Cloning And Activity Analysis Of The Transcription Regulation Region Of Odorant-binding Protein Gene GOBP2 And PBP2 In Spodoptera Litura

The olfactory sensory process in insects involves a variety of olfactory proteins,among which odorant-binding proteins(OBPs)play an important role in binding and transporting hydrophobic odorantmolecules to olfactory receptors(ORs)ondendritic membrane of olfactory nurons.In Lepidoptera,OBPs are devided into general odorant-binding protein(GOBP),pheromone-binding protein(PBP)and other OBPs.PBPs are mainly used for recognition and reception of pheromone components,and GOBPs are mainly used for the recognition of plants odorants.However,there is no study on the transcription regulation of OBPs.In the present study,transcriptional regulatory activity was determined in the 5’ flanking regions of GOBP2 and PBP2 of Spodoptera litura.Firstly,the 5’ flanking region of the two genes were cloned by genome walking,and the potential transcription factor binding sites in the 5’ flanking region of the two genes were predicted by online tools.Then,the 5’-end progressive deletion sequences of the two genes were cloned by PCR and constructed into pGL3-Basic vector.Finally,the transcriptional regulatory elements(enhancers and silencers)were suggested by the dual-luciferase reporter assay.The main results are as follows:1.Cloning and analysis of 5’ flanking region of SlitGOBP2 and SlitPBP2.The 5’ UTR of SlitGOBP2 was cloned by 5’RACE using the cDNA of antenna of adult S.litura.A 23 bp 5’UTR was obtained by sequencing,and thus the transcription start site was determined to be 23 bp upstream of the translation start site(ATG).The 5’ flanking region of 2025 bp of SlitGOBP2 was obtained by genome walking.The transcription factor binding sites were predicted by online tools,and the TATA box(TATAAA)of promoter was found at-20～-15.A total of 26 transcription factor binding sites belonging to 16 kinds were found in the 5’flanking region,and most of them are within-1000 bp.The 16 kinds of binding sites are Oct-1(Octamer binding proteins),Spl(Stimulatory protein 1),GATA-1,AP-1,D1,Hb(Hunchback),USF,NF-1,Ubx(Ultrabithorax),C/EBP(C/EBP like bZIP transcription factors),AhR,HSF,TATA,BR-C(Broad-Complex),TFIID,and CREB.Similarly,the 5’ UTR of SlitPBP2 was cloned by 5’RACE.A 28 bp 5’UTR of SlitPBP2 was obtained,and thus the transcription start site was determined at 28bp upstream of the translation start site.In addition,5’flanking region of 1079 bp was obtained by PCR according to the genomic sequence of S.litura(accession number:KJ956693.1).The transcription factor binding sites at 5’ flanking region were predicted by online tools,and 18 transcription factor binding sites of 15 kinds were found,including C/EBP,Oct-1,USF,Ubx,AP-1,Spl,GAGA,GATA-1,NF-1,BR-C Z,CREB,TATA,Hb,D1,AhR.2.Analysis and localization of the transcriptional regulation elements of SlitGOBP2 and SlitPBP2.The 5’-end progressive deletion sequences of the SlitGOBP2 and SlitPBP2 were cloned by PCR and constructed into pGL3-Basic vectors.Further,the ranscriptional regulation elements within-1000 bp in the 5’flanking region were analyzed by the dual-luciferase reporter assay.Results suggested a strong enhancer in the region from-243 to-207,two weak enhancers and a silencer in region-971～-891,-890～-811,and-315～-244,respectively in SlitGOBP2.For SlitPBP2,it was suggested at least one enhancer in each region of-1011～-887,-886～-775,-203～-140,and at least one silencer between-774～-204.These results provide basis for further clarifying the transcriptional regulation of SlitGOBP2 and SlitPBP2.