Striped Flea Beetle Acetylcholinesterase Purification And Cloning Of The Gene Encoding The Cdna Fragment,

Acetylcholinesterase (AChE, EC3. 1.1.7) played an important role in the function of nerve impulse transmission and was the target sit of organophosphorus and carbamate insecticides. Reducing AChE sensitivity was one of the key mechanism of insect pests resistance to organophosphorus and carbamate insecticides. Purification and cDNA cloning were helpful to further studies on AchE, and were good to insecticides rational design and application. As one of destructive insect pests, the resistance to Phyllotreta striolata (F.) insecticides was attracted more and more concern. However, there were no information about purification and cDNA cloning of P. striolata AchE. Reported the comparison of three purification methods in this paper, CEA was selected for AChE purification. The biochemical characteristics were also studied. In addition, RT-PCR was used to clone the AchE partial cDNA. cDNA sequence and its homologous analysis were dicussed subsequently in the paper.The results showed that the CEA affinity chromatography was the best method which accounted to the purification fold of 651.20 and yield of 41.80%. The purification fold and yield by the method of sephadex G-200 chromatography procedure were 12.60 and 48.50% respectively, and by ammonium sulfate method were 1.50 and 62.74% respectively. The separated fraction was detected by SDS-PAGE, and corresponded to molecular weights of 45kD and 37kD. Properties of crude extract and purified AChE were studied. The results showed the optimal temperature, pH and the substrate of ATChI of purified AChE were 37 C, 7.0 and 2500 M respectively. The crude extract conducted differently to varied temperature , pH and ATChI. Investigation revealed that crude extract and purified AChE had different characteristics. We infered that one or more factors in the crude extract could influence AChE activity.With RT-PCR, the partial cDNA sequence of AChE gene in the P. striolata was cloned and analyzed. Using a pair of primers based on the conserved gene sequence of AChE gene, the fragment of 750bp was amplified. Homologous analysis revealed that there was high homology degree of amino acid sequence between the P. striolata and other insects.