Molecular Cloning, Functional Characterization and Promoter Analysis of the Snakehead Fish (Channa Sp.) Elongase
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Date
2015-06
Authors
KUAH, MENG KIAT
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Abstract
In order to understand the molecular aspects of long-chain polyunsaturated
fatty acid (LC-PUFA) biosynthesis capability in a carnivorous freshwater fish, two
elongase genes were cloned from the striped snakehead and designated as elovl5 and
elovl4-like elongases. All characteristic features of a microsomal fatty acyl elongase
were found on the amino acid sequences of both striped snakehead elovl elongases,
and they shared high sequence identities with other fish counterparts. Striped
snakehead Elovl5 protein exhibited high elongation activities towards C18 and C20
PUFA substrates. It also demonstrated higher preferences towards n-3 PUFA than n-
6 PUFA. Unlike other typical Elovl4 proteins, the striped snakehead Elovl4-like
protein is capable in converting C18 PUFA substrates but displayed very low
elongation activities towards 22:5n-3 and 22:4n-6 substrates. Despite their similar
elongation preferences towards the C18 PUFA, both elovl5 and elovl4-like
transcripts possessed distinct profiles of tissue distribution and regulated by dietary
PUFA differently. In addition, the putative 2.5 kb promoter region of elovl5 gene
was cloned and functionally characterized to identify the transcription factor that was
involved in the regulation of the gene transcription. In silico analysis of the
promoter region of elovl5 gene revealed the presence of a putative binding site for
Srebp proteins. By using dual luciferase assays and EMSA, striped snakehead elovl5
elongase was confirmed as a target gene for the zebrafish Srebp1 protein. Moreover,
elovl5 promoter was fused to a GFP reporter gene and its in vivo promoter activity
was located to the yolk syncytial layer of developing zebrafish embryos. In
summary, the present study showed that striped snakehead is possible to synthesize
LC-PUFA de novo from C18 precursors with its well-functioning elongases and the
transcriptional regulation of its elovl5 gene is conserved with the mammalian
counterparts.
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Keywords
Molecular Cloning , Snakehead Fish (Channa Sp.) Elongase