Characterization, Coding Gene Cloning And Evolutionary Analysis Of LMW Glutenin Subunits In Common Wheat And Related Species

The storage protein is the main component affecting quality of wheat.It contains gliadin and glutenin.The glutenins are the major components of wheat storage proteins, which consist of high-molecular-weight glutenin subunits(HMW-GS)and low-molecular-weight glutenin subunits(LMW-GS).It is well known that the composition and content of HMW-GS and LMW-GS could influence the flour processing quality.In this study,we separated and identified some LMW-GSs in wheat and its related species by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).And then allele specific PCR primers were designed to amplify,clone and sequence these specific LMW glutenin genes,and phylogenetic analysis of storage protein gene family and secondary structure predication of glutenin were carried out.The main results were as the followings:1.Identification and molecular cloning of specific LMW-GS in commonwheat and related speciesLow molecular weight glutenin subunits of wheat and its related species were separated by SDS-PAGE in this study.The results showed that wheat low molecular weight glutenin subunits in SDS-PAGE pattern were mainly divided into two major regions:B subunits and C subunits.D subunits exist in some species.Genes encoding for these proteins were cloned in the next step.Four AS-PCR primers were designed according to the conservative sequences of 5'and 3' terminal domains to amplify LMW-GS genes from Triticum aestivum(AC and TR),Triticum dicoccoidesY 19 and Y21,Triticum monococcum(U7),Aegilops geniculata (PI287737,PI361880,PI573408,PI574475 and PI573409),Aegilops longissima(PI604108) and Triticurn zhukovskyi(PI352553,PI355706 and PI355707).Strongly amplified bands from all accessions were obtained,and then the amplified products were ligated into a pGEM-T easy vector(Promega)and sequenced by primer walking.The new cloned 23 genes were named and submitted to GenBank,respectively.2.Characterization analysis of LMW-GS genes in common wheat and related speciesTwo genes,TreLMW-ml and TmLMW-il,contain upstream,complete CDS and downstream,with 1509bp and 1274bp,respectively.In the upstream of LMW-GS genes, there are some conservative sequences,such as TATA Box,CAAT Box,Endosperm Box, which are important for the expression of them.Containing 386bp and 385bp nucleotides, the upstream of TreLMW-ml and TmLMW-il(partial sequence)had two TATA box at the position -76 to -71,and -59 to -54,and a CAAT box at -10 to -6.With a sequence of ACATGTAAA GTTAATAAGGTGAGTCAT,the Endosperm Box existed in both of the genes at -305 to -279.There were 193bp in the downstream of TreLMW-ml and TmLMW-il.Poly-adenosine signal,with the sequence of AAATAAA,presented at conservative position 72 to 77,130 to 136,and 141 to 147 under the stop codon in TreLMW-ml and in TmLMW-il.The coding sequences of TdLMW-ml,TzLMW-m2,AILMW-m2,TzLMW-ml, AILMW-ml,TreLMW-ml,AgLMW-ml,AgLMW-m2,AgLMW-m3,AgLMW-m4, AgLMW-m5,AgLMW-m6 were 1056bp,1056bp,1050bp,843bp,846bp,927bp,906bp, 903bp,903bp,903bp,912bp,903bp,respectively.TdLMW-ml,TzLMW-m2,AILMW-m2, AILMW-ml,TreLMW-ml,AgLMW-mI,AgLMW-m2,AgLMW-m6,AgLMW-m3, AgLMW-m4 had a double stop codon TGATAA,and encoding for 350,350,348,280,307, 300,299,299,299and 299 amino acid residues,respectively.Both TzLMW-ml and AgLMW-m5 had a stop codon TGA and and they encode for 280 and 303 amino acid residues,respectively.According to the deduced amino acid sequence,the molecular weight of the mature protein coded by the genes were 37.8KD,37.7KD,37.4KD,31.8KD, 31.8KD,32.8KD,31.7KD,31.6KD,31.8KD,31.6KD,31.9KD,31.8KD,respectively. Then,it could be presumed that all protein coded by the nine genes are C subunits.The coding sequence of AgLMW-il is 954bp,encoding 316 amino acid residues and ends at a double stop codon TGATAA.As the molecular weight of the mature protein deduced from amino acid residues was 34.2KD,the protein AgLMW-il encoded was regarded as C subunit.An extra cystenin and a poly-glutamine containing 27 glutamines occurred,which may have positive influence to flour quality.Only partial coding sequence of TmLMW-il was obtained as a small internal deletion occurred after ligated into a p^GEM-Teasy vector. The coding sequence of AcLMW-sl is 1074bp,encoding 356 amino acid residues and ends at a double stop codon TAATAA.As the molecular weight of the mature protein deduced from amino acid residues was 38.3KD.Analysis of sequence showed that this gene is LMW-s type.Some stop codons in the coding regions of AlLMW-il,pTzLMW-il, pTzLMW-i2,pAgLMW-ml,pAgLMW-il,pAgLMW-i2,pTreLMW-il and pTmLMW-ml makes these genes to be pseudo genes and unexpressed. 3.Multiple sequence alignment among proteins encoded by LMW-GS genesMultiple sequence alignment analysis between new genes and genes from GenBank was done by Bioedit software.LMW-m type genes were divided into three parts in order to compare with other nine LMW-m type genes.from GenBank.One new LMW-i type gene was compared to other nine LMW-i type genes from GeneBank.AcLMW-sl was aligned with other ten LMW-s type genes from GeneBank.Results showed that there were greater similarities among the same type of sequences.Except for AgLMW-il and AgLMW-m6 with one additional cystine residues in either the 47^thamino acid position of domainⅣor 63^thof DomainⅢ,the other sequences possess eight cysteine residues.We speculated that the emergence of the additional cysteine residues may increase the number of intre-molecular disulfide bonds,and thus increase visco-elastic of dough.There are lots of insertions and substitutions of amino acid residues in the repeated region of protein sequence deduced from TdLMW-ml,TzLMW-m2 and AILMW-m2.These insertions and deletions resulted in these proteins longer than others.Researches demonstrated that besides the number of cysteine residues,a long N-terminal repeat region also played a positive role in determine the quality of flour.4.Prediction for secondary structure of LMW-GSIn the present research,the new cloned genes were predicted with PSIPRED2.0.The results suggested that high content ofβturn existed in AlLMW-m2,TdLMW-ml and AgLMW-il,and these genes may have positive influences on the quality of flour.5.Evolutionary analysis of LMW-GSEvolutionary analysis among the new cloned genes and which published in Genbank showed that the LMW-m type and LMW-s type were more related compared that LMW-i type was separated earlier.The separation time calculated demonstrated that LMW-i type was separated with LMW-m and LMW-s in 15.22 million years ago(MYA),and the later two were separated in 9.149 million years ago(MYA)with each other.