Abstract
DNA photolyase is a photoreactivation enzyme which has a role in the mechanism of DNA repair. The existence of DNA photolyase gene could affect the success of mutation process. SM026H is a drought tolerance branching line of kenaf identified by Research Institute for Tobacco and Fibre Crops, Karangploso, Malang, Indonesia which has a potential as a source for mutation breeding. In this research, cloning and sequencing of the DNA photolyase gene of line SM026H were conducted. The DNA of SM026H leaves was isolated using DNeasy Kit, then was amplified by Polymerase Chain Reaction (PCR) using AC1 as a forward primer, paired with AC3R or AC4R as reverse primers and checked using electrophoresis on a 0.7% agarose gel. The AC1-AC3R primer pair produced a DNA fragment with a size of 750 bp, whereas the AC1-AC4R primer pair produced a DNA fragment of 1000 bp in size. Cloning of the AC1-AC3R and AC1-AC4R fragments was done prior to sequencing. Preparation for cloning was done by running and extracting the PCR products from a 0.7% Low Melting Agarose (LMA), and then ligating them to a pCR21 plasmid using an electroporation method. Two sub-fragments of each AC1-AC3R and AC1-AC4R fragments were identified. They were 600 bp and 500 bp, resulting from the AC1-AC3R fragment, and 1300 bp and 500 bp, resulting from the AC1-AC4R fragment. The sequencing result of those sub-fragments was analyzed using a Basic Local Alignment Search Tool (BLAST) program. It was shown that the sequences have a degree of homology to DNA photolyase sequence of Arabidopsis thaliana A. thaliana, Cucumis sativus, Spinacia oleracea, Stellaria longipes and Oryza sativa. These suggested that the kenaf line SM0026H possesses the DNA photolyase gene. However, further study needs to be done to ascertain that the gene was not the cryptochrome.
Highlights
Kenaf (Hibiscus cannabinus L.) is a fiber plant which belongs to the Malvaceae family whose bast fiber is usually utilized for making ropes, sacks, canvases and carpets
It was shown that the sequences have a degree of homology to DNA photolyase sequence of Arabidopsis thaliana A. thaliana, Cucumis sativus, Spinacia oleracea, Stellaria longipes and Oryza sativa
In the absence of kenaf genome sequence information, it was decided to utilize primers which were formerly used for cloning photolyase genes in soybeans which were designed based on the conserved region of the amino acid motif of Class II DNA photolyases of Arabidopsis thaliana and Chlamydomonas reinhardtii [24]
Summary
Kenaf (Hibiscus cannabinus L.) is a fiber plant which belongs to the Malvaceae family whose bast fiber is usually utilized for making ropes, sacks, canvases and carpets. Identification of DNA Photolyase Gene of a Drought Tolerance Branching Line of Kenaf (Hibiscus cannabinus L.) 811 energy source [12,16,17,18,19]. All the members of this family are flavoproteins that use near-ultraviolet (UV)/blue light photons as an energy source to mediate either DNA repairs or perceptions and signal transduction of external light signals. Whilst CPDs and 6-4 PPs act as agents for repairing DNA damage, Cryptochromes actas blue-light photoreceptors and exert various physiological functions, such as regulation of the circadian clock in animals and plants. Unlike photolyases, they generally lack of any kind of DNA-repair activity [18]. Two pairs of primers were designed based on the CPD photolyase sequence of the two organisms, and were used to detect the DNA photolyase gene of the kenaf line SM026H
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