Study On Structure And Function Of Acetylcholinesterase Type1Gene (ace1) From Silkworm (Bombyx Mori)

Acetylcholinesterase (AChE, EC3.1.1.7) is a kind of serine hydrolytic enzyme, located at thejunction place of neurons and neuromuscular. Acetylcholinesterase can maintain the normaltransmission of neural impulses in synaptic clefts through catalyzing the hydrolysis of neurotransmitteracetylcholine. AChE is a target enzyme for organophosphorus and carbamate pesticides. Thesepesticides can bind to AChE and reduce its activity, leading to massive accumulation of postsynapticmembrane acetylcholine, continuous stimulation, biological convulsion, and insect death. Meanwhile,AChE is related to cell apoptosis.To study the relationship between structure and function of Bombyx mori AChE, two ace’stranscriptional level of two types of AChE was compared after induced by the pesticide, phoxim.Furthermore, the ORF of ace1was expressed by prokaryotic expression system, and rabbit polyclonalantibody was successfully prepared. AChE1protein expression level in brain tissue after phoximstimulation was detected by Western blot. After that, a mutant (mace1) for three amino acids usingsite-directed mutagenesis was generated. The Baculovirus expression system was used for theexpression of the wild type ace1(wace1) and mace1. The expression products were purified todetermine the AChE activity and the inhibitory effects of physostigmine and phoxim. We used theDGE(Digital Gene Expression Tag Profiling) to measure the transcription level of the ace genes andtheir regulation related genes in the brain of B. mori by quantitative PCR. The main results are asfollows:1Transcriptional characteristics of ace in B. mori induced by phoxim.The Real-time qPCR method was used to analyze ace1, and ace2transcriptional level in the B.mori larvae tissues (midgut, silk gland, fat body, Malpighian tubules, hemolymph, testis, ovary, andbrain) and the transcription characteristics by the phoxim induced (4.0μg/mL). The results showed thatace1was highly expressed in brain tissue, and had weak expression in other tissues,with the levels ofonly0.21%-3.68%to normal brain tissue. In normal tissues, there was a relatively lower expressionlevels of ace2gene in the midgut, silk gland, and hemolymph, but a relative higher expression level inthe fat body and the ovarian. Comparing the two ace transcriptional levels, we found that, ace1 expression in various tissues were higher than ace2. The average expression level of ace1is13.50timeshigher to ace2. Specifically, the expression level of ace1was95.97times higher to ace2in the braintissue. Quantitative analysis of the ace expression level at different time points after induced by phoximin brain tissue showed that, ace1and ace2expressions appeared in the same trend: decreased firstly andthen increased. The changes of ace1were greater than ace2. Pesticide-induced ace2expression invarious tissues was relatively stable. These results indicated that ace1was the major AChE gene, and itwas more sensitive to phoxim. These studies lay the foundation for two aces in B. mori physiologicalprocesses.2Cloning and antibody preparation of ace1In order to further study ace1function, ace1ORF was cloned into the expression vector pET28a，and then the vector was transformed into the competent cells of E. coli BL21. The recombinant proteinwas prokaryotic expressed by0.1mmol/L IPTG induced. SDS-PAGE electrophoresis analysis showedthat the transformed ace1recombinant vector in E. coli BL21had a specific band at approximately76kDa size, while in the control groups (BL21bacteria and transformed with the vector pET28a) did nothave this specific bands at the same position. Western blottting analysis showed that6×His tag fusionprotein has been successfully expressed. These results illustrated that the ace1was correctly expressedin E. coli. Then a rabbit polyclonal antibody was successfully prepared by immunizing rabbits with thepurified protein. The titer of antibody was1:512000. These results lay the foundation for the futurefunctional studies.3The expression level of AChE1in B. mori exposed to phoxim.In order to further study the protein expression levels of ace1, we detected the expression levels ofthe AChE1in B. mori brain induced by organophosphorus pesticide, Phoxim. By western blot assay, theresults proved that AChE1expression induced for24h was increased to1.5fold, expression at48hshowed a reduced trend, and protein expression levels at72h rised again. These results are consistentwith the result by qPCR. It indicated that AChE1played an important role in the B. mori phoximresistance.4Site-Directed Mutagenesis of Bm-ace1and eukaryotic expression of the AChE1To study the relationship between mutations in ace1feature sites