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Expression and Functional Analysis of Ferritin Gene in Amphioxus Branchiostoma Belcheri

Author: LiMin
Tutor: ZhangShiZuo
School: Ocean University of China
Course: Cell Biology
Keywords: Amphioxus Ferritin LPS Iron
CLC: Q953
Type: Master's thesis
Year: 2008
Downloads: 119
Quote: 1
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Abstract

Amphioxus is considered to be the the extant vertebrates closest animal represents an important transitional stage in the process of evolution from invertebrates to vertebrates, classic experimental model for the study of the origin and evolution of the vertebrate. Using molecular biology methods to study the amphioxus gene structure, evolution and expression, from the molecular level to unlock the mystery of the origin of vertebrates. More importantly, linked to the evolution and development can be more clearly reveal the gene's role in development. Ferritin Ferritin main physiological function is to store the excess iron in the body, to avoid the generation of iron poisoning and iron release to the cells need iron for the iron-containing protein or enzyme in vivo biosynthesis, but also can prevent the process of oxidation of iron bis generating a radical damage. The gene encoding this protein from the people, animals, plants, fungi and bacteria are separated. So far, the the original cable animals ferritin has not been reported. Amphioxus ferritin gene sequences based on research by the slice in situ hybridization and immunohistochemistry the expression of the gene in the amphioxus body, and then evolution to study the amphioxus ferritin gene function and system. The amphioxus (BbFRT) ferritin cDNA sequence length of 1038 bp, containing a 519 bp open reading frame (ORF), encoding a 172 amino acid residue polypeptide consisting of a molecular weight of approximately 20.0kDa. In cDNA 5 'untranslated region of 116 bp 3' untranslated region of 403 bases, a terminator, and contains a polyadenylation signal AATAAA and a poly (A) tail. The initiation codon ATG is located 118 to 120 nucleotides, and in the 3 and 4 positions are purines, found typical kozak structure. This indicates that the cDNA encoding a full-length protein. Analysis BbFRT cDNA 5 'untranslated region found upstream ATG start codon 73 bp at the presence of an iron protein unique to the iron response element (iron Responsive Element IRE). Containing the components the conservative CAGUGN of the ring structure and a CYS mRNA secondary structure formation typical shank ring structure. BLASTp similarity searches against the NCBI database found that the amino acid sequence of the protein and the vertebrate ferritin H subunit amino acid sequence homology of 68% and 46-51%, with L subunit amino acid sequence homology, and some have been reported invertebrate ferritin amino acid sequence homology of 72-77%. Phylogenetic analysis showed that each of invertebrates and vertebrates ferritin together into one group, and BbFRT protein ferritin invertebrates clustered into one group, and invertebrates ferritin closer in the evolutionary status. Will be inserted into the pET28a amphioxus BbFRT gene fusion expression vector, to achieve a stable and efficient expression in the prokaryotic E. coli and purified the BbFRT fusion protein. A polyclonal antibody using the fusion protein preparation Westen blot hybridization with total protein in the body fluids of amphioxus, strong signal, indicating that in the amphioxus the body containing BbFRT protein and the antibody has the unique specificity of resistance. Slice in situ hybridization and immunohistochemistry the display BbFRT widely expressed in the amphioxus body. Of BbFRT function analysis and found that the bacterial endotoxin LPS treatment amphioxus, amount of BbFRT gene expression relative to the control at the level of transcription and translation levels were increased by 1.5 times and 1.6 times. Amphioxus after iron treatment, s BbFRT gene expression relative to the control at the translation level increased by 1.6 times. That BbFRT gene expression can be induced LPS and iron. This shows BbFRT not only be able to adjust the the amphioxus body iron metabolism, and LPS acute emergency response, since the immune function in vivo.

Expression and Functional Analysis of Ferritin Gene in Amphioxus Branchiostoma Belcheri

Abstract

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