Resistance To Phoxim And CDNA Cloning Of Acetylcholinesterase In Panonychus Citri McGregor

The resistance mechanisms of Panonychus cirri (McGregor) to phoxim were investigated on the basis of resistance selection of the mite in laboratory experiment with a series of techniques such as bioassay, resistance selection, biochemical analysis, and molecular biology. This research would help to completely understand the physiological and biochemical mechanisms of acaricides resistance of the mite in the future, and also possess great practical significance in the formulation of strategies for the delay and management of resistance. In addition, a fragment of acetylcholinesterase (ACHE) from P. cirri was amplified with degenerate primers by reverse transcriptase-polymerase chain reaction (RT-PCR) method and it provided some basic information for the further study about the complete sequence of the gene and the molecular mechanism of altered ACHE. After about two years' work, the main research results are summarized as follows.1 Resistance selection ofP. citri to phoximThe results of resistance selection of P. citri to phoxim indicated that the resistance level gradually rose with the increase of selection time and the resistance factors at the fourteenth generation amounted to 18.6-fold. In the process of resistance selection, there was a decreased tendency about the slope (b) value of LD-p lines, suggesting that P. citri developed obvious resistance to phoxim and resistance would further develop if the selection continue.2 Resistance mechanisms ofP. citri to phoxim2.1 Relationship between AChE and the resistance of P. citri to phoximCompared to the susceptible strain, the activity and specific activity of AChE from the resistant strain increased significantly. The results also showed that ACHE in the resistant strain possessed a significantly less affinity to the substrate acetylthiocholine iodide (ATChI). For the catalytic activity of AChE towards ATChI, the V_max value of the resistant strain decreased significantly compared to that from susceptible strain.The in vitro inhibition kinetics of phoxim on AChE activity showed that there was a strong linear relationship between inhibiting action and AChE activity from the two strains. The results suggested that there is target resistance in P. citri to phoxim.2.2 Relationship between Carboxylesterase (CarE) and the resistance of P. citri to phoximThe results showed that the susceptible and resistant strains differed significantly in the amount of protein per individual, and higher activity and specific activity of Care from the resistant strain were observed. For the catalytic activity of CarE towards the substrate, the K_m value of the resistant strain decreased significantly compared to that from susceptible strain.The inhibition of phoxim to CarE activity showed that CarE in the susceptible strain was more sensitive and it suggested that the change of Care activity in the resistant strain was an important reason for the resistance of P. citri to phoxim.2.3 Relationship between phosphatases (ACP,ALP) and the resistance of P. cirri to phoximThe activity and specific activity of ACP from the resistant strain were significantly higher than its susceptible counterpart. Although the activity and specific activity of ALP in the resistant strain were higher, no significant difference was observed.2.4 Relationship between SOD, POD, or CAT and the resistance of P. citri to phoximCompared to those of the susceptible strain, the specific activity of both SOD and POD from the resistant strain increased significantly while there was no significant difference of the specific activity of CAT between the two strains. These suggested that the resistance of P. citri to phoxim related to SOD and POD, and maybe have no relation with CAT.2.5 Relationship between GSTs and the resistance of P. citri to phoximThe GSTs activities of the resistant and susceptible strains were 0.55μmol/min.mg/pro and 0.3751μmol/min.mg/pro, respectively, and the difference was significant. The Km value of the resistant strain was significantly greater than that of the susceptible strain if GSH was used as substrate, while it was significantly smaller if CDNB was used as substrate. These suggested the affinity of GSTs from the resistant strain to GSH was less than that from the susceptible strain. All these implied that there was some relationship between the resistance of P. citri to phoxim and GSTs. 2.6 Relationship between MFO and the resistance of P. citriThe activities, specific activity and Km of MFO in the resistant strain were significantly higher than those in the susceptible strain, and the inhibition of phoxim to MFO was significantly greater in the susceptible strain. There was a significantly linear relationship between the inhibition rate of MFO activity and the inhibition concentration of phoxim. However, the inhibition of PBO to MFO in the resistant strain was significantly higher than that in the susceptible strain. The results suggested that the development of resistance to phoxim in P. citri might be related to the rise of the MFO activity.3 cDNA cloning of AChE in P. cirriA 151bp cDNA fragment of AChE gene in P. citri was amplified with the degenerate primers from the conserved peptide sequences of AChEs in insect and acari species by reverse transcription-polymerase chain reaction (RT-PCR) method, and the sequence analysis indicated there was a high degree of amino acid sequence homology between P. cirri and other species. In addition, it included several characteristic sequences of ACHE. Hence, it could be concluded that the fragment encoded the AChE in P. citri.