Promoter Activity Analysis Of Nachr Gene From Leptinotarsa Decemlineata

Colorado potato beetle(CPB),Leptinotarsa decemlineata(Say),is an important leaf-eating insects on potato.Since it invaded in Xinjiang in the early 1990s,the bettle has spread from west to east continuously and distributed in the majority region of the northern part of Xinjiang,which threating potato planting in China.Now,the leading insecticides for CPB control is neonicotinoids targeting on nicotinic acetylcholine receptor(nAChR).The study on structure and function of nAChR will provide important theoretical basis for effective management of CPB.Previous researchs have shown that CPB in Xinjiang has developed different levels of resistance to neonicotinoid insecticides,and adults and larvae exhibited different levels of tolerance to neonicotinoids and nAChR subunits expression.RNA interferencing of Ldal could lead to reduction of sensitivity to neonicotinoid insecticides in adults and larvae of CPB.On the basis of the results,we continued to clone remaining two nAChR subunits cDNA(Lda3 and Ld?1)from L.decemlineata and analyze the 5' flanking region sequences activities of Ld?1,Lda3 and Ld?1 by dual-luciferase reporter assay to reveal the expression regulation mechanism of nAChR in different development stages of L.decemlineata.The result explain the relationship between expression level of nAChR genes and insect susceptibility to insecticide.The results of this study are as follows:1.Cloning,sequence analysis and expression profiling of Lda3 and Ld?1In order to clarify the molecular structure and function of nAChR from CPB,the remaining two nAChR subunit genes were cloned and spliced by RT-PCR on basis of genome database of L.decemlineata.The genes were 1853 and 1828 bp,with an open reading frame(ORF)of 1674 and 1566 bp encoding predicted nAChR proteins of 558 and 522 amino acids respectively.They were named as Ld?3 and Ld?1(GenBank accession No.KP339869 and KP339870 respectively)according to their amino acid homology to corresponding subunits of Tribolium castaneum and other insects.The amino acid sequences of Ld?1 and Ld?3 had typical structures of insect nAChR.Phylogenetic analysis showed that Ld?1 and Ld?3 also had the nearest genetic relationship with the corresponding genes from T.castaneum.Quantitative real-time PCR was employed to investigate Ld?1 and Ld?3 genes expression levels during different developmental stages(egg,1-4 instar larvae,pupa and adult)and body parts in adults(head,thorax and abdomen).The results showed that Ld?1 and Lda3 were expressed in all developmental stages and body parts.The highest expression of Ld?1 was detectived in 1st and 2nd instar larvae,the lowest expression in egg,3rd,pupa and adult.The highest expression of Lda3 was detectived in egg,1st and 2nd instar larvae,the lowest expression in 4th instar larvae.The expression levels of Ld?3 and Ld?1 were significantly higher in head than in thorax and abdomen.The results established the foundation for further study of nAChR function and neonicotinoid insecticides resistance in L.decemlineata.2 Cloning and analysis on 5'flanking regions of Ldal,Lda3 and Ld?1Previous studies indicated that Ld?1,Ld?3 and Ld?1 had significant different expression levels between adults and 4th instar larvae,which affected their sensitivity to neonicotinoids.In this study,the molecular structures of Ldal,Lda3 and Ld?1 5'untranslated regions(5'UTR)and 5' flanking regions were analyzed to clarify the expression regulation mechanism of genes at the level of transcriptional regulation.The UTR sequences of Ldal,Lda3 and Ld?1 were obtained,consisted of 141,179 and 262 bp with 14314,74614bp and no intron inserted in 133,134 bp region from start codon upstream respectively.The obtained fragments of 5'flanking regions of Ld?1,Ld?3 and Ld?1 cloned by PCR were 1169bp,1606bp and 1522bp respectively.The transcription start sites of three genes were predicted on the website BDGP,and cis-acting elements were analyzed through website of gene regulation(http://www.gene-regulation.com/pub/progra-mms.html#match).The results may provide clues for further study on transcriptional regulation mechanism of the three genes.3 Dual-luciferase activity analysis of the three nAChR genes core promotersIn order to determine the exact sites of acting elements of Ldal,Lda3 and Ld?1 promoters from L.decemlineata adults and larvae,the activity of three gene promoters were evaluted by generating series of deletion constructs from 5' flanking sequence of Ldal,Lda3 and Ld?1 using dual luciferase reporter gene technology.The results showed that there was promoter activity in Ld?1 5'flanking sequences and core promoter region was located in the region from-167 to-199 bp.There may be cis-acting elements in the region of Ld ? 1 flanking between-405 to-804bp enhancing larva promoter activity,whereas transcription factor AP-1 located in the region from-308 to-340 bp inhibiting larva promoter activity.Nucleotide polymorphism of Ld ?1 regulation region were found between adults and larvae,which may result in the different expression level of the gene iamong them.The deletion constructs of 5'flanking region of Ld?3 and Ld?1 showed very low promoter activity and no core promoters were found.This study identified a number of cis-acting elements sites in Ld ?1 promoter,and the nucleotide polymorphisms of Ld ?1 regulatory sequences may be associated with the mRNA expression differences between adults and 4th instar larvae.The results lay foundation for further study on the regulatory mechanism of insect nAChR expression and resistance mechanisms of neonicotinoid insecticides in L.decemlineata.