The Study On Mutation And Mechanism Of Waxy Gene In EMS Mutagenized Wheat Populations

We reported the identification of waxy mutants with varied electrophoretic motilities using the method of polyacrylamide gel electrophoresis?SDS-PAGE?and two-dimensional electrophoresis?2D-PAGE?from a total of 1200 plants of two EMS populations of common wheat.And further explore the molecular mechanism of varied electrophoretic motilities by gene cloning and prokaryotic expression.The major results were described as follows:1.Three EMS mutant wheat lines with varied mobility of Wx were identified.In the mutant lines M2-97-8 and M2-104-2 from AS201642,Wx-B1 has varied mobility,and Wx-A1 in the mutant line M2-101-1 which is from Shumai126 has varied mobility.2.The results of 2D-PAGE indicated that Wx-B1 isoelectric point positions of the two mutants are relatively more basic compared with the wild type and support that the mobility changes in Wx-B1 subunit of M2-97-8 and M2-104-2 were accurate.However,the isoelectric point of Wx-A1 appears in 2D-PAGE,which contradicts the result of SDS-PAGE.Then we conducted modified SDS-PAGE by added 4M urea into the separation gel and found a weak band below Wx-D1 in M2-101-1.3.The Wx-1 genomic DNA sequences of were determined with a total of 2793bp for Wx-B1 in M2-97-8 and M2-104-2 and 2781bp for Wx-A1 in M2-101-1.The results of sequence alignment showed that M2-97-8 Wx-B1 had two single-base mutations,G2002A and G2715A.A G1860A and a C1448T mutation were found in M2-104-2 Wx-B1and M2-101-1 Wx-B1,respectively.In M2-97-8,SNP mutation of G2002A on 8^th exon is a nonsense mutation.The rest of all mutations have resulted in the amino acid mutations.4.The results of prokaryotic expression indicated that the Wx-A1 and Wx-B1 from the mutant lines and wild lines can express the protein bands and its molecular weight was around 66kDa.In the prokaryotic expression system,Wx-B1 protein of M2-97-8 also had slightly greater mobility than that of the wild line,while Wx-B1 protein of M2-104-2 and Wx-A1 protein of M2-101-1 also had greater mobility than that of the wild type.In spite of all the mobility of induced protein bands was consistent of the predicted results,they migrated slower than those of extracted from seeds.5.The determination of starch content showed that there were significant differences between the mutant and the wild type.In the Wx-A1 mutant lines,the total starch and amylose content was lower than the wild type Wx-A1a;the Wx-B1 mutant line has higher total starch contents and amylose contents than those of wild type.In addition,the agarose gel electrophoresis of starch showed same results.Compared with the wild-type,M2-97-8and M2-104-2 present higher content of amylose in electrophoresis.Compared with the wild-type,M2-101-1 has a lower content of amylose in the electrophoresis results.