Comparative Research Of Two Types Of AChE From Bombyx And Their Differences With The Silkworm Maggot AChE

Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme of the cholinergic system because it regulates the level of acetylcholine (ACh) and ter minates nerve impulses by catalyzing the hydrolysis of the neurotransmitter ACh in the synaptic cleft. The enzyme is encoded by the ace gene, and it represents the primary target of insecticides such as organophosphorous (OP) and carbamate (CB) insecticides. AChE coding sequences have been cloned so far from a range of evolutionarily diverse vertebrate and invertebrate species. Many insect species have been shown to have two different acetylcholinesterase genes. Although much work has been carried out on these genes, the precise functions of the two aces remain unanswered. This paper mainly includes the following research results:1 Polymorphism of ace in different silkworm strainsIn this study, we tested the LC50 values of chlorpyrifos and propoxur on 24 B. mori strains. The LC50 values of chlorpyrifos and propoxur on different silkworm strains were quite different. We cloned and sequenced aces from 31 silkworm strains. Sixty three and 13 amino acid mutations were found in Bmace1 and Bmace2, respectively. However, those mutations in aces are not likely involved in LC50 changes in the 24 strains of B. mori. The ace2 seems more conserved than ace1. The phylogenetic analysis based on the two types of aces nucleotide sequences from different strains showed two types of AChE were from independent origins.2 Realtime PCR and RNAi analysis of two types of AChE in Bombyx mori In this study, we analyzed the two types of ace's expression levels at different development stages of egg and larvae. We also compared the effects of dsRNA of Bmacel and Bmace2 on the development of silkworm. The results indicated that, at egg and lst-3rd instars of larvae, the mRNA levels of Bmace2 is higher than those of Bmacel, while at the 4th and 5th instars of larvae, the mRNA levels of Bmace2 is similar to those of Bmacel. At larva stage, the mRNA levels of Bmace2 decreased as the instar increased. The RNA interference experiments showed that feeding Bmacel or Bmace2 dsRNA reduced the expression of Bmacel or Bmace2, respectively. And the development of silkworm was partially blocked by the dsRNAs.3 The catalytic properties comparison and inhibition kinetics analysis of two types of AChE from Bombyx mandarinaOur research indicated that the amino acid identity between the two forms of lepidopteran AChEs is lower than 40%, and potential differences in the catalytic properties and inhibition kinetics of these two enzymes remain unknown. cDNAs encoding two different AChEs (Boma-AChEl and Boma-AChE2) from the wild silkworm, Bombyx mandarina, were cloned and expressed in insect cells to compare their enzyme properties and reactions with different OP and CB inhibitors in vitro. Expression of the two enzymes in insect cells was highly efficient. Recombinant Boma-AChEl and Boma-AChE2 enzymes had high specific activities for acetylthiocholine iodide, with a Vmax of 476.2,250×10-9mol/mg protein/min, and Km values of 29.2 and 53.9μmol, respectively. For both of the enzymes, the efficiency of hydrolysis for three kinds of substrates as indicated by Vmax values were ATC>PTC>BTC, and they were inhibited by high concentration of substrate. Inhibition assays results indicated that BW284c51, Eserine, iso-OMPA, and in 8 of the 9 insecticides tested, Boma-AChE1 was more sensitive to most of the inhibitors and insecticides than Boma-AChE2. The bimolecular reaction constant (Ki) of Boma-AChE1 was 1.3-10.56 times higher than that of Boma-AChE2, while the median inhibition concentrations (I50) of Boma-AChEl were only 0.01-0.80-fold those of Boma-AChE2. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos; the Ki of Boma-AChE1 was 0.08 fold as that of Boma-AChE2, while the I50 of Boma-AChEl was 21.54-fold higher than that of Boma-AChE2.4 Cloning, expression, purification, and inhibition analysis of an AChE from the silkworm maggot, Exorista sorbillansIn this study, we have isolated the cDNA encoding an AChE from the field Exorista sorbillans (Es). The predicted protein is 701 amino acids in length. One point mutation (Gly353Ala, Es-353G AChE, Es-353A AChE) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus-insect cell system, respectively. The enzyme properties analysis showed that the apparent Km of Es-353G AChE and Es-353A AChE for three substrates ATC, BTC, and PTC have no distinct differences, while the vmax of Es-353A AChE and the purified natural AChE were slightly higher than those of Es-353G AChE for the three substrates. The inhibition results showed that for eserine, dichlorvos, durshan and omethoate, Es-353A AChE was less sensitive than Es-353G AChE, hence, the results proved that this mutation is associated with Es AChE sensitivity. Meanwhile, comparison of the median inhibition concentrations (I50) of eserine, dichlorvos, durshan, and omethoate of recombinant and natural Es AChE with its host (Bombyx mori) AChEs indicated that, both Es-353G AChE and Es-353A AChE are more sensitive than B. mori AChEs, this results gives an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori. 5 Expression of the housefly ace in a bioreactor and its potential application in the detection of pesticide residuesThe Acetylcholinesterase gene was cloned from a housefly (Musca domestica) strain susceptible to organophosphate (OP) and carbamate (CB) insecticides and was expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.2 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to an OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used in the detection of pesticide residues.