Gene Clong And Funtional Analysis Of ATP-binding Cassette Transporter ABCG1 From Helicoverpa Armigera

Cry resistance developed in target pests has been caused by the sustainable planting of Bt crops.Previous work showed that the down-regulation of white(ABCG1)gene is tightly linked to Cry1 Ac resistance in Plutella xylostella,so we predict that the similar change of ABCG1 gene expression may be associated with the Cry1 Ac resistance in Helicoverpa armigera.The open reading frame(ORF)of HaABCG1 was cloned from H.armigera,the subcellular localization of HaABCG1 protein in TnH5 cells was detected by constructing expression vector of Ha ABCG1,the ABCG1 protein as the receptor of Cry1 Ac toxin with cytotoxicity of Bt toxin was confirmed by cytotoxicity tests,and whether the down-regulation of HaABCG1 could decrease the susceptibility of H.armigera to Cry1 Ac toxin was confirmed by RNA interference(RNAi)and bioassay.The results demonstrated that: 1,Gene clong and sequence analysis of HaABCG1 geneThe open reading frame of HaABCG1 is 1 896 bp,encoding 631 amino acid residues.The calculated molecular weight(Mw)and isoelectric point(pI)of the predicted protein are about 69.63 kDa and 8.76,respectively.Although N-terminal signaling peptides and potential O-glycosylation sites were not found,the HaABCG1 sequence contains four potential N-glycosylation sites.The deduced amino acid sequence of the HaABCG1 gene exhibits structural features characteristic of known insect white genes and belongs to ABC transporter subfamily G,whose members have one cytoplasmic N-terminal nucleotide-binding domain(NBD)with several conserved motifs followed by one transmembrane domain(TMD)with six transmembrane ?-helix segments at the C-terminus.2,Phylogenetic analysis of HaABCG1Phylogenetic analysis was performed by constructing a neighbor-joining(NJ)tree of white genes with MEGA 6.0 based on multiple alignments of amino acid sequences from 37 different species.The unrooted tree showed that the identified HaABCG1 proteins are more evolutionarily conserved,and the HaABCG1 proteins of insect are clearly homologous to non-insect.Additionally,the phylogenetic tree showed clear separations between white genes and ABCG1 genes,which indicated that these ABCG1 genes are homologous to ABCG4 rather than ABCG5 and ABCG8.3,Subcellular localization of HaABCG1-GFP and the relationship between HaABCG1 and Bt toxinsThe subcellular localization of HaABCG1-GFP protein in TnH5 cells was detected by constructing expression vector of HaABCG1,and we found that HaABCG1 mainly existed on nuclear membrane and endoplasmic reticulum membrane.TnH5 cells were treated with Cry1 Ac,Cry2Aa,Cry1 Ca and Cry1 Fa for 1 h after the expression of HaABCG1 for 24 h.It was showed that TnH5 cells did not change morphologically when the cells of positive control expressing HaABCC2-GFP and HaCad-GFP expanded and burst.And we can know that HaABCG1 was not the receptor of Cry1 Ac,Cry1Ca,Cry2 Aa or Cry1 Fa.4,Functional assays of the HaABCG1 gene by in vivo RNAi and bioassayTo further explore the potential involvement of the HaABCG1 gene in Cry1 Ac resistance of H.armigera,we used in vivo RNA interference(RNAi)to knockdown HaABCG1 expression by injecting siABCG1 into the early third-instar 96 s larvae.RT-PCR results revealed that injection of siABCG1 into larvae significantly reduced HaABCG1 transcript levels by more than 40% relative to controls.Subsequent bioassays performed at 48 h post-injection demonstrated that the body weight change had no significant difference between the control group and the experimental group reared on artificial diet with the Cry1 Ac concentration of 0.05 ?g/m L for 3 d after RNAi,and the silencing of Ha ABCG1 gene expression did not significantly reduced larval susceptibility to Cry1 Ac protoxin at 0.05 ?g/m L(the LC50 value).These results are consistent with the inference that the HaABCG1 gene cannot be involved in H.armigera resistance to Cry1 Ac.We concluded that the down-regulation of HaABCG1 is not associated with Cry1 Ac resistance in H.armigera,and HaABCG1 is not the receptor of Cry1 Ac.This is the first report that ABCG1 gene is not involved in the Cry1 Ac resistance of H.armigera.