Molecular Cloning, Functional Characterization And Development Expression Of A New Elongase With Potential Role Of Long-Chain Polyunsaturated Fatty Acid Biosynthesis In A Teleost (Danio Rerio)
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Date
2020-02
Authors
Goh, Pei Tian
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Elongase of very long-chained fatty acid (ELOVL) is very important in synthesising long-chained polyunsaturated fatty acid (LC-PUFA). At present, seven members of ELOVL family (ELOVL 1 – ELOVL 7) have been found in mammals. Recently, a putative new Elovl was found in teleost. In order to study and examine the function of this new Elovl, zebrafish (Danio rerio) was chosen. Zebrafish is a viable lipid metabolism model with a complete LC-PUFA biosynthesis pathway and also a very good biological development model. An interest has arisen to determine the elongation capacity and also expression of this elongase of interest during development and in adult tissues. The full length of elongase of interest A (NM_001076593.1) and elongase of interest B (BC095712.1) genes were successfully cloned from the sequence available in National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed and revealed elongases of interest were grouped as a distinct group from the others elongases (Elovl1-7). Therefore they were named as elovl8a and elovl8b based on the result obtained from the phylogenetic tree. Other than that, Elovl2, Elovl4, Elovl5 and Elovl8 were found originated from the same ancestor and Elovl2, Elovl4 and Elov5 could be the result of gene expansion from Elovl8. Functional characterization of both elongases showed zebrafish Elovl8b converted monounsaturated fatty acid (MUFA), together with C18 and C20 polyunsaturated fatty acid (PUFA) substrates while Elovl8a lacked in these activities. Result from Weblogo analysis showed the elongase motif region of Elovl8 is rigid and consistent. Expression of both elovl in adult tissues were determined by using
quantitative PCR (qPCR), where elovl8a showed high expression in eyes, followed by
muscle, gills, brain and intestine. In comparison, elovl8b showed high expression in
intestine followed by liver, muscle, gills and brain. In development, elovl8a showed
no significant different between stages while expression level of elovl8b showed
gradually increase and reached the highest in 120hpf. The result from spatio-temporal
expression pattern suggested that elovl8a gene involved in development of neuromast
starting from 72hpf to 120hpf while elovl8b gene involved in development of intestine
and liver from 72hpf to 120hpf. Both results from gene expression and spatio-temporal
expression were similar which showed the possibility of expression and importance of
elovl8a and elovl8b during development. Overall, the results from present study
strongly suggested that zebrafish elovl8 is distinct from current seven elovl family
member. Besides that, this research also reduced the possibility of elovl8 to be group
as paralog gene of elovl4 as suggested by other researcher. Therefore, this group of
elovl8 are prepared to be classified as new elovl member.
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Keywords
Natural history