| Backgroud:Housefly, Musca Domestic, is the most common flies in human dwellings and nearby. It is known that housefly is a pathogens spread carrier to human and animal. There are more than100pathogens that can cause disease in humans and animals have been concerned with the housefly, including cholera, typhoid, dysentery, anthrax ophthalmia, tuberculosis, infantile diarrhea and parasitic worms. Housefly is considered a pest to human health. Housefly is also an important species of flies in forensic entomology. It is mainly used to infer the time of death in crime scene investigation. It also has a relatively high value in the identified of abuse and disregard for children and old people.Housefly’s survival needs a warm environment. Within a certain temperature range, the more the warmth the higher its growth rate is. But in the winter, houseflies basically become extinct. In the following spring as temperature rise, a large number of houseflies reappear again. Then how do they survive through the cold winter? Scentific reaserch confirmed that they survive in the form of maggots or pupae in winter. Some studies showed that the protein aquaporin (AQP), water channel protein, may be relevant with low temperature tolerance in insects.AQP may be one of the key moleculars to insects’ wintering. Aquaporin is one member of major intrinsic protein (MIP) superfamily, and it has conservative structure in many insects. Previous studies on AQP in insects focused on two aspects including endocrine regulation and osmotic regulation. But the reported for the expression patterns of AQP and its function in houseflies is still not avilarble at present.Objective:1. Using bioinformatics methods to identify experimental fly species and lay the foundation for the subsequent experiments.2. Cloning the full-length of AQP gene of housefly and lay the foundation for the research of expression patterns of the gene. 3. Screening RT-qPCR reference genes for housefly and lay the foundation for the research of expression patterns of the gene.4. Studying the expression pattern of AQP in housefly and whether the gene has the function of low temperature tolerance.Methods:1. Using long-chain PCR approach combined with walking primers to clone the complete mtDNA sequence of housefly and using bioinformatics methods to identify Musca Domestic.2. Using RT-PCR method and RACE strategy to clone the full-length AQP gene of housefly. Using many bioinformatics tools to analyze the nucleic acid sequence of AQP gene and deduced amino acid sequence.3. Using GeNorm, NormFinder, and BestKeeper software to analyze the stability of common used internal reference genes of housefly under different experimental conditions. The stable reference genes under specific experimental conditions were selected to standardize its experimental data.4. Using quantitative PCR method to detect the relative expression levels of AQP gene in three parts of adult housefly including head, thorax and abdomen, and also cluding four developmental stages:egg, larva, pupa and adult. In situ hybridization method is applicated to analyze the distribution of AQP gene which is in the viscera of housefly.5. After silencing the AQP gene of housefly by RNA interference technique, using an experimental method to verify whether the AQP gene is a key molecule in low temperature tolerance.Results:1. The first almost full-length mtDNA sequence was upload to the GenBank database (accession number:EU154477) by other researchers. The whole sequence is approximately15200bp and has only900bp missed. So we quickly sequenced and assembled the full-length mtDNA of Sarcophaga peregrine and Megaselia scalaris, two routine cultured flies in our laboratory. The two sequences then were uploaded to the GeneBank database, and got accession number (KF921296and KF974742) respectively. The mtDNA sequence structure in the three kinds of flies are fully consistent with most insects’mtDNA sequence: they are about15000bp in length, including13protein-coding genes,22 tRNA, two rRNA and noncoded AT-rich region (generally considered to be the control area). Bioinformatics analysis suggested that full-length mtDNA sequences can be effectively identified species to genus.2. The AQP gene of housefly was successfully cloned. And the full-length sequence is1210bp (GenBank accession number:KJ599672), which has an open reading frame sequence of738bp, and can code245amino acid residues. The AQP gene sequences have higher conservatism compare with reported AQP gene sequences of other insects. The AQP gene has six transmembrane domains and two typical NPA motifs, which has the characters of amphoteric channel and belongs to the major intrinsic protein (MIP) superfamily.3. By GeNorm, NormFinder, BestKeeper software analysis, we found that the stability of reference genes of housefly are different under different experimental conditions. In general, EF-1and RPS18are relatively stable reference genes in the experimental conditions and can be used as an internal control to standardize the experimental data.4. The expression level of AQP gene of adult houseflies gradually increased high from head to chest, then to abdomen. The AQP gene is expressed in different developmental stages, incuding egg, larva, pupa and adult. While the expression level in late pupae stage is the lowest but in egg stage is the highest. The expression level of adults is equal with the level of early pupae, but higher than that expressed in late pupae. In the larval stage, the expression of AQP decreased gradually from first instar to third instar, and until to the late pupae stage which reached the bottom. In situ hybridization showed that, the mRNA of AQP gene is expressed in almost the entire visceral tissues, but the distribution in the salivary gland was the most obvious, which present in piece or strip shape. The midgut tissues have high sporadic expression. But in Malpighian tubule has suspected positive expression only, in which had common highly expression in other insects.5. After the third instar larvae of housefly was injected dsRNA24hr,48hr and72hr respectively, the expression level of mRNA on AQP gene significantly decreased in48hr and72hr, as compare to RNase-free water-injected and non-injected control groups. This indicated that the dsRNA interfered the expression of AQP gene with time passed. RNase-free water-injected group and and non-injected group displayed a very close expression intensity, and this demonstrated that the RNase-free water had no significant effect on AQP gene expression. The low temperature tolerance experiment showed that the mortality about third instar larvae of housefly in the three expermental groups were very close. After statistical analysis, the different mortality between the two groups had no statistically significant diference in statistics.Conclusion:1. For the first time, the aquaporin gene from housefly and the full length mtDNA sequence from both Sarcophaga peregrine and Megaselia scalaris were successfully cloned.2. The most stable reference genes were selected under specific experimental conditions and can be used to standardize the experimental data. EF-1and RPS18are relatively more suitable in this experimant.3. The in vivo microinjection of RNA interference system in housefly larvae was successfully established for the first time.4. In our experiments, the AQP gene of housefly larvae has not yet been confirmed as a key molecule to low temperature tolerance. |
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