Preliminary Study On The Expression Of Midgut Tissue Specific Genes In Spodoptera Frugiperda Sf9 Cell Line Induced By GATAe

Insect midgut epithelial cells play an important role in digesting food and absorbing nutrients.In addition,they can be used as targets for a variety of biological control microorganisms.There is currently a lack of cell lines that carry out the interaction between insect pathogenic microorganisms and insect midgut-epithelial like cells.It’s of great significance to induce insect cell lines to have partial characteristics of midgut cells,which may break the bottleneck.Spodoptera frugiperda,a serious pest posting a real threat to agriculture,has invaded most areas of China in recent years.In this study,we found that the chromatin remodeling factor GATAe can induce the high expression of some midgut tissue-specific genes in Sf9 cells,which can be used to study the interaction between intestinal pathogenic microorganisms and cells of some insects,and provide a basis for theoretical research on biological control of this pest.The main research results are as follows:The coding sequence of SfGATAe gene was cloned successfully.Its open reading frame is 1827 bp and encodes 608 amino acids.The predicted molecular weight is 65.14 KD and the theoretical isoelectric point is 6.01.It is closely related to the GATAe gene of Spodoptera litura and Helicoverpa armigera in the same branch of the phylogenetic tree.Immunofluorescence assay showed that SfGATAe recombinantly expressed in Sf9 cells was localized in the nucleus.When the final concentration of IPTG is 0.4 mM,the induction temperature is 37℃,and the induction time is 6 h,SfGATAe in the form of inclusion body could be detected.Transcriptome analysis showed that the gene expression profile of Sf9 cells transfected with GATAe changed significantly.There are 169 differentially expressed genes,including 169 up-regulated genes and 0 down regulated genes.The functions of the main differentially expressed genes are involved in Carbohydrate transport and metabolism,Lipid transport and metabolism,Amino acid transport and metabolism.The metabolic pathways of differentially expressed genes can be divided into three types:Metabolism,Cellular Processes and Environmental Information Processing.The results of real-time PCR showed that the expression levels of 7 midgut tissue-specific genes including Chitin synthase,Chitin-bindingproteins,Lipase,Parenzyme,Toll-like receptor,α-Chymotrypsin and Tetraspanin in Sf9 cells transfected with SfGATAe are significantly increased by 20,108,466,4,12,498 and 5 times,respectively.This indicates that Sf9 cells transfected with SfGATAe are similar to midgut epidermal cells,with higher expression levels of genes involved in material transport and digestion.The sensitivity of Sf9 cells transfected with GATAe to five kinds of Bt toxins was determined.The results showed that cry1Ac,cry1Ab,cry1F,cry2Aa and Vip3Aa had no obvious toxicity to Sf9 cells that were not transfected with GATAe.The EC50 of Cry1Ac,CrylAb and Cry1F toxin on cells transfected with SfGATAe were(6.47±2.13)μg/mL,(6.35±2.38)μg/mL and(34.82±1.74)μg/mL,respectively.CrylAc,CrylAb and Cry1F toxins are more toxic to Sf9 cells transiently expressing HaGATAe,and their EC50 is(1.86±0.81)μg/mL,(1.29±0.28)μg/mL and(8.90±1.96)μg/mL,respectively.Cry2Aa toxin had lower toxicity to Sf9 cells overexpressing SfGATAe and HaGATAe,respectively.The toxicity of Vip3Aa toxin to Sf9 cells overexpressing SfGATAe and HAGATAe,respectively,was not observed.Then the endogenous expression of the toxin receptor genes SfABCC2 and SfABCA2 in Sf9 cells transfected with SfGATAe was detected,which increased by 38 and 26 times,respectively.These results indicate that Sf9 cells transfected with GATAe are similar to midgut epidermal cells and are more sensitive to multiple Bt toxins.Finally,the influence of SfGATAe protein on the formation of AcMNPV polyhedrons in Sf9 cells was explored,and it was found that the volume of polyhedrons formed by Sf9 cells transfected with SfGATAe was smaller.In short,GATAe can induce Sf9 cells to express a series of midgut tissue-specific genes at a high level,and significantly improve the sensitivity of Sf9 cells to the Bt toxins.These findings expand the application range of Sf9 cells,provide the foundation for the study of the interaction between the intestinal pathogenic microorganisms and host cells,and benefit theory research on pests biological control.