Identification And Functional Analysis Of Cry2Ah1 Binding Protein In The Midgut Of Beet Armyworm Spodoptera Exigua

With the global warming and the change of planting system in the field,the beet armyworm Spodoptera exigua has become more and more serious on a variety of crops in China.At present,the main control method is still using chemical pesticides,but the chemical pesticides have significant negative impact on beneficial insects and the environment.Spodoptera exigua is one of the most important pests on cotton.The monovalent transgenic cotton expressing only Bacillus thuringiensis(Bt)Cry1Ac protein has been planted in large areas in China.Although it can effectively control major target pests such as cotton bollworm Helicoverpa armigera and pink bollworm Pectinophora gassypiella in cotton fields,but its toxic effect against Spodoptera exigua is low.Moreover,with the increase of transgenic cotton planting time,H.armigera and other pests have developed initial resistance to transgenic cotton under continuous selection pressure.So,to develop efficient new Bt gene has important significance for the development of new biocides and candidate genes of transgenic insect-resistant crops.Cry2Ah1 is a new protein with independent intellectual property rights in China.In order to better utilize Cry2Ah1 protein,it is very important to further understand the mechanism of Cry2Ah1 against Spodoptera exigua.In this paper,the ligand fishing,in vitro binding and RNAi interference were used to identify the Cry2Ah1 binding proteins in the midgut of in Spodoptera exigua,and the roles of APN2 and ABCC2 binding proteins in the insecticidal mechanism of Cry2Ah1 were analyzed.The main research results are as follows:1.Extraction and purification of Cry2Ah1 protein.The insecticidal effect of Cry2Ah1 protein was determined by bioassay,which indicated that Cry2Ah1 had good insecticidal activity against Spodoptera exigua.2.The proteins in the midgut of Spodoptera exigua binding with Cry2Ah1 were identified by Pulldown,ligand blot,mass spectrometry and other molecular biology techniques.The results showed that APN1,APN2,APN3,APN5,APN6,ABCC2,ABCC3,Polycalin,ALP and some other unknown proteins on BBMV could bind with Cry2Ah1 insecticidal protein.3.Cloning APN2 gene of Spodoptera exigua,and the expression of APN2 in the midgut of larvae at different instars was detected by fluorescence quantitative PCR.The results showed that the expression of APN2 gene increased with the increase of instars before the fourth instar larvae,and the expression level was the highest level at the fourth instar,and the expression level decreased after the fourth instar.Western blot analysis showed that the APN2 protein expressed by prokaryotic system could specifically bind with Cry2Ah1.The expression of APN2 was significantly increase after larvae feeding Cry2Ah1 protein.Therefore,we speculate that APN2 is a binding protein of Cry2Ah1 and can be induced by Cry2Ah1,but whether it is a functional receptor need to be further verified.4.Cloning ABCC2 gene of Spodoptera exigua,and phylogenetic tree analysis results showed it had high homology with that of Spodoptera frugiperda.showed that The expression level of ABCC2 in larvae at different instars was detected by RT-PCR,it was found that the expression level of ABCC2 gradually increased with the growth of the larval instar.The ABCC2 genewas silenced by RNA interference technique,and the sensitivity change of the larvae to Cry2Ah1 insecticidal protein was analyzed further.The results showed that the expression of ABCC2 gene and its sensitivity to Cry2Ah1 insecticidal protein obviously decreased after treated 48 hours.Therefore,the ABCC2 in the midgut of Spodoptera exigua may be an important receptor protein binding with Cry2Ah1 and play an crucial role in the insecticidal process of Cry2Ah1.In this study,the toxic effect of Cry2Ah1 insecticidal protein on Spodoptera exigua was clarified,and the types of Cry2Ah1-binding proteins on BBMV were identified.The ABCC2 in Spodoptera exigua was determined as an important functional receptor for Cry2Ah1 protein to kill insects.The results can provide a foundation for further revealing the mechanism of Cry2Ah1 insecticidal protein on Spodoptera exigua and for better application of Cry2Ah1.