and insecticide resistance of the B.mori, by site-directed mutagenesis method, a mutant of three amino acids at the conserved sites in theAChE was generated, with mutation sites of Ala (GCG)303Ser (TCG), Gly (GGA)329Ala (GCA), Leu(TCT)554Ser (TTC). The Baculovirus expression system was used for the eukaryotic expression of thewild type ace1(wace1) and the mutant ace1(mace1). First, the gene s (wace1and mace1) weresubcloned into the transfer vector pFastBacTMHT B, and then transformed into the Ac DH10Bac E coli strain to give the transfer plasmid Bacmid, finally transfected Sf9cells to produce the recombinantprotein. The sequences were confirmed. The result laid the foundation for the structure and function ofgenes ace1. SDS-PAGE and Western blotting were used to detect the targeting proteins with expectedsize of about76kDa. These results showed that target protein was correctly expressed.5Expression product purification and biochemical properties determination.We purified the expression products of wild-type the AChE (wAChE) and mutation of AChE(mAChE) in the baculovirus expression system using Ni-NTA purification system. The products wereused for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. Nosignificant differences were observed in the overall activity of the wild type and mutant AChEs. TheKms of wAChE and mAChE were0.0305mM and0.0284mM, respectively. The Vmax values were0.731and0.743μM min1mg1, respectively. However, the remaining activity of wAChE wassignificantly lower than that of mAChE after physostigmine and phoxim inhibition. These resultsindicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine andphoxim, which laid the foundation for future in vivo studies onAChE’s roles in pesticide resistance.6Using DGE technology to detect gene expression in brain tissue induced by phoxim.We used DGE technology to study the changes of gene expression in brain tissue induced byphoxim (4.0μg/mL)48h. We analyzed influence of phoxim pesticide on the B. mori brain tissue fromthe nerve conduction, apoptosis and so on. The method of DGE was used to sequence control group(CK), add poison group (OP) two library, which produced3588285and379576clean tags,respectively. The type of clean tags were67679, and59278, respectively. Genomic alignment showedthat previous tag types were12173and10116, respectively, countting for17.99%and17.07%of thetotal clean tag types. There were315differentially expressed genes, which including74up-regulatedgenes, and241down-regulated genes. Expression of ace1showed a down trend. There was aup-regulation in mitochondrial oxidative phosphorylation related gene expression, and adown-regulation in DNA damage repair, base synthesis and metabolism related genes. Transmissionelectron microscopy (sem) results showed that phoxim caused mitochondria swelling, after induction invacuolated, chromatin agglomeration. This lay the foundation for our further study of B. mori and otherlepidoptera insect metabolism, and molecular mechanism of organophosphorus pesticide.7The AChE regulation related gene transcription characteristics by phoxim inducedIn order to study relationship between phoxim and Bm-ace1, we used q RT-PCR method todetermine the transcription level of the five genes related to AChE regulation in B. mori brain afterinducted by phoxim: cJun-N-terminal kinase, activating transcription factor2, calpain protein,calcineurin A and calcineurin B. The results showed that the expression of cJun-N-terminal kinase and activating transcription factor2rised with the passage of time after induced by phoxim. cJun-N-terminal kinase showed an obvious increase at the48h. Atf2expression levels increased fastest at72h.Calpain protein, calcineurin A and calcineurin B expressions increased firstly and then decreased. Theseresults provided a reference for the elaboration of the mechanism of apoptosis, insecticide choice andinsecticidal mechanism’s clarifies.This study proved an important role of ace1phoxim after induction from the gene transcriptionlevel and protein expression levels for the first time. Referring to the mutations of Lepidopteradrug-resistant pests AChE, ace1was mutated by site-directed mutagenesis. We found a decline in thesensitivity of the product of mutation to specific inhibitors. These results provided an importantbreeding material for genetically modified to cultivate the new varieties resistant of B. mori. Meanwhile,the gene expression profile of the B. mori brain tissue were studied after phoxim induction.Thetranscription characteristics of pesticide metabolism related gene were analyzed. The transcriptionalcharacteristics of Ace1transcription-regulated genes were analyzed. These studies lay the foundation forthe regulation mechanisms of the B. mori ace1expression.