Increasing the amylose content of durum wheat through silencing of the SBEIIa genes.
BACKGROUND
High 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.


RESULTS
Amylose 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).


CONCLUSIONS
We 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.
Sestili
Francesco
F
Department of Agrobiology & Agrochemistry, University of Tuscia, Viterbo, Italy.
Janni
Michela
M
Doherty
Angela
A
Botticella
Ermelinda
E
D'Ovidio
Renato
R
Masci
Stefania
S
Jones
Huw D
HD
Lafiandra
Domenico
D
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics* / Q000378:metabolism*; D000688:Amylose / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D056625:Endosperm / Q000737:chemistry / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D006801:Humans; D010940:Plant Proteins / Q000032:analysis; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012231:Rhizobium / Q000235:genetics; D013213:Starch / Q000032:analysis; D014908:Triticum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D010940:Plant Proteins; D013213:Starch; D000688:Amylose; D015061:1,4-alpha-Glucan Branching Enzyme