Analysis of high pI α-Amy-1 gene family members expressed in late maturity α-amylase in wheat (Triticum aestivum L.)

Cong-Rong Cheng,Daryl Mares,Kolumbina Mrva,Klaus Oldach

doi:10.1007/s11032-013-9968-z

Abstract

Late maturity α-amylase (LMA) is a genetic defect involving the synthesis of high pI isozymes of α-amylase encoded by α-Amy-1 genes during the later stages of grain development. The aims of this investigation were to determine both the number of expressed α-Amy-1 genes and their relative transcript abundance. Sub-cloning and sequencing of expressed high pI α-amylase genes in developing wheat seeds revealed three insertion/deletion patterns in the 3′ untranslated region and numerous single nucleotide polymorphisms at the 3′ end of α-Amy-1. The genetic variations defined 36 α-Amy-1 gene sequences that were expressed on the onset of LMA in doubled haploid progenies (SpM25, SpM52 and SpM127) derived from the cross Spica (LMA)/Maringa (non-LMA). Five isoelectric point groups were predicted based on the translated partial coding sequences. The potential application of quantitative real-time RT-PCR in screening wheat genotypes for LMA is discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9968-z) contains supplementary material, which is available to authorized users.

Highlights

Alpha-amylase (EC number 3.2.1.1: 1,4-a-D-glucan glucanohydrolase), an endo-hydrolase which belongs to glycoside hydrolase family 13, acts on a-1,4glycoside linkages of starch (Davies and Henrissat 1995)
No high pI a-amylase protein was detected at any stage in the non-Late maturity a-amylase (LMA) lines but the time courses of change in grain moisture and grain dry weight were very similar to the LMA lines (Barrero et al 2013)
With the limited information available on the genomic DNA, cDNA and protein sequences of wheat high pI a-amylases in the National Center for Biotechnology Information (NCBI http://www.ncbi.nlm.nih.gov/) and Expert Protein Analysis System proteomics server (ExPASy http://expasy.org/) online databases, the known genomic and protein sequences of barley (Hordeum vulgare) a-amylase were used to search for high pI a-amylase genes in wheat

Summary

Introduction

Alpha-amylase (EC number 3.2.1.1: 1,4-a-D-glucan glucanohydrolase), an endo-hydrolase which belongs to glycoside hydrolase family 13, acts on a-1,4glycoside linkages of starch (Davies and Henrissat 1995). The major a-amylases in wheat (Triticum aestivum L.) are the high and low isoelectric point (pI) aamylases, encoded by the a-Amy-1 and a-Amy-2 genes, respectively. They have previously been referred to as germination and developmental or green isozyme, respectively, due to the appearance of the former during the germination stage and the latter during early grain development (Gale and Ainsworth 1984). Other methods involving Southern Blot analysis have yielded similar results (Lazarus et al 1985) In another similar experiment, up to 27 allelic variations of a-Amy-1 and a-Amy-2 genes have been proposed (Ainsworth et al 1985). Due to an inconsistent nomenclature of high and low pI a-amylase gene products in wheat and barley literature, we will refer to high pI or low pI a-amylase isozymes rather than AMY-1 and AMY-2

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