Study The Genetic Gene Character With Anopheles Spreading The Malaria And Cloning And Sequencing Of Antibacterial Peptide

Objective To analyze the genomic DNA polymorphism of Anopheles dims and Anopheles stephensi of different susceptible to Plasmodium yoelii, explore the relationship of the genomic DNA between Anopheles and Plasmodium. Furthermore, to study innate immune of Anophele dims against exogenous antigen and clone the defensins genes from Anophele dims and conduct sequence analysis.Methods The genomic DNA extracted fiom the different stages of An. dims and An. stephensi were amplified by random amplified polymorphic DNA (RAPD) with 5 random primers, and then amplified products were carried on agarose gel electrophoresis. Moreover, the genomic DNA and total RNA were extracted from An. ditus infected Plasmodium yoelii and bacteria, respectively. The cDNA in An. dims was acquired by Reverse transcriptase-polymermed chain reaction (RT-PCR). PCR and RT-PCR were followed with 2 pairs of primers designed and synthesized on the basis of reported defensins gene sequence of Anopheles gambiae. The product of PCR were purfied and the target fragments were cloned into the compatible sites of the T -vector pMD-18T, and then transferred into the Escherirchia coli DH5 a. Finaly, the genetically engineered bacteria was sequenced and analyzed.Results The genomic DNA of An. dirus and An. stephensi were amplified by RAPD with 5 different primers. The primer P5 was better than others to An. Stephensi, however, the primer P4 was better than others to An. dims. No bands were obtained to An. dims with the primer P1.An. dims and An. stephensi were then amplified with primer P3, the bands quantity and their types were the same, however, their product was difference in normal and infected mosquito. The different bands quantity was expressed in adult mosquito and larva Furthermore, the targeted products were about 219 bp from Anophele dims infected Plasmodium yoelii with degeneracy primer, it had homology with 95% compared with the defensins sequence from Aedes aegypti. 308 bp targeted fragment had homology with 100% compared with the defensins sequence from An.gambiae, which was amplified fiom the genomic DNA with specific primer. 333 bp targeted fragmen from total RNA of the An. dirus infected by bacteria with specific primer. It had a high homology with the defensins sequence from An. gambiae. 188bp was obtained of the An. dims infected by bacteria with degeneracy primer. It had homology with 87% compared with the defensins sequence from An. gambiae and 88% compared with the defensins D of Aedes albopictus.