The Verification of DNA Sequences Adjacent to LB of T-DNA in Transgenic Dendrobium ‘Sonia’ Earsakul Genome Using AL-PCR Technique
DOI:
https://doi.org/10.14456/thaidoa-agres.2021.11Keywords:
Transgenic plant, AL-PCR, insertion positionAbstract
In Agrobacterium-mediated genetic transformation, the integration site of T-DNA in plant genome was non-predictability. The inserted position of transgenes may affect gene expression and subsequent physiological traits. This research explained the utilization of Adaptor Ligation-Polymerase Chain Reaction (AL-PCR) technique to verify the sequence adjacent tothe leftborderofT-DNA (LB T-DNA)of the transgenic Dendrobium ‘Sonia’ Earsakul possesses antisense CP-ACO1. The genomic DNA of transgenic orchid was digested with restriction enzyme PsiI to generate blunt ended DNA strands prior to ligating with the blunt end adapter. The PCR was performed using adapter ligated DNA fragments as the templates. The forward primer was designed to be complimentary with adapter sequences and reverse primer was specific to known sequence of hpt gene that was inserted into the plant genome. The analysis of amplicons resulted from AL-PCR technique revealed 3 DNA bands at the sizes of approximately 750, 1,500 and 2,000 base pairs. The DNA sequence analysis of 750 bp band revealed that part of hpt gene in T-DNA was connected to LB sequence that fused to the 160 bp upstream of vector backbone sequences. For the bands of 1,500 and 2,000 base pairs, the DNA sequence analysis could not be achieved due to their low purity and amount
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