Functional Analysis Of ABCC Gene Subfamily On Spodoptera Exigua Under Ethanol Extract Of Eclipta Prostrate And Cry1Ca Stress

The Spodoptera exigua is a serious harm agricultural pest in many areas of the world.It causes great harm to vegetables and crops.In recent years,the harm to cotton is becoming more and more serious.The insecticidal active ingredient of the plant extract can be used as a source of botanical insecticide.Botanical insecticides can safely and effectively control agricultural pests,and pests are not prone to produce resistance.Ethanol extract of Eclipta Prostrate has insecticidal activity against insects.Cry1Ca is a kind of biogenic insecticide,and S.exigua is more sensitive to Cry1Ca.The aim of this study was to determine insecticidal activity of ethanol extract of E.Prostrate（EEEP）on S.exigua,the synergistic effect of EEEP on toxicity of Cry1Ca against S.exigua,and the changes of expression of SeABCC,to provide a theoretical basis for molecular mechanism of resistance and resistance managemen for S.exigua.The main results are as follows:1.Bioactivity determination of EEEP and WEEP to S.exigua.EEEP had significant insecticidal activity against S.exigua larvae.The virulence was determined by surface contamination.The 2nd instar larvae of S.exigua feeding 5.26 mg/cm² of WEEP for 7 days,the larval mortality were 7%.So the insecticidal activity of WEEP was weak on S.exigua larvae.Larval mortality and inhibition rate of weight were positively correlated with the concentration of EEEP.When the 2nd instar larvae of S.exigua feeding 5.26 mg/cm² of EEEP for 7 days,larval mortality and inhibition rate of weight were 58.35%and 71.17%.According to the virulence regression equation of EEEP on 2nd instar larvae of S.exigua,50%lethal concentrations(LC₅₀)were 4.55 mg/cm².With the increase of Cry1Ca concentration which feed 2nd and 3rd instar larvae of S.exigua,larvae mortality and inhibition rate of weight also increased gradually.According to the virulence regression equation of Cry1Ca on 2nd and 3rd instar larvae of S.exigua,LC₅₀ were 145.72 and 434.34 ng/cm².2.Analysis of midgut transcriptome of S.exigua under Cry1Ca stress.Midgut transcriptome of S.exigua showed that:There were 2962 differentially expressed genes（DEGs）,in which 1454 genes were obviously up-regulated and 1508 genes were obviously down-regulated.Gene Ontology analysis of DEGs found,882 DEGs participated catalytic activity,accounting for 51.07%.159 DEGs participated catalytic activity,accounting for 9.21%.Pathway enrichment analysis of DEGs found,DEGs were mainly enriched in the PPAR signaling pathway.Generating statistics from the database of results,ABCA,ABCB,ABCC,ABCD,ABCE,ABCF,ABCG subfamily had 42,39,27,6,3,10,17,3 Unigenes respectively.Blast analysis of ABCC subfamily Unigene fragments.Collation and verification,finally got eleven ABCC sequences.3.Identification and spatial-temporal expression of ABCC subfamily genes in S.exigua.The full length sequence of SeABCC was obtained by RACE technique.The spatial-temporal expression of SeABCC were analyzed by qPCR.SeABCC1 were higher expression in intestinal tract of S.exigua larval.SeABCC2,SeABCC3 and SeABCC7 were highest expression in midgut of S.exigua larval,and higher expression in malpighian tubules secondly.SeABCC4,SeABCC5,SeABCC6,Se ABCC11were most abundant expression in malpighian tubules of S.exigua larval.The expression of SeABCC12was significantly higher in intestinal tract,fat body and malpighian tubules than in other tissues.There was no significant difference in the expression of SeABCC13 in different instars and tissues.4.Synergism of EEEP and Cry1Ca and the effect of ABCC mRNA expression in S.exigua.There was synergistic effect of EEEP and Cry1Ca on S.exigua.When S.exigua ingested the admixture of of EEEP(LC₃₀)and Cry1Ca(LC₃₀),larval mortalities were 59.72%,73.61%and 83.33%after 3 d,5 d and 7 d.However,after feeding with EEEP(LC₃₀)or Cry1Ca(LC₃₀)for 7 d,the mortalities were 30.06%or 31.94%.Thus,the EEEP can obviously enhance the control effect of Cry1Ca against S.exigua.Farther,the sublethal dose of admixture significantly inhibited the body weight,pupal weight,pupation rate,emergence rate,fecundity per female and egg hatchability of survival larvae.The relative expression levels of SeABCC were analyzed by q PCR.The expression level of SeABCC1,SeABCC2,SeABCC3,SeABCC4,SeABCC5,SeABCC7,SeABCC12 was significantly increased under the treatment of sublethal dose of admixture.The increased expression of SeABCC could cause S.exigua larvae more susceptibility to Cry1Ca.In summary,EEEP had significant insecticidal activity against S.exigua larvae,and had synergistic effect of Cry1Ca against S.exigua.Thus,EEEP as botanical pesticide could be mixed with Bt toxin,to improve the control effect against S.exigua.