Study On Binding Affinity Between Receptors Of Midgut In Helicoverpa Armigera (H(?)bner) And Spodoptera Exigua(H(?)bner) And Bt Toxins

Cotton bollworm Helicoverpa armigera(H(?)bner) and beet armyworm Spodoptera exigua(H(?)bner) are both important noctuidae pests which have wide range of hosts. Transgenic insect-resistant cotton have been planted in China since 1997, the planting of Bt(Bacillus thuringiensis) cotton not only effectively controls the population development of H. armigera, but also may reduce the presence on other host crops. But this kind of Bt cotton(expressing Cry1Ac protein) has low efficiency to S. exigua. Bt cotton expressing Cry1Ac+Cry2Ab can delay the resistance evolution of target pest and have wider insecticidal spectrum which can control several kinds of lepidoptera pests including S. exigua. It has been widely planted in some countries. It is assumed that Cry2 A has different action mode compared with Cry1 A, but there has less research on mechanism of Cry2 A action mode. In this study, we compared the binding abilities between Cry1Ac, Cry2Aa with different receptor proteins(cadherin, aminopeptidase N and alkaline phosphatase of) on midgut of H. armigera. Alkaline phosphatase2(ALP2) gene on midgut of S. exigua was cloned, and it was confirmed as a binding protein with Cry1Ac. The differences of binding ability between Cry1Ac, Cry2Aa with ALP2 from H. armigera and S. exigua were further compared. The results will provide the basis for further determine the mechanism of Cry2 A and rational planting Cry1A+Cry2A cotton. The results as follows:1. The binding affinity of Cry1Ac and Cry2Aa toxins was compared and the results showed that:the binding affinity between the expressed cadherin, aminopeptidase N and alkaline phosphatase with two insecticidal proteins. The binding affinity with Cry1Ac is 0.2519 nM, 121.7 n M and 121.7 nM, respectively, while the binding affinity with Cry2Aa ability 0.836 nM and 9.813 nM and 47.91 nM.2. The full-length cDNA encoding ALP2 protein was cloned from the midgut of S.exigua by the homology cloning and RACE methods. The results showed that the alp2 gene in S.exigua was 1 629 bp(registered in Gen Bank with the accession number KP420013) and encoded 543 amino acid residues, containing 21 amino acid residues signal peptide in the N-terminus region, existing a GPI anchorsite site in the C-terminus region.3. The expression level of ALP2 in different larval development periods was compared using real-time quantitative PCR analysis. The ALP2 could express in whole larval periods of S.exigua, but there are significant differences among various instars. The lowest expression level appeared in 1st instar larvae, while 4th instar larvae was the highest The binding of ALP2 with Cry1Ac tested by Ligand blot analysis showed that recombinant ALP2 could bind with Cry1Ac insecticidal protein.4. The results of binding affinity of the receptor of ALP with Cry1Ac and Cry2Aa in Helicoverpa armigera and Spodoptera exigua measured by the ELISA showed the binding affinity of the receptor with Cry2Aa of ALP in the two insects is higher than with the toxin of Cry1Ac. Second, our results also found that the binding of ALP to Cry1Ac toxin is non- special, in contrast to Cry2Aa being special. The toxicity of Cry2Aa is far higher than the toxicity of Cry1Ac.