| The waxy protein composition of Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and was further verified by two-dimensional gel electrophoresis.In addition,the molecular markers specific for null Wx-1 alleles were used to investigate the molecular characteristics of this locus.The results indicated that three accessions of Chinese wheat landraces exhibit inconsistent results between protein analysis and molecular marker detection.We speculate that it may have new allele mutations.The strategy of Genome Walking was used to isolate the unknown region,and the full gene sequence of 9160-bp was determined,reveal the molecular mechanism of gene silencing.The main results are as follows:1.The waxy protein composition of Chinese wheat landraces was analyzed using SDS-PAGE;of these,10 did not show the expression of Wx-A1(four accessions)or Wx-B1(six accessions)protein.We investigated the genotype of the accessions lacking Waxy protein subunits by using molecular marker detection.The results showed that four Wx-A1 alleles and three Wx-B1 alleles deletions were Wx-A1 b and Wx-B1 b,respectively.However,the rest three accession of Xiaobaipi,Maohongmai and Youmangyangmai showed inconsistent results between protein analysis and molecular marker detection.We further verified the Waxy protein composition of three accessions using two-dimensional electrophoresis and confirmed that their Waxy protein was absent in truth.2.The determined full Wx-B1 n sequences of the 9160-bp covering the Wx-B1 coding region and 3?-downstream region revealed that a 2170-bp fragment had been inserted at 2462 bp within the tenth exon of its open reading frame,leading to the absence of Wx-B1 protein.According to international nomenclature,we named it as Wx-B1 n.3.Sequence analysis indicated that the insertion fragment possessed the structural features of invert repeat and target repeat elements,which were the typical features of transposon.Further PCR analysis revealed that this fragment had moved,but not copied itself,from 3B chromosome to the current location in Wx-B1 n.4.The reason for the inactivation of Wx-B1 n was considerably different from those for the inactivation of Wx-B1 b,Wx-B1 k,and Wx-B1 m.The deletion including the waxy gene,the total size of the deletion is approximately 67-kb is the reason of Wx-B1 protein deficiency in the Wx-B1 b null allele.However,transposon insertion resulted in the silencing of Wx-B1 n.So,this 67-kb sequence is retained in Wx-B1 n.To our knowledge,this kind of structural mutation has never been reported in Wx-B1 alleles.It is possible that the deletion of a portion of this Wx-B1 b allele gene may affect endosperm and starch properties. |
