecessors results showed that Cy1Ab and Cry1Ah have cross resistance to Cry1Ac-resistant CBW, the difference of different Bt binding proteins between susceptible- and resistant-CBW were lower expression of novel protein, maybe this was reason for cross resistance or other reasons need further study.