Construction the rnc mutant strain of Asaia the symbiont of An. Stephensi for producing the dsRNA to fight against Malaria

Background: Malaria is the most important protozoan infectious disease that in humans caused by the genus of Plasmodium. World Health Organization (WHO) in 2011 requested the scientists to advise their scientific solutions to reach the global malaria eradication till 2050. One of the scientific groups was vector control consultative group. Those recommended different strategies to restrict the role of mosquitoes in malaria emergence and transmission which of them was paratransgenesis. Paratransgenesis is using the different symbiotic microorganisms to inhibit pathogen development in the vector or affect the vector fitness. Recently, using the bacterial produced dsRNA has been introduced for gene knock down in insect. Furthermore, it has been revealed that RNase III mutant bacteria are more efficient for dsRNA production. With regard that Asaia is a potent paratransgenesis candidate against malaria, we decided to use homologous recombination to for knock-out rnc gene in this bacterium. Objectives: using the homologous recombination to for knock-out rnc gene in Asaia spp.Materials and Methods: Asaia bacteria were isolated from the larval and adult’s midgut of An. stephensi. For molecular characterization of the rnc gene the conventional PCR used to amplify the middle part of the gene and the genome walking (GW) method was performed to characterization of upstream and downstream of the gene. Then, with help of homologous arms (HAs) the site directed recombination was performed by Lambda Red Recombination system.Results: Full length sequence of the rnc gene was achieved by using the genome walking method. Characterization of this gene is fundamental step to create the RNase III mutant bacteria as a suitable strain for construction of dsRNA to target different 2 nd International Congress of Vector-Borne Diseases & Climate Change; 4 th National Congress of Medical Entomology. Shiraz University of Medical Sciences, Shiraz, IRAN; Nov. 13-15, 2019 gene in the vector based on RNAi mechanism. The recombination system was capable to knock-out the rnc gene of Asaia spp.Conclusion: As mentioned, RNase III mutant strains are more efficient in dsRNA production. Therefore, by performing this study, the rnc mutant strain of Asaia was achieved. This step is very important to producing the large scale dsRNA substrates to targeting different genes in the vector orparasite life cycle. Keywords: Asaia, Malaria, paratransgenesis, Anopheles, Rnase III, RNAi, dsRNA production, Vector control

کلیدواژه ها:

Asaia ، Malaria ، paratransgenesis ، Anopheles ، Rnase III ، RNAi ، dsRNA production ، Vector control