Post-transcriptional gene silencing of GBSSI in potato: effects of size and sequence of the inverted repeats.
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.
Heilersig
H J B
HJ
Graduate School Experimental Plant Sciences, Laboratory of Plant Breeding, Department of Plant Sciences, Wageningen University, P.O. Box 386, 6700 AJ, Wageningen, The Netherlands.
Loonen
A
A
Bergervoet
M
M
Wolters
A M A
AM
Visser
R G F
RG
D015152:Blotting, Northern; D016373:DNA, Antisense / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D030821:Plants, Genetically Modified; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013214:Starch Synthase / Q000235:genetics* / Q000378:metabolism
D016373:DNA, Antisense; D004269:DNA, Bacterial; D018076:DNA, Complementary; D018749:RNA, Plant; C036483:T-DNA; D013213:Starch; C434607:granule-bound starch synthase I; D013214:Starch Synthase