| Background:Mosquito is an important medical insect. It not only bites humans, but also transmit various diseases, such as dengue fever, malaria, filariasis, yellow fever and Japanese encephalitis, etc., which cause a great harm to human health. Mosquito olfactory system mainly includes the odorant-binding proteins (OBPs), odorant receptors (ORs) and the olfactory receptor neurons (ORNs), which are mainly distributed in tentacle, mandible and beak containing various forms of the smell sense sensors. The odorant binding protein and odorant receptor play a key role in sense of smell. Therefore, the research of mosquito odorant receptors is of great significance in mosquito-borne infectious diseases prevention and control.Recent researches suggest that the Anopheles gambiae contains79odorant receptor genes, Aedes aegypti contains131and Culex pipiens contains180. Studies of Aedes albopictus odorant receptors came out lately, but it is still a blank area in China now. Not until May2012was the Aedes albopictus olfactory receptor2(AalOr2) firstly reported. The coding region of AalOr2includes six exons divided by5introns. Its cDNA is1556bp long with the final protein products containing376 amino acids, and there are seven transmembrane areas.Orco gene is a member of the mosquito olfactory receptor gene families, it can identify odor molecules cooperating with the odorant receptors. Orco genes identified in flies, Anopheles gambiae, Culex pipiens, and Aedes aegypti respectively are Or83b, AgOr7, CqOr7, and AaeOr7. When odors get into mosquito olfactory systems, odor molecules will firstly combine with OBPs, by which they will be transported to ORs. As a consequence of the stimulation of OR genes expressed on the membranes of ORN, the ion channels could turn open or closed together with a change of voltage difference. The transmission of the voltage change from the ORN membrane to the central nervous system causes repellent or attractive behaviors to odors of mosquitoes. Whether Orco-genes could enhance the ability of OR genes to recognize odors remains to be further researched. Current researches show that it is selective in the cooperation of OR genes and Orco genes in recognizing odors, as in the process of recognizing partial odors, the function of some OR genes may be strengthened by Orco genes, while some are may not.DEET (N, N-Diethyl-meta-toluamide) is a common repellent to mosquitoes and could disrupt the abilities of mosquitoes to recognize odors of human sweats,which has a history of nearly60years. However, indole is an important kind of attractants to mosquitoes in human sweats accounted for nearly30%. Among the stimulations of nearly30kinds of odor molecules of Aedes albopictus olfactory receptor gene Or2together with Drosophila Melanogaster Orco gene in HEK293(Human embryonic kidney) cells, the intracellular calcium concentration reached the highest level under the stimulus of indoles, which suggests that indoles have a strong attraction to the olfactory system of mosquitoes, so as to the larvae. Based on what has been dicussed, we use DEET and1-methyl-3-indole to stimulate the HEK293cells where the Aedes albopictus Or7and the Anopheles gambiae Orco genes are co-expressed. In the end, the intracellular calcium concentration gradually increased to a peak with the stimulation of1-methyl-3-indole; however, the intracellular calcium concentration fell to the valley with the stimulation of DEET.It is popular to study mosquito OR genes in the identification of the changes of odor molecules concentrations. When stimulating oocytes of clawed frogs cloned with Aedes aegypti Or7(AaeOr7) co-expressed with Aedes aegypti Or2(AaeOr2) with five odors at a concentration of10-3M, they found that DEET caused greatest reactions. The same result also appeared in oocytes of clawed frogs cloned with Aedes aegypti Or7co-expressed with Aedes aegypti Or8genes. Dramatically, the cells have a stronger response to the stimulation of indole at10-7M in these groups, which was far stronger than that of DEET at10-3M. However (In fact), the reactions increased gradually with the increase of DEET concentration from10-10M to10-5M and the largest concentration got a peak response as well, Just like the trend of indoles. What's more, the clawed frog oocyte where AaeOr10and AaeOr7co-expressed got a the same result, which furthermore confirms that AaeOr2, AaeOr8, AaeOr10co-expressed with AaeOr7have a similar response spectrum to DEET.Objective:In my study, I chose Aedes albopictus Or7olfactory receptor (AalOr7) gene as the object to analyze its potential function and corresponding molecular mechanism with kinds of technologies and methods, which will help build theoretical basis of prevention and control of the mosquito-borne infectious diseases. Methods:1. The recognition and coding regions of the Aedes albopictus odorant receptor gene Or7(AalOr7) full-length sequence. Based on the genome sequence data of Aedes albopictus in our laboratory, we predicted partial sequence of AalOr7after alignment with the gene sequences and amino acids of Aedes aegypti Or genes using tBLASTn, and got the AalOr7coding sequence after RT-PCR amplification. Later, I analyzed corresponding amino acid sequence bioinformatically.2. Analysis of profile expressions of AalOr7in different tissues and stages of Aedes albopictus using RT-PCR technique.3. Building recombinant plasmid pME18S-AalOr7with primers based on AalOr7ORF sequences and pME18S polyclonal sites and pEASY-AalOr7plasmid as templates.4. Transfection of eukaryotic expression vector pME18S-AalOr7into HEK293cells and using RT-PCR to identify its expression.5. Taking commonly used repellent/attractant agents such as indole and DEET to stimulus the HEK293cells to preliminary study the function of odors recognition of AalOr7Results:1. The prokaryotic expression vector pEASY-AalOr7was built successfully and showed that AalOr7is with full-length of1395bp and the amino acid sequence length is465aa correspondingly. Through bio-informatics technology analysis, I found that the deduced amino acid sequence contained six transmembrane areas, which may belong to the membrane protein. The homology between Aedes albopictus Or7and Aedes aegypti Or7is98%with amino acid sequence alignment.2. The profile expressions of AalOr7were detected in different development periods:larvae, pupae and adult mosquitoes, etc. It's highly expressed in females'olfactory (beak, antenna, the palate), but lowly in males.3. I built the eukaryotic expression vector pME18S-AalOr7.4. pME18S-AalOr7expressed evidently in HEK293cells which was identified with RT-PCR.5. The calcium imaging technology with HEK293cells showed that along with the stimulation of1-methyl-3-indole and DEET, the intercellular Ca2+concentration changed a lot. The phenomenon suggests that AalOr7has a certain regulation function of Ca2+, thus affecting cell in odor identification capability.Conclusion:1. Constructing eukaryotic expression vector of the Aedes albopictus olfactory receptor gene pME18S-AalOr7successfully.2. AalOr7in different stages of Aedes albopictus are expressed, and especially evidently in female mosquito olfactory.3. Preliminary analysis with calcium imaging technology of AalOr7shown that it has the function of odor identification. |
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