Abstract
BackgroundHigh amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter.ResultsAmylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR).ConclusionWe have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.
Highlights
High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food
Biolistic transformation and selection of transgenic plants In total, 1954 immature embryos obtained from the Triticum durum cv
No significant differences in morphology and growth were observed between transgenic lines and either the corresponding "null" genotype that had lost the transgene by segregation, or untransformed plants
Summary
High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. The inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. Genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). The processing and end-use quality of wheat-based products depends on different factors such as protein content and composi-. Reserve starch is accumulated in the amyloplast organelles and is composed of two different glucosidic polymers, amylose and amylopectin. Amylopectin is the major constituent of starch in wheat endosperm and comprises about 70-80%; with amylose constituting the remaining 20-30%. Amylose and amylopectin are synthesized by two different pathways having a common substrate (ADP-glucose). A granule bound starch synthase (GBSSI) is involved in amylose synthesis, whereas amylopectin is produced by the concerted action of starch synthases (SSI, SSII, SSIII), starch branching enzymes (SBEI, SBEIIa and SBEIIb) and starch debranching enzymes of isoamylase- and limit dextrinase-type (ISA and LD) [2,3]
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