| Wheat grain hardness is one of the main characteristics that determine the quality, the end-use and international trade through directly affecting both the milling and bread-making qualities. The Ha (Hardness) locus located on the short arm of chromosome 5D contains major genes controlling grain hardness in wheat, encoding Friabilin, which is on the surface of starch granules and the matrix proteins and regarded as biochemical marker of wheat grain hardness. Friabilin mainly contains Puroindolines (Pins, including Pin A and Pin B) and grain softness protein (GSP). Puroindolines bind with starch granules through their tryptophan motif, act as a "non-stick"protein, change adhesion between the starch granule and the gluten matrix and finally affect grain hardness. Many mutants of Pins have been characterized and different mutations had different effect on grain hardness, especially the mutations in tryptophan motif affected grain hardness greatly. In this study a number of Aegilops species, wild relatives of wheat, were analyzed on Pin a, Pin b and GSP in order to identify further allelic variants of all three genes at the Ha locus and expand the reservoir of wheat genetic resources. Through gene cloning and sequencing, all three genes were verified to occur in the 10 diploid, tetraploid and hexaploid Aegilops accessions with the C, D, S, M and U genomes. Altogether 7 new Pin a alleles, 9 new Pin b alleles and 5 new GSP alleles were identified with each containing two or more mutations. The new Pins' alleles shared high homologies with Pins from T. aestivum. They contained the tryptophan motif and five disulphide bonds formed by ten cysteines, which were specific to Pins in cereal crop. There existed three important new allelic variants of Pins, two Pin a mutations within the tryptophan motif and one Pin b mutation adjacent to the tryptophan motif. The differences between the Pin a alleles were observed at 14 positions, between the Pin b alleles at 24 positions and between the GSP alleles at 19 positions. The differences in the number of new alleles and frequency of substitutions between the Pin a and Pin b alleles are consistent with studies of bread wheat where Pin b has at least six allelic forms but Pin a has only a single form. The biological role of GSP remains uncertain. Residues 1-20 may form a signal peptide. If so, residues 21-35, which correspond to the arabinogalactan-binding peptide (AGP), may also correspond to the N-terminus of the mature protein. It is possible that the Ala/Asp mutation present in alleles 2 and 5 could affect glycosylation. Southern blot results showed that there were multiple copies of grain hardness-controlling genes in Aegilops. Four copies of Pin a in Ae. sharonensis and 5 copies of Pin b in Ae. triuncialis were verified. There were two copies of all the three genes in all the three Ae. kotschyi accessions. Pins' expression in endosperm at both transcriptional and translational levels was confirmed by RT-PCR and Western Blot analysis. Grain hardness of Aegilops was determined by scanning electron microscopy (SEM) of freeze-fractured grain. It was demonstrated that all except Ae. sharonensis were soft textured. This difference may be accounted for by the sequences and multiple copies of Pin a allele in this accession. The results showed that Aegilops contain grain hardness-controlling genes quite different from those of T. aestivum. It's possible that Aegilops provide an extensive reservoir of wheat genetic resources and lay a fundamental basis for further improvement of grain hardness of wheat variety through genetic engineering.
|
PDF Full Text Request