Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

Abstract

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.

EVALUATION OF OIL PALM LEAF-SPECIFIC PROMOTER (LSP1) ACTIVITY FOR EXPRESSING PHB GENES IN Arabidopsis thaliana

“…The T 3 generation of A. thaliana plants that have been transformed with pLSP15 vector (Figure 1) (Masani et al, 2009) by the Agrobacterium-mediated floral-dip method (Clough and Bent, 1998), were used in this analysis. The pLSP15 vector carried a Basta resistance gene (bar) driven by the CaMV35S promoter and each of the PHB genes (phbA, phbB, phbC) was driven by an oil palm leaf-specific promoter (LSP1).…”

“…Total RNA was isolated from leaves of A. thaliana according to Zeng and Yang (2002) with Masani et al, 2009). modification. Isolated RNA were quantified spectrophotometrically (NanoDrop ND-1000; NanoDrop Technologies Inc. USA) with absorbance at 260 nm.…”

“…No PCR products were observed in untransformed A. thaliana plants. The presence of the bar gene indicated that the PHB genes (phbA, phbB, phbC) might also be present in the transgenic A. thaliana because these genes are linked in tandem in pLSP15 plasmid (Masani et al, 2009).…”

“…This study also demonstrated that the use of RT-qPCR and Nile blue staining could be applied to oil palm genetic engineering programme especially on the production of PHB in oil palm. Through this programme, thousands of embryogenic calli have been bombarded with PHB genes and hundreds of the putative transgenic plants have been obtained (Parveez et al, 2008;2015;Yunus et al, 2008;Masani et al, 2009). By using RT-qPCR and Nile blue staining, the putative transgenic oil palm producing PHB could be efficiently evaluated in shorter time compared to conventional molecular analyses such as Southern and Western blot hybridisation.…”

“…In transgenic flax, the use of a stem-specific promoter (14-3-3 promoter) to produce PHB does not affect the plant stem height (Wrobel et al, 2004). The use of a tissue-specific promoter will facilitate the production of PHA in an easily harvested tissue without disturbing the normal growth and development of plants (Masani et al, 2009).…”