Results
Design of the RNAi vector
The PMT gene family is well conserved among the Solanales. There are five genes in the PMT gene family (PMT1 to PMT5) which differ in the exon 1 sequence due to the insertion of a variable number of 33 nucleotide repeats. This makes the first exon as an ideal region for the design of gene specific RNAi trigger sequence (Fig 1A). Full-length sequences are available for four PMT genes (PMT1 to PMT4) whereas only the sequence of exon1 is known for PMT5 which shares 95% identity with PMT1. The PMT4 contains 6 insertions of 33 nucleotide repeats (198 bases) in exon 1. The exon 1 sequences of the five PMT genes were analyzed to design a trigger sequence that is specific to the major PMT gene, PMT2. A 162bp sequence …show more content…
The sequence characteristics of the RNAi trigger sequence used for silencing plays major role in both. The 162bp RNAi trigger sequence used in the present study was derived from the PMT2 gene, which is the major gene (highly expressed) in the 5-member PMT gene family. The trigger sequence was derived from the first exon, which showed the maximum divergence primarily due to the insertions of a variable number of 33 nucleotide repeats (Fig 1). It was designed to target the major PMT2 gene, but cross silencing of the minor PMT4 genes was also expected. PMT1, PMT3 and PMT5 genes may also be cross silenced to some extent due to the sharing of 22 nucleotide perfect match sequences with the RNAi trigger. The 162bp RNAi trigger sequence contained guide siRNA sequences with preferred thermodynamic properties for efficient silencing. The spacer sequence used to generate hairpin dsRNA of the trigger sequence also play a crucial role in the silencing of the target genes [24, 25]. RNAi trigger with intronic spacers provides a dsRNA template for efficient Dicer processing, and thereby, increases the efficiency of the RNAi vectors [25, 26]. We have used the native intron including the conserved 5’ and 3’ splice sites as a self-cleaving spacer. This may be one of the reasons that we have observed the highest level of silencing as evident from the low nicotine content found …show more content…
In the present study, constitutive silencing of PMT genes was targeted to block or reduce the expression of putrescine methyl transferase that catalyzes the conversion of putrescine to methyl putrescine. As a consequence, accumulation of putrescine and nicotinic acid derivatives was expected. It was reported that silencing of PMT genes resulted in an increased level of putrescine and spermidine in Nicotiana sylvestris [14] and anatabine in Nicotiana tabacum [15]. Silencing of ADC, which converts arginine to putrescine and ODC which converts ornithine to putrescine also produced low nicotine plants with increased levels of anatabine in Nicotiana tabacum [16, 17]. Anatabine is a toxic alkaloid derived from nicotinic acid and is a cholinergic agonist. MS, ESI spectra of the low nicotine RNAi plants in positive and negative mode did not show accumulation of anatabine. Instead, we report for the first time that reduction of nicotine content results in a concomitant increase in the content of chlorogenic acid. Chlorogenic acid is an ester formed between caffeic acid and quinic acid with proven antioxidant properties [14, 33]. Silencing PMT genes accumulate putrescine that can combine with hydroxycinnamates to produce hydroxy cinnamate-putrescine conjugates such as caffeoyl putrescine,
Design of the RNAi vector
The PMT gene family is well conserved among the Solanales. There are five genes in the PMT gene family (PMT1 to PMT5) which differ in the exon 1 sequence due to the insertion of a variable number of 33 nucleotide repeats. This makes the first exon as an ideal region for the design of gene specific RNAi trigger sequence (Fig 1A). Full-length sequences are available for four PMT genes (PMT1 to PMT4) whereas only the sequence of exon1 is known for PMT5 which shares 95% identity with PMT1. The PMT4 contains 6 insertions of 33 nucleotide repeats (198 bases) in exon 1. The exon 1 sequences of the five PMT genes were analyzed to design a trigger sequence that is specific to the major PMT gene, PMT2. A 162bp sequence …show more content…
The sequence characteristics of the RNAi trigger sequence used for silencing plays major role in both. The 162bp RNAi trigger sequence used in the present study was derived from the PMT2 gene, which is the major gene (highly expressed) in the 5-member PMT gene family. The trigger sequence was derived from the first exon, which showed the maximum divergence primarily due to the insertions of a variable number of 33 nucleotide repeats (Fig 1). It was designed to target the major PMT2 gene, but cross silencing of the minor PMT4 genes was also expected. PMT1, PMT3 and PMT5 genes may also be cross silenced to some extent due to the sharing of 22 nucleotide perfect match sequences with the RNAi trigger. The 162bp RNAi trigger sequence contained guide siRNA sequences with preferred thermodynamic properties for efficient silencing. The spacer sequence used to generate hairpin dsRNA of the trigger sequence also play a crucial role in the silencing of the target genes [24, 25]. RNAi trigger with intronic spacers provides a dsRNA template for efficient Dicer processing, and thereby, increases the efficiency of the RNAi vectors [25, 26]. We have used the native intron including the conserved 5’ and 3’ splice sites as a self-cleaving spacer. This may be one of the reasons that we have observed the highest level of silencing as evident from the low nicotine content found …show more content…
In the present study, constitutive silencing of PMT genes was targeted to block or reduce the expression of putrescine methyl transferase that catalyzes the conversion of putrescine to methyl putrescine. As a consequence, accumulation of putrescine and nicotinic acid derivatives was expected. It was reported that silencing of PMT genes resulted in an increased level of putrescine and spermidine in Nicotiana sylvestris [14] and anatabine in Nicotiana tabacum [15]. Silencing of ADC, which converts arginine to putrescine and ODC which converts ornithine to putrescine also produced low nicotine plants with increased levels of anatabine in Nicotiana tabacum [16, 17]. Anatabine is a toxic alkaloid derived from nicotinic acid and is a cholinergic agonist. MS, ESI spectra of the low nicotine RNAi plants in positive and negative mode did not show accumulation of anatabine. Instead, we report for the first time that reduction of nicotine content results in a concomitant increase in the content of chlorogenic acid. Chlorogenic acid is an ester formed between caffeic acid and quinic acid with proven antioxidant properties [14, 33]. Silencing PMT genes accumulate putrescine that can combine with hydroxycinnamates to produce hydroxy cinnamate-putrescine conjugates such as caffeoyl putrescine,