Isolation, characterisation and functional analysis of root-specific promoters from mining of oil palm transcriptome data

Root-specific promoter is a valuable genetic engineering tool to improve oil palm resistance to diseases, malnutrition, or abiotic stresses that cause extensive losses in the oil palm industry. Since the availability of root-specific promoters from oil palm is still limited, this study was conducted to identify and characterise a strong promoter in a strict root-specific or preferential manner that may have great potential for root-system modification. Mining the transcriptome of various oil palm tissue-specific data generated from RNA Sequencing technology has resulted in discovering seven candidate genes for root-specific promoters. The expression pattern of these candidate genes was further validated through real-time quantitative polymerase chain reaction (RTqPCR) analysis using various oil palm tissues, including root tissues at different developmental stages. Two transcripts, namely RSP-2 and RSP-3, that showed high expression in roots were selected for further research. RSP- 2, annotated as an oil palm metallothionein gene (EgMT), was significantly upregulated at 7 to 170-fold in roots. Likewise, RSP-3, which belongs to the proline-rich protein (EgPRP1) gene, was significantly upregulated at 7 to 55- fold. DNA vectors carrying a full-length promoter region and 5' promoter deletion fragments were constructed for the promoter functional study. Deletion analyses were performed according to the density of the cis-acting elements related to the root expression present in the promoter sequences. The promoter activity in transient expression was studied using the biolistic method in various oil palm tissues. The study indicated that EgPRP1 and EgMT promoters could drive the expression of red fluorescence protein (DsRED) in oil palm root tissue. Since oil palm transformation was hampered by a long regeneration time, the stable promoter functional study was investigated further using tobacco as a model plant. The promoter activity was evaluated in seedlings, vegetative and reproductive tissues of T1 generation using GUS histochemical assay. Promoter activity was evaluated based on the intensity of blue GUS staining. The RSP-3A construct (2000 bp upstream of EgPRP1 start codon) showed root-preferential or dominant promoter activity in all the root developmental stages. Fine dissection revealed that the shortest promoter construct, designated as RSP-3D (665 bp upstream of EgPRP1 start codon), conferred root-specific expression in mature plants. For EgMT, only the RSP-2D (1107 bp upstream of EgMT start codon) deletion construct directed strong GUS expression from early to mature root developmental stages. GUS expression directed by RSP-2A and RSP-2C constructs were detected in the roots of mature plants, with intensities lower than RSP-2D. However, the activity of EgMT promoters was also detectable in seed pods and immature seeds, albeit at lower levels than the Cauliflower Mosaic Virus 35S promoter (CaMV35S). Interestingly, the GUS expression driven by RSP- 3A, RSP-2C and RSP-2D was detected at the flower and leaf-cutting sites, suggesting the promoters' activity was inducible by wounding. Results also showed that the promoters consist of cis-acting elements that act as negative regulators, which might be responsible for root specificity. The results further indicated that the 5' UTR and ATATT sequences defined as ROOTMOTIFAPOX1 cis-acting elements are essential for strong promoter activity. Overall, the functional study conclusively demonstrated that the genes identified through the mining of transcriptome data and RT-qPCR analysis that were upregulated during root developmental stages have a high potential to regulate a strong expression of transgenes in a root-specific or preferential manner. The results suggest that promoter regions of RSP-3A and RSP-3D from EgPRP1 and RSP-2D from EgMT are potentially helpful in facilitating oil palm genetic engineering, notably improving crop yield through root-traits modification.

Text 124817.pdf Download (1MB) Official URL or Download Paper: https://ethesis.upm.edu.my/id/eprint/18782

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