Expression of Recombinant Alcohol Dehydrogenase in Escherichia coli Strain BL21 (DE3) and In Planta Agrobacterium Transformation of Tomato Seeds

• Main Authors: Mastura, Binti Sani, Hairul Azman, Bin Roslan
• Format: E-Article
• Language: English
• Published: King Mongkut's Institute of Technology Ladkrabang 2020
• Subjects: QH426 Genetics
• Online Access: http://ir.unimas.my/id/eprint/29489/1/sani%20roslan%202020.pdf; http://ir.unimas.my/id/eprint/29489/; https://li01.tci-thaijo.org/index.php/cast/article/view/241941
• Institution: Universiti Malaysia Sarawak

Alcohol dehydrogenase is an enzyme that is involved in various roles in plant such as in plant development, growth and plant responses to abiotic and biotic stresses. A recombinant alcohol dehydrogenase 1 (Adh1) cDNA (r-msAdh1) from Metroxylon sagu has been previously isolated, containing 20 nucleotides derived from Elaeis guineensis at the 5’-end, with a molecular weight of 1.14 kb. The objective of this study is to determine the function of r-msAdh1 via analyses in prokaryotic and eukaryotic hosts. For expression in prokaryotic system, pET-41a(+) with a 8x His tag at the C terminal was used for r-msAdh1 protein purification and expression was achieved using IPTG for four to six hours in Escherichia coli strain BL21 (DE3) incubated at low temperature. The induced BL21 strain produced a small amount of soluble r-msAdh1 protein while large amount was present as insoluble aggregates. Subsequently, the r-msAdh1 cDNA was transformed into tomato seeds (Solanum lycopersicum cv. MT1) via Agrobacterium-mediated in planta transformation. The integration of r-msAdh1 cDNA and the selectable marker were detected in transformed seedlings, T0, using polymerase chain reaction technique. The transformation efficiency was determined to be 33% for r-msAdh1 cDNA and 46% for the selectable marker. For stability analysis of the transgene, eleven T1 generation randomly selected from the transgenic T0 were analyzed for the presence of the cDNA, and all seedlings were found to contain the full length of r-msAdh1 cDNA. However, out of eleven T1 transgenic lines produced, only four seedlings were used for expression analysis using the reverse transcriptase PCR (RT-PCR). Two transgenic lines, T19 and T111, were determined to contain r-msAdh1 cDNA and verified by nucleotide sequencing. Although only a small number of T1 transgenic seedlings was obtained, this study shows that tomato seeds could be used as a target tissue for Agrobacterium-mediated in planta transformation primarily because the protocol is easy, rapid and cheaper compared to tissue culture-based method.