Study On The Founction Of ABCA2 Gene Of Spodoptera Frugiperda (J.E.Smith) Mediated By CRISPR/Cas9

The fall armyworm(Spodoptera frugiperda)is a lepidoptera noctuidae pest native to the tropical and subtropical regions of the Americas,has strong migratory and reproductive abilities.It invaded Yunnan province at the end of 2018 and has spread to many provinces and cities,threatening Chinese food production security.Planting pyramid Bt corn is an effective means to control S.frugiperda.Cry2 Ab toxin is an important toxin in pyramid Bt corn.The previous study showed that the ABCA2 transporter proteins in several lepidoptera species is the receptor of Cry2 Ab toxin,mediating the production of Cry2 Ab resistance;but whether ABCA2 in S.frugiperda perform the same function or not reminds to be studied.Based on that,this study used CRISPR/Cas9 system to edit the ABCA2 and ABCA2-Like genes of S.frugiperda to study the function of these two genes in S.frugiperda,and conclusions from this study would provide important theoretical guidance for the monitoring and management of resistance Bt maize.The main contents of this study are as follow:(1)Mutant populations with gene function deletion of ABCA2 and ABCA2-Like genes of S.frugiperda were successfully constructed by using CRISPR/Cas9 system,named SfABCA2-ko and SfABCA2-Like-ko respectively.The mutation type of SfABCA2-ko population is to insert 6 bp and delete 2 bp at the 177 th bp of SfABCA2 gene exon 1,resulting in frameshift mutation and premature termination of coding protein;The mutation type of SfABCA2-Like-ko population is a 171 bp deletion in exon 2,and 56 bp deletion in exon 3 of SfABCA2-Like gene,resulting in frameshift mutation and premature termination of coding protein.(2)Bioassays for the newly-hatched larvae of SfABCA2-ko population,SfABCA2-Like-ko population and Sf-WT population were carried out by using the artificial diet surface covering method.The results of data analysis showed that,compared with Sf-WT population,the resistance ratio of SfABCA2-ko population and SfABCA2-Like-ko population to Cry2 Ab toxin was 1.04-folds and 0.27-folds respectively,suggesting no obvious resistance;and they also showed no obvious resistance to Vip3 Aa,Cry1Ac,Cry1 Ab and Cry1 Fa.The above results suggest that the SfABCA2 and SfABCA2-Like genes may not be involved in the process of Cry2 Ab toxin exercising toxicity on S.frugiperda.At the same time,these two genes may not participate in the toxicological process of Vip3 Aa,Cry1Ac,Cry1 Ab,and Cry1 Fa,too.(3)In order to determine the cost of fitness caused by the mutation of ABCA2 and ABCA2-Like genes in S.frugiperda,the life tables of SfABCA2-ko strain,SfABCA2-Like-ko strain and Sf-WT strain were constructed In this study,and the fitness of three laboratory strains were compared.The results showed that SfABCA2-ko and SfABCA2-Like-ko strains were significantly lower than Sf-WT strains in terms of the eggs produced per female and hatch rate.Compared with Sf-WT wild-type strain,the egg produced per female in SfABCA2-ko strain was reduced by 1168,the hatching rate was reduced by 11.4% and the larval duration was increased by 1.79 days.Compared with Sf-WT strain,the eggs produced per female in SfABCA2-Like-ko strain was reduced by 456 eggs,the hatching rate was reduced by50.33%.According to the fitness analysis of the above three laboratory strains,the fitness of SfABCA2-ko and sf ABCA2-Like-ko strain relative to Sf-WT strain is 0.24 and 0.23 respectively.The results showed that the mutations of ABCA2 and ABCA2-Like genes had adverse effects on the fitness of individuals of S.frugiperda,and the mutations of these two genes would lead to the decline of survival and fecundity in S.frugiperda.This study has proven that the CRISPR/Cas9 system is an effective gene editing tool for S.frugiperda,providing a good reference for research work related to the gene function of S.frugiperda.Moreover,this study has found that the ABCA2 and ABCA2-Like genes in the living body of S.frugiperda may not be involved in the toxicological process of these five toxins: Cry2 Ab Vip3Aa,Cry1 Ac,Cry1Ab,and Cry1 Fa,which is important for research related to the resistance of S.frugiperda to Bt toxin.Meanwhile,this study has found that the ABCA2 and ABCA2-Like genes of S.frugiperda may be related to it’s fecundity,This may be a new target for the study of genetic population regulation of S.frugiperda.