Cloning,Expression,and Functional Analysis Of Two Acetylcholinesterase Genes In Spodoptera Litura

Spodoptera litura,commonly known as common cutworm,which considered the most important pest that causes widespread economic damage to vegetables and ornamental plants.The function of acetylcholinesterase(AChE,EC3.1.1.7)ends synaptic transmissions by hydrolyzing the neurotransmitter acetylcholine(ACh)in all animals.Organophosphate(OP)and carbamate(CB)insecticides primarily target AChE genes.Management challenges of this insect include some features such as wide host range,capability to migrate for longdistance,numerous generations,higher fecundity,and highly resistance to pesticides makes it very difficult to control.At the molecular level,insect management requires a high comprehending of the essential neurotransmitter genes functions.Formerly,a molecular investigation,particularly quantitative real-time polymerase chain reaction method was extremely utilized for the gene expression in S.litura.The aims of the recent research were to investigate two Ace genes of the cutworm S.litura,focusing on the cloning,phylogenies,and determination of the transcript level of SlAce1 and SlAce2 in different developmental stages and tissues.Moreover,their expression patterns were analyzed,and biological functions were compared using RNA interference.The main results are as follows:Herein,two acetylcholinesterase genes(SlAce1 and SlAce2)were identified in S.litura.Our results showed that SlAce1 and SlAce2 cDNA contains 2085 bp and 1917 bp nucleotides encoding proteins of 694 and 638 amino acid residues,respectively.The SlAce1 and SlAce2 both have characteristic amino acids,including the catalytic triad,anionic choline-binding site,an oxyanion hole,acyl pocket,peripheral anionic subsite,the characteristic “FGESAG” motif surrounding the active serine residue,and the conserved 14 aromatic amino acids in the lining of the catalytic gorge.The SlAce1 model has an active site that is almost identical to those of A.gambiae and S.graminum APAChEs,and it has Cys432 at the opening of the active-site gorge.Furthermore,the deduced amino acid sequence of SlAce1 showed the highest homology to Helicoverpa assulta(87%)and from 65-69% to other insects.Deduced amino acid sequences of SlAce2 exhibited high homologies to ace2-type genes from Helicoverpa assulta(97%),and from 49-78 to other insects.Phylogenic analysis indicated that the lineage of SlAce genes was collected in two major clusters(AChE1 and AChE2),and SlAce1 was completely different from SlAce2.SlAce1 is similar to proteins from Helicoverpa assulta(HaAce1)and Bombyx mandarina(BmAce1)while Ace2 was more closely associated with the proteins of Bombyx mandarina(BmAce2)and Bemisia tabaci(BtAce2).qRT-PCR analyses indicated that both SlAce genes were expressed in all developmental stages and tissues,but a higher expression was observed in the brain.The expression levels of the two SlAce genes were suppressed by inserting their related dsRNA in the 6th instar larvae,which led to 47.3%(SlAce1)and 37.9%(SlAce2)mortality.Interestingly,the suppression of the SlAce2 transcripts led to significant reductions in the fecundity,hatching,and offspring in the parental generation of S.litura.When male and female coupled in case of control,100 % egg laying and 90% hatching were observed under control conditions.However,when dsSlAce2 injected to both male and female,found 27.9%,36.5%,0.94% and 7.54% egg laying,hatching,pupae and adults,respectively.While dsGFP(male)and dsSlAce2(female)injected to couples,we found that 47.5%,40.5%,1.3% and 10%,egg laying,hatching,pupae and adults,respectively.However,for male and female treated with dsSlAce2 and dsGFP resulted in 88.6% egg laying and 88.4% egg hatching.It is concluded that SlAce2 is responsible for the hydrolysis of acetylcholine and non-cholinergic function in female breeding,embryo progress,and the development of progeny,whereas SlAce1 may have an additional role in the non-cholinergic function.Considerable larval mortality was observed after AChE genes were silenced in S.litura confirms its insecticidal effectiveness,which provided a molecular basis in biological pest control approach.