Isolation and cloning of ABCE1 gene in Rasbora sarawakensis

Lim, Whye Kit Leonard (2016) Isolation and cloning of ABCE1 gene in Rasbora sarawakensis. [Final Year Project Report] (Unpublished)

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Abstract

The ABCE1 gene is one of the members of ABCE/ABCF subfamily in the ATP-binding cassette (ABC) transporter superfamily. The loss-of-function mutation of ABCE1 gene is a proven potential human cancer therapy for prostate, esophageal, oral and lung adenocarcinoma. The current zebrafish ecotoxicological studies may not be applicable domestically. The aim of this research is to isolate and clone ABCE1 gene from Rasbora sarawakensis. To examine this, total RNA was isolated from this fish using TRIzol and reverse transcribed. A pair of degenerative primers (forward primer: 5'-GCCAAYTCCTTCAARCTGCA-3'; reverse primer: j'-ACAGCAGATGAAGTCNGACA-3') were designed for RT-PCR amplification of this gene based on conserved sequences from multiple alignment of six selected freshwater fishes. The size of the gene amplified is around 580 bp in length which is close to its expected size (600 bp). Sequencing result and BLASTn analysis revealed that this gene from R. sarawakensis have high similarity of 94% to that of Danio rerio with E-value of 0.0. pGEM®-T Easy Vector was utilized as biological delivery vehicle for this gene in cloning, and cloning had yielded 30 white and 11 blue colonies with transformation efficiency of 2.02 x 105 transformantslJ.lg. Not! restriction enzyme digestion was done to prove plasmid propagation of the insert DNA. 100% homology was obtained after BLASTp pairwise alignment between ORFs of R. sarawakensis and D. rerio. Phylogenetic analysis revealed great resemblance between this gene from these two fishes in the maximum likelihood tree. The e results may suggest divergent evolutionary relationship between these two fish species which is beneficial to future establishment of this fish as a model in domestic ecotoxicological studies.

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