The Molecular Mechanism For The Mutation Of Wx-a1 In EMS Wheat Mutant And Its Effects On Starch Properties

An EMS mutagenized population was generated using common wheat cv.SM126 consisting of 10,600 M2 plants.The Waxy protein composition was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),one Wx-A1 null mutant M2-31 was identified.The strategy of PCR amplification and gene cloning was used to reveal the molecular mechanism of gene mutation.Effect of the mutation on starch properties was studied by methods of starch contents testing and granule morphology scanning.The main results are as follows:1.The complete coding region of Wx-A1 gene of SM126 and M2-31 mutants was cloned.The results of sequence alignments showed that a single-base changed from G to A at nucleotide sequence position 2168 bp downstream of the start codon,was located at the splice donor site within the 8th intron.2.The SNP in Wx-A1 of M2-31 did not occur in the exon region,but appeared at RNA splicing site.Thus,we focused on the transcripts of Wx-A1 in M2-31 mutant.Totally 52 c DNA clones derived from M2-31 were sequenced,all of them were found to be incorrectly spliced and can be summarized as five types of variations.The deletion of the exon and the exclusion of intron were structural features in abnormal splicing RNA.3.The results of starch content examination indicated that the lack of Wx-A1 has led to changes in starch properties in the M2-31 mutant,with the decreased of amylose and total starch contents.The results of granule morphology scanning showed that protein matrix is tightly bound to the starch granules in M2-31 mutant.This is the characteristic of hard wheat.The grain hardness of M2-31 was significantly higher than that of SM126.4.We looked further into the main gene controlling the wheat hardness(Pina-D1 and Pinb-D1).Pina-D1 of SM126 and M2-31 were common null Pina-D1 b,the genotype of Pinb-D1 were Pinb-D1 a,while the expression level of Pinb-D1 in M2-31 was lower than that of M2-31,We hypothesized that be correlated with the increased grains hardness of M2-31.