Screening And Analyzing The Differentially Expressed Genes From Anopheles Stephensi Induced By Infection With Plasmodium Yoelii After 24 Hours

Objective:Malaria is the most important mosquito-borne infectious disease in the world. For a long time, drug prevention and treatment are the primary means of malaria control . Scientists now focus on the relationship between mosquito and malaria parasite, because of the development of parasite resistance to drugs and of vector resistance to insecticides, as well as the lack of an effective vaccines. In the future, transforming the malaria-transmitting mosquito into harmless tends to be a new malaria control strategy. Much of immune-responsive genes are activated to interfere the development of Plasmodium parasites in mosquito after infection. Therefore, to find these genes as a target of controlling malaria transmission is the hot research area.We established the Anopheles stephensi's infection model and constructed the bi-directional cDNA library of Anopheles stephensi after 24 hours'infection. From the library , we selected differentially expressed genes which were related to the development of Plasmodium parasites in Anopheles stephensi, then compared them with the sequences from other mosquito which has been published. Then more bioinformatical sofeware were used to annotate and predict these sequences which could not meet any similarities from NCBI.Methods:Suppression subtractive hybridization technology were used to build the infection of Anopheles stephensi's bi-directional cDNA library. The clones from the two libraries were sequenced and their sequence homologues in Genbank datebase were searched with blast by Internet. Then more bioinformatics software were used to analyze the sequences which could not meet any similarities from NCBI, such as: signal peptide prediction software,SignalPv3.0,protein predition software Targetp v1.1,non-classical secreted protein predition software,SecretomeP,transmembrane helix prediction sofeware TMHMMv2.0and ProtFun.Results:(1)54 sequences from forward cDNA library and 67 from the reverse library were got. (2)All these sequences were compared with sequences in NCBI/tblastx/ESTs. The blast search of these sequences revealed that 51 single homologous sequences could be found in the two library. But there were no homology sequences with the rest of 66. (3)The results of these 66 sequences showed that 20 sequences contain the signal peptide region; only 1 sequence might be non-classical secreted protein; 11 sequences belong to secretory pathway signal peptide; 27 sequences belong to mitochondrial target site-peptide;and 10 sequences contain both signal peptide and transmembrane helix structure. The ProtFun showed that most sequences were involved in Amino acid biosynthesis, Energy metabolism and Translation. (4)Gene Ontology category showed that these sequences might be Growth_factor, Receptor, Ion_channel, Transcription_regulation, Structural_protein and Immune response.Conclusions:(1)We successfully construct the bi-directional cDNA library of Anopheles stephensi after 24 hours'infection. (2)The ProtFun result of the 66 sequences which could not meet any similarities from NCBI shows that the majority of these sequences might be Growth_factor, Receptor, Ion_channel, Transcription_regulation, Structural_protein and Immune response.