Cloning, Molecular Evolutionary Analysis And Genetic Transformation Of Novel High Molecular Weight Glutenin Genes From Aegilops Tauschii

High molecular weight glutenin subunits(HMW-GS) are one group of the major components of storage proteins in wheat seeds.Variation in the amount and composition of HMW-GS would affect and determine the flour processing quality.Aegilops tauschii (DD,2n=2x=14,syn.Ae.squarrosa) is the D genome donor of hexaploid wheat. Extensive allelic variation occurs in HMW-GS composition in Ae.tauschii more than in the subunits encoded by the D genome of modern wheat cultivars,and many new allelic variation does not exist in modern bread wheat.Therefore,cloning HMW-GS genes from Ae.tauschii would not only provide useful resources for wheat quality improvement by increasing the genetic diversity of the D genome,but also offer more genetic information on the evolutionary relativeship between bread wheat and Ae.tauschii.The objectives of this work were to isolate and characterize novel HMW-GS genes from Ae.tauschii.By comparing their deduced amino acid sequences with these previously characterized Dx or Dy type HMW-GS,it can help us to understand the evolution of Glu-D1 alleles.Besides, in order to investigate effect of 1Dx1.1~t on flour processing quality,1Dx1.1~t gene was transferred into E.coil and wheat.The main results are as follows:(1) Molecular cloning,bacterial expression and phylogenetic analysis of a novel x-type HMW-GS geneA novel x-type HMW-GS possessing a slightly slower mobility than that of subunit 1Ax1 in SDS-PAGE,which was designated 1Dx1.1~t in Ae.tauschii was identified.Its gene was amplified with allele-specific PCR(AS-PCR) primers.The open reading frame (ORF) of the gene was 2628 bp,encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1~t had high similarity with other 1Dx subunits but has two unique characteristics.Firstly,a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits is absent from subunit 1Dx1.1~t.Secondly,three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence(GQL) are present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1~t preferentially clustered with other known 1Dx subunits and at least three Ae.tauschii genotypes may have been involved in the polyploidization process during the origin and evolution of bread wheat.(2) Isolation,secondary structure predicton and phylogenetic analysis of two novel y-type HMW-GS genes Two novel y-type HMW-GS genes were cloned with AS-PCR method,designated 1Dy12.5~t and 1Dy9.5~t in Ae.tauschii,respectively.1Dy12.5~t encoded a protein that has much faster mobility than that of subunit 1Dy12 in SDS-PAGE.1Dy9.5~t encoded protein that has faster mobility than that of subunit 1By9,but slower mobility than that of subunit 1Dy10 in SDS-PAGE.A nucleotide sequence of 2,650 bp containing an open reading frame of 1,788 bp and upstream sequence of 862 bp was obtained.Nucleotide sequence comparisons showed that the 5'flanking region shared the conservative domains of y-type HMW-GS gene promoters,indicative of its transcriptional activity.However,there was a 5 bp(ACGAG) insertion before ATG(start codon) in 1Dy12.5~t subunit gene,which had not been observed in other known HMW-GS genes.1Dy9.5~t and 1Dy12.5~t had the typical primary structure of y-type HMW-GS.The deduced amino acid sequence of subunit 1Dy12.5~t showed greater similarity to subunit 1Dy13~t,differing by only nine amino acid residues.The deduced amino acid sequence of subunit 1Dy9.5~t displayed greater similarity to subunit 1Dy12.4~t and 1Dy,differing by only two and four amino acid residues, respectively.Compared the nucleotide acid sequence of subunit 1Dy9.5~t with that of subunit 1Dy12.4~t,we found that two 16 bp direct repeats(DR) existed in sites 806 and 1478 in the 1Dy9.5~t subunit gene,resectively,and inferred that these two 16 bp DR could make 224 amino acid residues deletion of 1Dy9.5~t which made 1Dy9.5~t to 1Dy 12.4~t.Five single nucleotide polymorphisms(SNPs) were detected in the coding region of the subunit 1Dy12.5~t gene,three of which resulted in the residue substitutions.Three SNPs were detected in the coding region of the subunit 1Dy9.5~t gene,among which two SNPs resulted in residue substitutions.Secondary structure prediction results showed that subunit 1Dy9.5~t could have a good bread processing quality while subunit 1Dy12.5~t could have a poor bread processing quality.Phylogenetic analysis showed that at least two genotypes of Ae.tauschii might be involved in the formation of bread wheat and Ae. tauschii could be also be involved in the formation ofAegilops cylindrica.(3) Transformation 1Dx1.1~t gene into wheat by particle bombardmentWe constructed the transgene expression vector of 1Dx1.1~t gene and transformed this vector into wheat by particle bombardment,which provided with the basis for further analyzing the function of this gene in influnceing flour processing quality.