Screening And Resistance Function Of ABC Gene In Midgut Of Spodoptera Frugiperda After Feeding On Bt Protein

Fall armyworm,Spodoptera frugiperda(J.E.Smith),native to the America,is an important migratory agricultural pest.It was found in cornfields and infested corn in January 2019 in China.S.frugiperda has posed a serious threat to corn production and food security since its invasion.Parasporal crystal proteins and vegetative proteins produced by Bacillus thuringiensis(Bt)have insecticidal effects on insects and are widely used as biological insecticides for control of agricultural and forestry insect pests.Transgenic insect-resistant crops that express the Bt protein are already widely used in many countries.However,large-scale cultivation of transgenic insect-resistant crops will lead to high resistance of target pests to Bt protein,and affect the control effect for target insects and also the sustainable use of transgenic crops.It has been reported that specific binding of ABC transporters plays an important role in the mechanism of insect resistance to Bt proteins.The down-regulation or deletion of the target receptor ABC gene for Bt protein in the target insect affects the insecticidal action of Bt protein.Exploration of potential target genes and functional analysis of different Bt proteins are therefore effective measures to delay the development of resistance.In this study,the fourth instar larvae of S.frugiperda were fed a diet containing Cry1Ab,Cry1F,and Vip3Aa19,respectively,and transcriptome sequencing was used to compare the differences in the expression of midgut genes.Differentially expressed ABC genes were screened and validated by fluorescence quantitative PCR.Finally,the ABCD3 gene was specifically down-regulated after screening for S.frugiperda larvae fed on Bt protein.The ABCD3-KO knockout strain of S.frugiperda was established using the CRISPR-Cas9 gene knockout technique.The survival rate and life table parameters of the knockout strain and the susceptible strain of S.frugiperda were compared.The ABCD3 protein is expressed by the Escherichia coli prokaryotic expression system,and the binding properties of ABCD3 and Bt proteins are verified by the Pull-down protein interaction test.The main results are as follows:1.qPCR results show that the expression of ABCD3 gene was significantly down-regulated after Cry1Ab and Vip3Aa19 protein treatment,suggesting that ABCD3 gene was involved in the insecticidal process of Bt protein in S.frugiperda.2.After knocking out ABCD3 gene,the mortality of S.frugiperda larvae fed on diet containing Cry1Ab,Cry1F and Vip3Aa19 decreased significantly and the resistance level increased,which indicated that ABCD3 gene participate in the production of Bt protein resistance.We also found that after ABCD3 gene deletion,the pupal weight of S.frugiperda increased significantly,the survival time of female moths increased markedly,and the oviposition numbers per female decreased obviously,indicating that ABCD3 gene also be involved in the reproductive process.3.Through the Pull-down protein interaction test,we found that the prokaryotic expression of ABCD3 protein had a good binding affinity with Cry1Ab protein,but it did not bind to Cry1F protein,and the binding effect with Vip3Aa19 protein was weak,indicating that ABCD3 protein was the binding receptor of Cry1Ab,but not the binding receptor of Cry1F and Vip3Aa19 protein.