MOLECULAR CHARECTERIZATION OF THE MIDDLE PART AND 5′-END OF THE SEMAPHORINE (SEMA-1A) OF ANOPHELES STEPHENSI AS A NOVEL TARGET FOR VECTOR CONTROL

Background: Malaria is the deadliest infectious diseases in the world. Therefore, it has been considered as one of the priorities of world health organization (WHO). Despite ongoing efforts to battle the disease by using vaccines, various insecticides, developing new drugs and treatment, the crisis continues to worsen. Vector control is introduced by WHO as an effective strategy for eradicating Malaria. Previous studies have shown that the genetic manipulation of insect vectors is a potential method for controlling the arthropod-borne diseases. Semaphorine (sema1a) is a member of the family of axon guidance molecules which is expressed during developing central nervous system (CNS). sema1a is essential for proper developing of the visual and mosquito’s olfactory systems. Hence, sema1a was candidated as a novel target for vector control. In this investigation, the middle part and the 5´-end of sema1a of An. stephensi was identified and characterized to provide the basic required information for transgenic and paratransgenic studies.Objectives: Identification and characterization of the middle part and 5´-end of sema1a gene of Anopheles stephensiMaterials and Methods: First, full length of sema1a of the Aedes aegypti and Anopheles gambiae were aligned by MEGA 10.0 software. Then, the conserved regions were selected to design primers for determining of the middle part region in An. stephensi. Total RNA was extracted from mosquitos and cDNA was synthetizes by the outermost specific primer. Next, the middle part of the gene was amplified by combination of the deigned primers. In the next step, to achieve the 5-´end of the gene, genome walking method was used. Single stranded DNA (ssDNA) molecules were synthesized by the outermost gene specific primer in the firststep with regard to the 5′-end of the middle part sequence. Then, Genome Walking primers (GWPs) (A-G) were used for dsDNA production in seven separate reactions. Next, UAP-N1 and UAP-N2 primer pair combinations were used for the nest-1 and nest-2 reactions, respectively. Finally, the related amplicons with the favorite lengths were TA-cloned in pTG19 and sequenced.Results: The middle part of sema1a with 700bp length and 5´-end with length 951bp were sequenced and it was acknowledged by BLAST tool and submitted to GenBank: 1902170 and GenBank: 2038372 respectively.Conclusion: According to the role of sema1a during vector mosquito embryonic ventral nerve cord development and previous results on genetic manipulating of sema1a, it can be considered as a new potential and effective genetic target in order to eliminate and eradicate malaria. Our results provide the fundamental information for full length sequence identification of sema1a and intervene to development of Anopheles setephensi via knock downing the function of gene.

Keywords:

Malaria , semaphorin , Anopheles stephensi , vector control