Degenerate Primers Polymerase Chain Reaction Clone And Identification Of The Acetylcholinesterase Gene Fragment From The Mosquito,Aedes Albopictus

AIM The development of insecticide resistance has long been the most serious problem in chemical control of vector. AChE is the major molecular target for organophosphate and carbamate insecticides which inhibit enzyme activity and kill the pests. Insensitive AChE is one of the major reason of the insecticide resistance to organophosphate and carbamate for DrosophiLa, Mosquito and other insccts.Several point mutation of the insensitive AChE gene (Ace) that associated with resistance in Drosophikz melanogaster and other insects are identified, but it has not been described in mosquitoes. Furthermore, other information about the molecular structural of Ace from mosquitoes is I not known, except the Anopheles Stephenst.. 4 In this study, The Ace fragment and its DNA sequence from the Mosquito, Aedes albopictus would be gotten by PCR and cloning. It would be beneficial to searching for the relation between mosquito acetylchotinesterase and insecticide resistance. METHOD 1. degenerate primers polymerase chain reaction The genome DNA which extracted from the Mosquito, Ae. albopictus were used for degenerate primers polymerase chain reaction (PCR) and the two pairs of oligonucLeotides encoding the highly conserved protein sequences according to the conserved amino acid sequence of Ace in D. melanogaster and An. step hensi were used as primers. 2. cloned and identified The reaction products were cloned to T-vector and transfected into E.co/i JM 109. The replicative form DNA of recombinant vector extracted from F. co/i JM 109 by the method of alkali lysis were identified by the methods of digested with restriction enzymes (EcoR I and Sal I) and PCR. 3. Sequence analysis Several positive clones were selected for sequencing. The deduced amino acid sequences were undertaken to homologous analysis by using the internet and pcgene soft. RESULT 1. The Ace fragment of Ae. albopictus was obtained by degenerate S primers polymerase chain reaction. 2. The clone which belongs to the Ace fragment of the Mosquito, Ae. albopictus was obtained by cloning and identification. 3. The DNA sequence of the Ace fragment of the Mosquito, Ae. alb~pictus was obtained. A conserved feature of the sequence of the Ae. albopictus is the six amino acid consensus sequence FGESAG surrounding the active site serine. This peptide motif is common to all vertebrate and invertebrate cholinesterase studied to date. 4. Compared with other organisms, the deduced amino acid sequences of Ae. aibopiclus AChE has a high degree of homology .with Ae. aegypli. The homology overall is 96%. It is 87% similar to An. stephensi and 69% similar to D. melamogaster. CONCLUSION It is the first time to describe the identified clone and the DNA sequence of the Ace fragment from Ae. albopictus in the world.