EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP‐ or CB‐resistant strains have been generated. In this study, wild‐type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21‐fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.     

Functional study on the mutations in the silkworm (Bombyx mori) acetylcholinesterase type 1 gene (ace1) and its recombinant proteins

The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1’s characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 μM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 μM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE’s roles in pesticide resistance.     

杆状病毒凋亡抑制基因IAP1促进家蚕CyclinB的核内积累

[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

Pharmacogenetic Regulation of Acetylcholinesterase Activity in Drosophila Reveals the Regulatory Mechanisms of AChE Inhibitors in Synaptic Plasticity

We conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer’s disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. We found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. Our study suggests that exposure to AChE inhibitors for 6–12 months may induce altered synaptic architectures in human brains with Alzheimer’s diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.

     

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down‐regulation of BmPP2A expression. Furthermore, up‐regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down‐regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti‐BmNPV mechanism, and a possible target for the breeding of silkworm‐resistant strains.     

Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

Expression of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endo-β-glucanase II in silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> L. by using BmNPV/Bac-to-Bac expression system and its bioactivity assay

The silkworm, Bombyx mori, was used to produce recombinant endo-β-glucanase II (rEGII). The EGII gene (egl2) was cloned from the cellulolytic fungus Trichoderma reesei and inserted into B. mori nucleopolyhedrovirus (BmNPV) genome using BmNPV/Bac-to-Bac expression vector. For expression of rEGII, both the BmN cells and B. mori larvae were infected with the recombinant virus. The putative rEGII yield was about 386 μg per larva and the enzyme activity of the purified rEGII was approx 352 U/mg of rEGII. The optimal activity of this purified protein was observed at 55°C and pH 4, respectively.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis (IAP) and nucleopolyhedrovirus IAPs

As an important insect immune response, apoptosis plays a critical role in the interaction between baculoviruses and insect hosts. Previous reports have identified inhibitor of apoptosis (IAP) proteins in both insects and baculoviruses, but the relationship between these proteins is still not clearly understood. Here, we found that insect IAP proteins were clustered with baculovirus IAP3, suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera. We demonstrated that Bombyx mori inhibitor of apoptosis (Bmiap) gene had an inhibitory effect on apoptosis in silkworm cells. Further analysis of the effects of Bmiap genes on the proliferation of B. mori nucleopolyhedrovirus (BmNPV) showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells. Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells, implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection. Taken together, our results provide important insights into the functional relationships of iap genes, and improve our knowledge of apoptosis in baculoviruses and insect hosts.     

Effects of overexpression and inhibited expression of thymosin,an actin‐interacting protein from Bombyx mori,on BmNPV proliferation and replication

Previous study showed that exogenously applied recombinant thymosin from Bombyx mori (BmTHY) reduces B. mori nucleopolyhedrovirus (BmNPV) proliferation in silkworm. Which stands to reason that BmTHY in B. mori is crucial for the defense against BmNPV. However, little is known about the effect of endogenously overexpressed or repressed BmTHY on B. mori resistance to virus infection. To study this issue, we constructed an overexpression and inhibited expression systems of BmTHY in BmN cells. The viral titer and the analysis from the quantitative real‐time polymerase chain reaction (PCR) revealed that overexpression of BmTHY decreased the copies of BmNPV gene gp41, which goes over to inhibit the proliferation of BmNPV in BmN cells, while the inhibited expression of BmTHY significantly enhanced viral proliferation in infected BmN cells. These results indicated that endogenous BmTHY can inhibit BmNPV proliferation and replication in infected BmN cells. Furthermore, Co‐IP showed that BmTHY could bind to actin in BmN cells. Also, the overexpression or inhibited expression of BmTHY shifted the ratio of F/G‐actin in infected BmN cells. Lastly, the BmTHY, an actin‐interacting protein, might be one of the key host factors against BmNPV, which inhibits viral proliferation and replication in BmN cells.     

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.     

Comparative analysis of the genomes of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> and <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviruses

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.     

Invertebrate acetylcholinesterases: Insights into their evolution and non-classical functions

Acetylcholinesterase (AChE) plays a pivotal role in synaptic transmission by hydrolyzing the neurotransmitter acetylcholine. In addition to the classical function of AChE in synaptic transmission, various non-classical functions have been elucidated. Unlike vertebrates possessing a single AChE gene (ace), invertebrates (nematodes, arachnids, and insects) have multiple ace loci, encoding diverse AChEs with a range of different functions. In the field of toxicology, AChE with synaptic function has long been exploited as the target of organophosphorus and cabarmate pesticides to control invertebrate pests for the past several decades. However, many aspects of the evolution and non-classical roles of invertebrate AChEs are still unclear. Although currently available information on invertebrate AChEs is fragmented, we reviewed the recent findings on their evolutionary status, molecular/biochemical properties, and deduced non-classical (non-neuronal) functions.     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.     

Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, V_max and K_m, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in K_i ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.