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Cloning of GPR30Gene and the Effect of EE2on Its Mrna Expression in Paramisgurnus Dabryanus

Author: ZhangXia
Tutor: WangZaiZhao
School: Northwest University of Science and Technology
Course: Fisheries
Keywords: endocrine disrupting chemicals Paramisgurnus dabryanus estrogen receptor GPR30 gene cloning quantitative real time PCR
CLC: S917.4
Type: Master's thesis
Year: 2013
Downloads: 6
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Abstract


Endocrine disrupting chemicals (EDCs) exists extensively and has the potential to damage the health of other animals. The biological effect of EDCs has attractd considerable attention. As higher organisms in water, fish are important objects of enrichment of various EDCs. It is significant to explore the effection of EDCs on fish. Now, a lot of results have demonstrated that EDCs can bind to estrogen receptor (ER) and take effect on growth and development of fish. Due to these researchs, more and more scientists devote themselves to studying the function of ER. The hotspot about research program of ER is focus on the relationship of ER and membrane estrogen receptor (mER), like GPR30, as well as the regulating pathway and molecular traits. At present, a lot of researchers have suggested the fact that G protein-coupled receptor30(GPR30) is a kind of mER, which is different from the classical estrogen receptor, ERa and ERβ. GPR30is located on the cell membrane or endoplasmic reticulum. Numerous studies have shown that GPR30has overexpression in human malignant tumor tissues, such as breast cancer, endometrial cancer and ovarian cancer, what’s more, it is clear that GPR30has a closed contact with the growth and development of organisms. Therefore GPR30has become the focus of international attention. For nearly two decades, many scholars dedicated to the study of GPR30functional characteristics, mechanism of action and the relationship among ERa, ERβ and GPR30. The result seemed to live up to expectations. These work has acquire considerable progress.Paramisgurnus dabryanus (Cypriniformes; Cobitidae) as a common carp shape mesh cobitidae fish, which is distributed in Changjiang River and nearby bodies of water. It is not only one of freshwater fishes with better nutritive value and raising value, deserving of exploitation and utilization, but also discussed as a new model of experimental animal. According to those reports, the results advanced that P. dabryanus is worth future application of its similar characteristics to fish in laboratory animal science, just like Gobiocypris rarus, Oryzias latipes and Danio rerio. It belongs to multi-oviposition type and has a relative longer time to reach sexual maturation, it has a longer life-time than those commonly small model fish, likeGobiocypris rarus, Oryzias latipes and Danio rerio. Nay, compared to other wild fish, its smaller body size is convenient to be raised in lab. Its sickness agent and breed technique problem have been solved in a certain degree effectively. Therefore, Paramisgurnus dabryanus is gradually becomeing a model laboratory animal, which iswidely used in research of aquatic toxicology. It is expected to be a perfect model organism of studying EDCs.In order to study out the disruption of EDCs on Paramisgurnus dabryanus’ GPR30gene and the mode of action of EDCs on GPR30gene, this experiment cloned GPR30of Paramisgurnus dabryanus (Cobitidae, Cypriniformes) by reverse transcription PCR method and rapid amplification cDNA ends (RACE) method. The full length cDNA of GPR30was2152bp, includeing a1062bp open reading frame (ORF) coding for (encode)353amino acids. Sequence analysis found that PdGPR30included a seven-transmembrane domain and a DRY (Asp-Arg-Tyr) sequence, which are the structural characteristics of a G protein coupled receptor (GPCR). In addition, it has three potential N-glycosylation sites in its N-terminal domains, two of which are at conserved positions like other species.Alignment of the PdGPR30amino acid sequence with the countparts in other vertebrates showed that PdGPR30shared high similarity with the other amino acid sequences, especially Danio rerio (92.9%). The result of qRT-PCR suggested that the expression of GPR30mRNA was highest in ovary, which1404times more than in intestine of male Paramisgurnus dabryanu.Two-month female Paramisgurnus dabryanus was exposure by EE2in different concentration, including1ng/L,5ng/L,25ng/L for21days, we made detect the expression of ERa,(also known as Esrl) and GPR30mRNA. With contrast, The result showed that GPR30mRNA expression slightly increased in ovary of lng/L EE2and liver of5ng/L EE2. However, theexpression of ERa mRNA increased signally as the concentration of EE2grew.The result suggests that the amino acid sequence of PdGPR30is highly similar to other vertebrates’, as well as the structural domain and functional domain of GPR30. However the expression of GPR30mRNA is dramatically different in gonad of Paramisgurnus dabryanu, which is about702times in ovaries more than testes, it suggests that PdGPR30has an affinity with estrogen effect which has been confirmed by previous researches in mammal. With EE2treatment, the expression ofGPR30mRNA shows no significant change, on the contray, the expressionof ERa mRNA was dramatically increased in the liver and ovary of Paramisgurnus dabryanu. The present result shows that it may have no direct and distinct interaction between PdGPR30and ERa in transcriptionallevel. The relationship of diverse estrogen receptors is necessary to be explored with more extensive experimental strategies and test methods.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
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