| Much of immune-responsive genes were found to be activated by the invading and developing of Plasmodium parasites in mosquitoes. Therefore, to find a new candidate molecule as a target of controlling malaria transmission is the hot research area. However, most of the researches were mainly focused on Anopheles gambiae.Anopheles sinensis is a major vector with a vast distribution and big population in China. Although its vectorial capacity is lower than A. lesteri (an important malaria transmiting vector), it still confers to much malaria transmissions in vivax-malaria endemic areas. In this study, we first constructed the bi-directional subtractive cDNA library, from which, we selected differentially expressed genes which might inhibiting or promoting the development of Plasmodium parasites in A.sinensis mosquito,and compare them with the sequences from other mosquito organisms which has been published, then predict their function. The results might provide data for further researches to find candidate targets used in mosquito controls.Suppression subtractive hybridization (SSH) technology was used to construct bi-directional subtractive cDNA library between infected mosquito group (24 hours after P.yoelii-infected blood-feeding) and control group (24 hours after non-infected blood-feeding). For constructing forward cDNA library 1, we used infected-mosquito group as tester and control group as driver. Theoretically, it represents the up-regulateted expressed cDNA library. And the reverse cDNA library 2 represent the down-regulated expressed cDNA library. Fifteen single sequences from library 1 and eighteen from library 2 were sequenced. All these sequences were compared with sequences in the NCBI/tblastx/ESTs,NCBI/blastx and NCBI/blastn database. Nineteen single homologous sequences could be found in the two library. But other fourteen sequences could not meet any similarities from the databases. Then more bioinformatical softwares were introduced to annotate and predict the sequences, including the signal peptide prediction software, Signal Pv3.0, transmembrane helix prediction software TMHMMv2.0, SecretomeP, protein targeting prediction software targetp v1.1 and non-classical secreted protein prediction software. The preliminary results showed that 4 sequences contain the signal peptide region,6 contain the transmembrane helix structure, and 2contain both signal peptide region and transmembrane helix structure. Twelve sequences might be non-classical secreted protein; 3 sequences belong to mitochondrial target site-peptide (mitochondrial targeting peptide, mTP), and 5 sequences belong to secretion pathway signal peptide (secretory pathway signal peptide, SP). In addition, ProtFun 2.2 predictions software were used for functional annotation. The results showed that the majority of sequences were involved in protein translation, energy metabolism, transcriptional regulation, protein transport, binding adhesion and other processes. Gene Ontology category analysis showed that the proteins encoded by related sequences may be growth factors, receptors, carriers, structural proteins or proteins involved in immune response, and so on. It is clear that these differentially expressed genes are not just simply immnue-related genes, they contained many genes involved in other processes.Some of the sequences were chosen based on the above analysis for further validation and full-length sequences. The mosquito infection experiment were repeated in the same conditions as above. The interested differentially expressed genes were validated by Real-time PCR, Some of those which had been verified were amplified the full-length cDNA with the RACE technology. We got full-length cDNA sequence of heat shock protein 40 (HSP40) and an unknown gene. A 896 bp sequence of 3'end of Enoyl CoA hydratase (ECAH) involved in fatty acidβ-oxidation and another two 3'end sequences of unknown genes with the length of 911 bp and 662 bp, respectively, had been amplified.This is the first and initial molecular study in interactions and relationship between A. sinensis and P. yoelii, The results we obtained need to be further functional verification. Systematic molecular study of immune-related, metabolism and even other function-related genes will take a long time of exploring.The sequences we obtained, in particular, unknown genes are need to be well studied. Further results might provide the new candidate targets in developing the new strategy for vector control.
|
PDF Full Text Request