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Molecular Cloning and Expression of Apolipoprotein E Gene in Annual Fish Nothobranchius Guentheri

Author: ShangXiaoMei
Tutor: ZhangShiZuo
School: Ocean University of China
Course: Biological Engineering
Keywords: aging Nothobranchius guentheri ApoE RACE Real-time Quantity PCR
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 22
Quote: 1
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Abstract


In recent years, the issue of aging has been more and more attractive to researchers. The determinants of life span are various and complex, most of which have not been completely understood yet. Some advances have been made in recent years in understanding molecular mechanisms that influence aging and lifespan. However, to some extent, genetic and pharmacological researches on aging are still impeded by the relatively long lifespan of available vertebrate models. Previous study informed us that lipofuscin, senescence associatedβ-galactosidase and neurodegeneration in teleost species express in an age-dependent manner. Besides, the precocious expression of those phenotypic markers above coincide with the notion that short life span in N.furzeri is due to accelerated aging. We started a aging related study on Nothobranchius guentheri, a species kept in our laboratory with a maximum life span of one year, which might represent a very useful model for comparative genomics of aging. The components of plasma lipoproteins are known as apolipoproteins, which are mainly synthesized in the liver and intestine. Apolipoproteins bind to lipids and play important roles in lipid transportation and lipid admission in the circulation system. In human, the major physiological roles of ApoE are mediating the cellular recognition and internalization of lipoproteins with members of the low-density lipoprotein receptor. ApoE was also found in the peripheral and central nervous systems. ApoE as a aging gene may play a role in several diseases, such as Alzheimer’s disease, gardiac and vasular diseases and diabetes Mellitus Complications.In this study, a full-length cDNA for ApoE, denominated NgApoE, was cloned from the N. guentheri. This cDNA sequence is 1018bp in length, and codes for a polypeptide of 262 amino acid residues, which contains a signal peptide with 18 amino acid residues. Mature NgApoE polypeptide consists of 245 amino acid residues, including a 33-codon block and tandem repeat units of 11 or 22 amino acid residues predicted to form amphipathicα-helical secondary structures. The deduced protein has the complete conserved domain of Apolipoprotein. Comparative analysis of the amino acid sequence in NgApoE and other vertebrates revealed that NgApoE showed higher levels of identity with their corresponding orthologs from teleosts than from mammals. NgApoE is 60.7% and 48.8% identical to ApoE-1 of Fugu rubripes and ApoE of Northern pike; and shows 30.4%, 27% and 26.2% identity to frog, mouse and human, respectively. The overall secondary structures of ApoE were also generally conserved among vertebrates, despite of modest or low levels of amino acid sequence identity in different species. The phylogenetic tree showed that NgApoE formed an independent cluster with Fugu rubripes ApoE-1. In the teleost group, three ApoE-1s and one ApoE clustered together while three ApoE-2s and other ApoEs are grouped in another clade. The present study characterizes an apoE gene homologous to mammalian apoEs from the Redtail notho. Consequently, the NgApoE may be ApoE-1 .The tissue localization of the ApoE has been studied in some species of teleost. In this study, Real-time Quantity PCR showed that the transcripts are specifically expressed in liver, with weak expression in heart. This is consistent with the results in human, which indicate that two-thirds to three-quarters ApoE of plasma derive from the liver secretion. Our study on relationship between age and expression in N. guentheri shows that ApoE gene expression decreases with aggravating aging process. The trend of the expression of ApoE is also similar to that of human aging-related markers. So we could preliminarily predict that ApoE can serve as an aging marker in N. guentheri, which will possibly be useful for aging reaseach.In conclusion, ApoE can be used as a kind of aging phenotypic marker in N. guentheri. While N. guentheri is a very useful model for comparative genomics of aging, in which many molecular mechanism of aging gene can be explored,ApoE will undoubtedly serve to enhance the reseach on aging.

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