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.
Both high-and low-molecular-weight glutenin subunits (LMW-GSThe glutenin fraction of the gluten proteins is primarily responsible for the viscoelastic properties of wheat (Triticum aestivum L.) flour doughs. It consists of various types of protein subunits that are linked together by intermolecular disulfide bonds. These form a polymeric mixture that has a broad molecular-weight distribution, with component polymers ranging from the dimeric forms with molecular weights as low as 60,000, to polymers containing many subunits with molecular weights in the millions (for review, see Kasarda, 1989; Wrigley, 1996). Variations in the types and amounts of subunits correlate with quality variations among wheat cultivars, probably by affecting the molecular-weight distribution of the glutenin polymers (Gupta et al., 1993(Gupta et al., , 1995. There are two main types of subunits, the HMW-GS and the LMW-GS, with the former having been much more extensively characterized than the latter.Difficulties in characterization of LMW-GS arose because they derive from many more genes than HMW-GS and because the subunits are somewhat insoluble after reduction of the intermolecular disulfide bonds (which is necessary for their purification, but which also breaks down intramolecular disulfide bonds to expose buried hydrophobic regions). Until recently, almost all attempts at cloning lmw-gs genes led to DNA sequences corresponding to similar protein products that are not representative of the major LMW-GS types; almost all had the apparent N-terminal sequence METSCIPGL-, relatively low molecular weights of about 35,000 or less, and a total of eight Cys residues, including the Cys at position 5 (for review, see Shewry and Tatham, 1997; Cassidy et al., 1998).In contrast to the apparently single type (with very minor variations) of the LMW-GS indicated by the DNA sequencing, two main types of LMW-GS have been defined on the basis of N-terminal amino acid sequences: the LMW-s and LMW-m types, with the former starting with the sequence SHIPGL-, and the latter represented by the METSHIPGL-, METSRIPGL-, or METSCIPGL-N-terminal sequences (Kasarda et al., 1988; Tao and Kasarda, 1989; Lew et al., 1992). The LMW-s types are predominant. They also tend to have higher molecular weights, in the approximate range of 35,000 to 45,000 relative to the LMW-m types, which seem to fall into the wider molecular-weight range of about 30,000 to 45,000 (Lew et al., 1992). In bread wheat cultivars, the LMW-m type with the METSHIPGLsequence was the next most abundant type of LMW-GS, followed by the METSRIPGL-type, whereas the METSCIPGL-N-terminal sequence, typical of the cloned sequences, appeared to be somewhat rare among the types defined by direct protein sequencing (Lew et al., 1992). Both LMW-s and LMW-m types are coded by genes present at the complex Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3 in hexaploid wheat). Only partial sequence information has been available for the LMW-s types because 1
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