Clone And Functional Identification Of Deltamethrin Resistance-associated Chymotrypsin Gene Of Culex Pipiens Pallens

Insecticides have played an important role in the control of pests since the early 2 Oth century. Insecticides will remain a vital part in the integrated control programs of vectors for the foreseeable future. Unfortunately, the vector populations in some areas have developed remarkable resistance to eveiy classes of insecticide. Now it has been recognized that a change in the genetic composition of a population is a direct result of the selective effects of a toxicant. An understanding of the insecticide-resistant mechanisms can lead to better management of pest resistance to pesticides.In this paper rapid amplification of cDNA ends (RACE) was used to screen the constructed cDNA library and the full-length sequences of (3 -actin gene and chymotrypsin gene were gotten. Then LightCycler fluorescent quantification system, (3 -actin gene as control, was used to validate the different expression of chymotrypsin mRNA between deltamethrin-resistant and susceptible strains of Cx pipiens pallens.1. The acquirement and analysis of full-length (3 -actin cDNA of Cx. pipiens pallens.Two primers were designed according to the (3 -actin genes of Anopheles gambiae and Drosophila melanogaster from GenBank RACE were used to screen the constructed cDNA library and the 5' and 3' RACE fragments of the (3 -actin cDNA of Cx. pipiens pattern were gotten and cloned into pGEM-T vector ,then sequenced. After splicing, a sequence of 1290 base pairs including an open reading frame of 1132 basepairs was gotten (GenBank/NCBI AY 100005 ) . The deduced protein had 377 amino acids and the theoretic molecular weight and PI were respectively 41.7KD and 5.4. Aligned to Swissport, it shared 91% and 91% homology respectively with (3 -actin gene of An. gambiae and D. melanogaster. The (3 -actin gene will be used as the internal control in several experiments of Culex mosquitoes.2.The acquirement and analysis of full-length chyinotrypsin cDNA of Cx. pipiempallens.Two primers were designed according to the chymotiypsin gene fragment obtained from SSH. RACE were used to screen the constructed cDNA library and the 5' and 3' RACE fragments of the chymotrypsin cDNA of Cx. pipiens pallens were gotten and cloned into pGEM-T vector ,then sequenced. After splicing, a sequence of 977 base pairs including a open reading frame of 813 base pairs was gotten (GenBank/NCBI AF468495 ). The deduced protein with 271 amino acids shared 73% and 50 % homology respectively ,aligned with Aedes aegypti chymotiypsin and Glossina morsitans chymotrypsin With LightCycler fluorescent quantification system, we detected a higher expression of chymotiypsin mRNA in deltamethrin-resistant strains of Cx. pipiens pallens, indicating that the chymotrypsin gene was associated with the deltamethrin resistance of Cx. pipiens pallens. The chymotrypsin gene is very important for studying the molecular mechanisms of insect resistance to pesticides.