Study On Expression Regulation Of Two BT Toxin-receptor Genes ABCC2 And ABCC3

Insecticidal crystal protein produced by Bacillus thuringiensis or transgenic crops is very toxic to the target pest and is a very important environment-friendly biological pesticide which provides well protection to crops.However,due to its widespread use,laboratory or field pests have produced significant resistance,which becomes a serious threat to the sustainable use of Bt resources.The decrease of Bt toxin receptor expression is one of the important mechanisms of insect resistance,though,while there is little research on the regulation mechanism of Bt toxin receptor.This study focused on the regulation of the expression of Bt toxin receptor ABCC2 and ABCC3 genes in insect,and FOXA regulates susceptibility of insect and cells to Bt toxins.1.Establishment of Helicoverpa armigera midgut cell line and its susceptibility to Bt toxinBrush border membrane(BBM)of insect midgut epithelium of insects is the target of Bt toxin.Only few of insect midgut cell lines was established.Most of them were non-susceptible to BT toxin,which suggests BT toxin receptor genes were not expressed or expressed at low level in these cell lines.In this study,a novel cell line was initiated from midgut tissue of the fourth instar lavae of Helicoverpa armigera designated as HNU-Ha-MG1,and a serious identification was conducted.The size of the cells was 13.8 ± 1.8 ?m in diameter,and the maximum density reached(2.40 ± 0.15)×106 cells/ml.The population doubling time during logarithmic growth phase was 58.6±7.0 h at 28?.The number of chromosomes was about 90-130,which exhibited typical chromosome characteristics of lepidopteran cell lines.The patterns of random amplified polymorphic DNA and esterase isozyme analysis of HNU-Ha-MG1 were different from those of S1-HP and Hi5 cell lines which were frequently used in our laboratory.Cell apoptosis was observed after induced by 20-hydroxyecdysone,while no significant autophagy was detected by WB.HNU-Ha-MG1 is susceptible to a variety of baculoviruses and Bt Cry1Ca toxins,but not to Bt Cry1Ac toxins.ABCC2 and ABCC3 are Cry1Ac toxin receptors.Other insect cell lines were chosen to study the regulation mechanism of these receptor genes.Our previous study showed that ABCC3 gene was expressed at a high level in S1-HP cell line.Spodoptera ltura ABCC3 promoter and Sl-HP cell line were selected to investigate regulation mechanism of the Bt toxin receptor genes.2.Cloning and Regulation of ABCC3 Promoter from Spodoptera litura2.5kb upstream flanking sequences of Spodoptera litura ABCC3 was isolated by genome walking and the transcriptional start site was identified using 5'RACE.FoxA,Foxj2,Kruppel and BR-C Z1 motifs within SIABCC3 sequence were predicted by online software.Luciferase reporter recombinant plasmid fused with whole length SlABCC3 promoter was constructed.Relative luciferase activity in 3 insect cell lines was detected by dual luciferase reporter assay.5' and 3' deletion was conducted base on the whole length promoter,of which relative activities were analysed in Sl-HP and Sf9.Result showed promoter activity reached the peak while 5 promoter was deleted at-1064nt.Two putative positive regulation element was identified as Kruppel(274nt?283nt),FOXJ2(659nt?672nt),and a putative negative transcriptional fator binding site asBR-C Zl(1267nt-1239nt).5'UTR was proved necessary to SIABCC3 promoter.3 Cloning and functional identification of Helicoverpa armigera ABCC2 promoterIn this study we cloned whole length sequence of H.armigera ABCC2 promoter by two different methods.And silicon analysis and cis-acting elements prediction was performed with online software.Results as follow:We isolated 1.8kb upstream flanking sequences of HaABCC2 with genome walking and direct PCR.Transcription start site was identified with 5' RACE.Bioinformatic analysis revealed 2 putative Kruppel motifs and 17 putative FoxA motifs by which HaABCC2 promoter might be regulated.This study suggested Lepidoptera ABCC2 and ABCC3 might share some common regulation mechanism.4 Influence and molecular mechanism of transcription factors on BT toxin susceptibility of insect cell lines and larvaeSequences analysis revealed multiple putative FoxA binding sites on ABCC2 and ABCC3 promoters,so we cloned FoxA,of which mechanism to regulate promoter activity and gene expression was studied.Software prediction showed the molecular weight of SIFoxA was 38.9 kDa,the theoretical isoelectric point was 8.74,the instability coefficient was 55.62,the average hydrophilicity was-0.731.Two classic NSL sequences was found at both end of SIFoxA DBD,which suggested SIFoxa locate in the nucleus.Swiss-Model homology modeling indicated conserved domain of SIFoxA was consist of 2 wings(W1,W2),3a-helix(H1,H2,H3)and 2?-sheet(S1,S2).Homology comparison of protein sequence showed FoxA was highly conserved and forkhead domain was highly consistent.Phylogeny tree showed SIfoxA was closely related with Lepidoptera FoxA.Cotransfection with S1ABCC3 promoter demonstrated SIFoxA and HaFoxA transactivated ABCC3 promoter in Sl-HP with 17 and 12 times of relative activities increased,responsively,while no transactivation was observed among S1Foxj2,S1Kuppel and BmBR-C Z1.Similar results were determined in Sf9.Transient expression of GFP fused SIFoxA indicated FoxA locates in nucleus during mitotic and bind to chromasomes during metaphase.Real-time qPCR was used to determine the effect of FoxA over-expression on BT receptor gene expression in sf9.Expression level of ABCC2 and ABCC3 was significantly increased by 2.2 and 3.7 times to control,respectively.Susceptibility of Sf9 to Cry1Ac was significantly raised by over-expression of SlFoxA.About 32%Sf9 cells were observed swollen and broken after 5?g/ml toxin treatment for 1 h(transfection efficiency of 40-60%).Silence of FoxA using RNAi led to 55.7%decrease in lavae midgut in 72h.The expression of ABCC2 and ABCC3 were reduced by 35.8%and 48.8%,respectively,and larvae fed with CrylAc toxin were more tolerant than control group with heavier weight and significantly reduced mortality.In a word,our study first reported pioneer factor FoxA increased susceptibility of insect cell and larvae through up-regulating BT toxin receptor ABCC2 and ABCC3.The result suggested down regulation of FoxA might be a potential BT resistance mechanism.