Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

Abstract

In the past, silencing of granule-bound starch synthase (GBSSI) in potato was achieved by antisense technology, where it was observed that inclusion of the 3' end of the GBSSI coding region increased silencing efficiency. Since higher silencing efficiencies were desired, GBSSI inverted repeat constructs were designed and tested in potato. First, large inverted repeats comprising the 5' and the 3' half of the GBSSI cDNA were tested. The 5' IR construct gave a significantly higher silencing efficiency than the 3' IR construct. Since it was not known whether the observed difference was due to the sequence or the orientation of the inverted repeat, the GBSSI cDNA was divided into three regions, after which each region was tested in small inverted repeats in two orientations. To this end large numbers of independent transformants were produced for each construct. The results suggested that there was no effect of inverted repeat orientation on silencing efficiency. The percentage of transformants showing strong inhibition varied from 48% for a 3'-derived construct to 87% for a 5' as well as a middle region-derived construct. Similar to the large inverted repeats, the 3' sequences induced the least efficient silencing implying that the observed differences in silencing efficiency are caused by sequence differences. The small inverted repeat constructs with a repeat size of 500-600 bp and a spacer of about 150 bp were more efficient silencing inducers than the large inverted repeat constructs where the size of the repeat was 1.1 or 1.3 kb whilst the size of spacer was 1.3 or 1.1 kb. The results presented here show that size and sequence of the inverted repeat influenced silencing efficiency.

Presence of an intron in inverted repeat constructs does not necessarily have an effect on efficiency of post-transcriptional gene silencing

“…Figure 3B shows that no signal was present in transformants of IR MA-S, while transformants of IR MS-A did show hybridisation of siRNAs with probe A. From previous work, it was known that all these transformants, except the non-silenced transformant IR MA-S-26, showed accumulation of GBSSI siRNAs after hybridisation with the MF1+MR1 PCR product of the GBSSI cDNA as a probe (Heilersig et al 2006). Therefore, the lack of hybridisation with the spacer probe in transformants of IR MA-S indicates that no siRNAs of spacer sequences are formed.…”

“…GBSSI cDNA inverted repeat constructs were made as described by Heilersig et al (2006). The GBSSI cDNA (Visser et al 1989; sequence deduced from the genomic sequence published by van der Leij et al 1991) was divided in three regions; the 5¢, the middle and the 3¢ region.…”

Epimutant Induction as a New Plant Breeding Technology

“…However, although transformants obtained using antisense or invertedrepeat constructs show strong silencing of GBSSI, GBSSI mRNA remained present (Kuipers et al 1994, Heilersig et al 2006a. To obtain a transformant with no GBSSI mRNA, TGS technology has been used, while to induce allele-specific silencing, TGS could also be a good approach, since the four classes of GBSSI alleles are highly homologous in the coding region but vary in their promoter sequences (van de Wal et al 2001).…”

Correlation between computed equilibrium secondary structure free energy and siRNA efficiency

“…al. 14 showed that size and sequence of inverted repeats sequences had an effect on silencing efficiency. Ichihara et.…”