The Screening Of Mutants About Starch Synthase And Molecular Mechanism Of Wx Mutants From Wheat

Starch properties are important factors to determine wheat quality. The mutation at Wx-A, Wx-B, and Wx-D alleles of hexaploid wheats may cause lack of Wx-A, Wx-B, and Wx-D proteins, and influence obviously the wheat quality. In this report, iodine-dyeing-seed-section and 1D-SDS-PAGE were employed to survey the mutation of starch synthase in the progeny seeds of commom wheat cv. 8901 which were induced and handled by EMS (Ethyl methane sulphonate). Molecule mechanism and starch properties of mutants carrying different null Wx alleles were also recorded. The major results were presented below:2 Waxy mutants of 8901(signed in Wx-1,Wx-2)were isolated by screening M2 seeds derived from EMS mutagenized materials with iodine-dyeing of endosperm starch. 5 mutants of Wx-A null(signed in Wx-A-1,Wx-A-2,Wx-A-3,Wx-A-4,Wx-A-5)and 7 mutants of SGP-1B null(signed in SGP-1B-1,SGP-1B-2,SGP-1B-3,SGP-1B-4,SGP-1B-5,SGP-1B-6, SGP-1B-7)were isolated by screening M2 seeds derived from EMS mutagenized materials with 1D-SDS-PAGE of proteins bond to starch granules.The single plant of Wx-1, Wx-A-5, SGP-1B-3 and SGP-1B-4 were gained from above mutants. 1D-SDS-PAGE were employed to survey, the result showed that Wx-1 was the Wx-A, Wx-B and Wx-D null; Wx-A-5 was the Wx-A null; SGP-1B-3 and SGP-1B-4 were the SGP-1B null. There were no back mutations in M3 and M4 seeds.The mutations in Wx-1 had been characterized in molecular level in this study. 13 primer pairs of special primers were designed to distinguish the different genes among Wx-A, Wx-B, and Wx-D alleles in common wheat. Subsection cloning for three Wx genes in Wx-1 and wild-type was carried out.We got the Genbank number of Wx-A, Wx-B, and Wx-D gene from Wx-1(EU719609,EU719611,EU719613) and Wx-A, Wx-B, and Wx-D gene from wild-type (EU719608,EU719610,EU719612).Comparison of the genomic sequences between the null and wild-type.We have found that a 16bp detection at the beging of intron1 of Wx-A gene is responsible for a null allele at the Wx-A locus in Wx-1, whereas four missense mutations in the Wx-B locus and a nonsense mutation at exon4 in Wx-D allele, respectively.The relative RT-PCR was carried out to detect the express level of wx null genes. The lack of Wx-A, Wx-B, and Wx-D proteins in the mutant Wx-1 appeared to be largely due to nonsense-mediated mRNA decay.The correlation among waxy protein and wheat quality were studied. The waxy protein was responsible for amylose synthesis. Wild-type possessed the highest amylose content (17.22%) and the lowest in the Wx-1(0.22%), the amylase content of Wx-A-5 was lower than wild-type(15.18%). There were no detectable differences in the granule-size and granule morphology among Wx-1, Wx-A-5 and wild-type through scanning electron micrographs of starch granules isolated from the three wheat varieties.