Abstract
Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5′ flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5′UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8–21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5′-UTR significantly enhances the level of gene expression. We termed this phenomenon as “microsatellite mediated enhancement” (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids.
Highlights
GAGA elements have been most thoroughly examined in Drosophila, where they have been shown to be present in the promoter regions and are involved in the regulation of developmental genes by binding to a protein called the GAGA factor, which results in local nucleosome disruption thereby allowing gene expression[17]
The Tryptophan decarboxylase (Tdc) gene, which catalyses the conversion of tryptophan to tryptamine in the the alkaloid producing pathway (TIA) pathway, had been cloned and characterized from C. roseus[32] and shown to possess (CT)n repeats in the 5′-untranslated mRNA leader sequences (UTRs) region[32,33]
In the present study, primers P1 and P3 were designed from the regions flanking the (CT)n motifs in the 5′UTR of Tdc gene (Fig. 1)
Summary
GAGA elements have been most thoroughly examined in Drosophila, where they have been shown to be present in the promoter regions and are involved in the regulation of developmental genes by binding to a protein called the GAGA factor, which results in local nucleosome disruption thereby allowing gene expression[17]. While analyzing the Catharanthus roseus transcriptome we have demonstrated that GA/CT and GAA/CTT repeats were most frequent in 5′-flanks of genes that are known to be involved in enzymatic, regulatory and housekeeping functions[7] Such preferential distribution and conservation of SSRs in the 5′-UTRs1,7 strongly suggests that they may be strong contenders for being characterized as a regulatory element. Tryptophan decarboxylase (TDC) catalyses the first committed step of indole alkaloid synthesis by decarboxylation of tryptophan to form tryptamine[29] and is a key enzyme in the biosynthetic pathway by virtue of its position at the interface of primary and secondary metabolism It has been characterized in C. roseus[28,29,30] but its complex, coordinated regulation needs to be investigated in order to exploit it for genetically engineering plants with enhanced levels of useful indole alkaloids
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