Biomolecular Analyses of Starch and Starch Granule Proteins in the High-Amylose Rice Mutant Goami 2

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• 作者：Vito M. Butardo ; Jr. ; Venea Dara Daygon ; Michelle L. Colgrave ; Peter M. Campbell ; Adoracion Resurreccion ; Rosa Paula Cuevas ; Stephen A. Jobling ; Ian Tetlow ; Sadequr Rahman ; Matthew Morell ; Melissa Fitzgerald
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2012
• 出版时间：November 21, 2012
• 年：2012
• 卷：60
• 期：46
• 页码：11576-11585
• 全文大小：458K
• 年卷期：v.60,no.46(November 21, 2012)
• ISSN：1520-5118

文摘

Elevated proportions of amylose in cereals are commonly associated with either the loss of starch branching or starch synthase activity. Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound proteins revealed that SSI and SSIIa accumulate inside the mature starch granules of Goami 2, which is similar to the amylose extender mutant IR36ae. However, unlike the amylose extender mutants, SBEIIb was still detectable inside the starch granules of Goami 2. Detection of SBEIIb after protein fractionation revealed that most of the SBEIIb in Goami 2 accumulates inside the starch granules, whereas most of it accumulates at the granule surface in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound proteins failed to detect any peptide sequence mutation or major post-translational modifications in Goami 2. Moreover, the signal peptide was found to be cleaved normally from the precursor protein, and there is no apparent N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between exon and intron 11, which could potentially introduce a premature stop codon and produce a truncated form of SBEIIb. It is suggested that the mutation responsible for producing high amylose in Goami 2 is not due to a defect in SBEIIb gene as was observed in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic basis of this mutation will be important in identifying novel targets for increasing amylose and resistant starch contents in rice and other cereals.

Keywords:

amylopectin; ion trap tandem MS; Goamy 2; mass spectrometry; matrix-assisted laser desorption/ionization MS; resistant starch; single-nucleotide polymorphism; Suweon 464