Heterologous expression of recombinant alcohol dehydrogenase gene from sago palm in bacterial and plant system

Mastura, Sani (2016) Heterologous expression of recombinant alcohol dehydrogenase gene from sago palm in bacterial and plant system. Masters thesis, Universiti Malaysia Sarawak(UNIMAS).

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Abstract

Alcohol dehydrogenase (ADH) is an enzyme involved in pathways that respond to various environmental stresses such as osmotic, wound and anaerobic condition. In this study, the recombinant Metroxylon sagu Adh1 cDNA (r-msAdh1) was cloned into an expression vector; pET-41a(+) and expressed in Escherichia coli (E. coli) strain BL21 (DE3). SDS-PAGE analysis of the E. coli lysate revealed that large amount of expressed protein formed insoluble aggregation, non-active protein. However, soluble r-msAdh1 protein was successfully produced at low-growth temperature and showed catalytic activity when spectrophotometrically assayed. Second part of this study involves the in-planta transformation of r-msAdh1 cDNA into tomato seeds using Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404. The result showed that, out of 15 putative transformed plants (T0) were analysed, five putative transgenic plant lines were determined to carry r-msAdh1 cDNA in their genome. Further analysis also showed that r-msAdh1 cDNA have passed into all T1 transgenic lines and expression analysis at transcript level confirmed the presence of r-msAdh1 in transgenic tomato genome.

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