| A major class of wheat storage proteins is the high-molecular-weight(HMW) glutenins,which play a crucial role in determining the quality of wheat due to its contribution to the dough visco-elastic properties.The visco-elastic properties of bread wheat flour are associated with the formation of disulphide bridge-linked aggregates, through several cysteines(Cys)of HMW gluten peptides in the terminal,non-repetitive domains of the polypeptide,and the central domain composed of repeats of short peptide motifs.It is well established that there were multiple forms of proteins active on exogenous or endogenousα-amylases in wheat kernel,and proteinaceous dimericα-amylase inhibitors with inhibitory activity were againstα-amylase of various origins.It was known that the bulk of wheat albumins consisted of a few amylase isoinhibitor families very likely phylogenetically related and coded by a small number of parental genes.For weevil control,α-amylase inhibitors could be used through plant genetic engineering.However,many insects have severalα-amylases that differ in specificity,and successful utilization of a food source is dependent on the presence of aα-amylase for which there is no specific inhibitor.The structure and function of dimericα-amylase inhibitor genes with different cSNPs by direct sequencing,which is the most direct way to identify SNP polymorphisms, from cultivated wheat varieties and its diploid putative progenitor,were investigated. Fusarium head blight(FHB)is a major disease of cereals associated with at least seventeen Fusarium species,although there is no strong evidence for race-specific resistance.The disease can cause significant yield losses and reduce grain quality due to the production of mycotoxins.Breeding for resistance has taken a high priority worldwide,as genetic resistance,although partial,is considered to offer the most promising tool for the control of associated mycotoxin contamination in harvested grain.The mechanisms of resistance to FHB are complex and as yet,not fully understood and consequently breeding has been hampered by the difficulty of incorporating resistance into adapted high-yielding cultivars. The precise reasons for the lack of progress in incorporating exotic resistances into well-adapted commercial cultivars are not known.However,this lack of progress has led to a greater interest in understanding FHB resistance mechanisms.Our results about storage proteins were described as follows:1.Four high-molecular-weight glutenin subunit(HMW-GS)genes from Elytrigia elongata(Host)Nevski were characterized by determining the coding sequences of two x-type subunit genes Ee2.1 and Ee1.9,and two y-type subunit gene Ee1.8 and Ee1.5 with 2082,1938 1788 and 1488bp,respectively.The numbers of amino acids in the central repeat domains of these E genome glutenin subunits were considerably fewer than the other known HMW-GSs with substitutions,insertions and/or deletions involving a single or more amino acid residues.In spite of the high similarity at both 5' and 3' regions,Ee1.5 had only 1492 bp with several major deletions in its middle region.Thus,Ee1.5 is one of the smallest known HMW-GS genes.The N-terminal domain of Ee1.5 has 105 amino acid residues,while the other known y-type HMWGSs all have 104 amino acid residues. Comparison of Ee1.5 and 1Dy10 showed that Ee1.5 had a Cys residue at the middle of repetitive domain,while the Cys at the last of repetitive domain,which was common in other y-type HMW-GSs,was lost in Ee1.5.The altered Cys at the repetitive domain may likely have impact on the inter- or intra-molecular disulphide bonds.Moreover,an extra cysteine(Cys)was found in the repeated domain of x-type subunit Ee2.1.The difference in the number and position of Cys residues might be associated with the good dough quality. The extra Cys in the good-quality subunit Dx5 was at the beginning of the repetitive domain,while the extra Cys in Ee2.1 was at the central repetitive domain.To investigate the molecular evolution of the high-molecular-weight glutenin subunits (HMW-GSs)in Triticeae,multiple sequence alignment was carried out on the N-terminal sequences of 77 HMW-GSs.The sequence of N-terminal domain showed high homology in both x- and y-type subunits.Sequence alignment of the N-terminal domain of y-type subunits suggested that they shared similar primary structure with each other,not only in the length variation of the N-terminal domain but also they have less variation in amino acids in this domain than those of x-type subunits.Analysis of the x-type HMW-GSs genes revealed that there were three conservative HMW-GSs gene groups with distinct fragments at the beginning of the N-terminal domains of ORFs,defined by the presence or absence of 24 bp fragment,and the presence of 15 bp fragment,respectively.The frequency of mutations in the x-type and y-type HMW-GSs genes was 1.89 and 1.25 out of 10 bases, respectively.Sequence analysis revealed that the y-type HMW-GSs did not contain enough variation for evolutionary analyses.It is obvious that the HMW-GSs encoded by genes of A,B and D genomes in common wheat formed three clusters.The x-type HMW-GSs from Aegilops bicornis,Aegilops comosa,Aegilops cylindrica,Aegilops umbellulata and Aegilops uniaristata were more closely related to the subunits from the D genome rather than those from the B genome. 2.α-Amylases inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source.For weevil control,α-amylases inhibitors and their genes could be used to genetically engineer weevil resistant seeds.Seventeen new genes encoding 24 kDa family dimericα-amylases inhibitors had been characterized from cultivated wheat and its diploid putative progenitors.And the differentα-amylases inhibitors in this family,which were determined by coding regions single nucleotide polymorphisms(cSNPs)of their genes,were investigated.The amino acid sequences of 24 kDaα-amylases inhibitors shared very high coherence(91.2%).It indicated that the dimericα-amylases inhibitors in the 24 kDa family were derived from common ancestral genes by phylogenetic analysis.Eightα-amylases inhibitor genes were characterized from one hexaploid wheat variety,and clustered into four subgroups, indicating that the 24 kDa dimericα-amylases inhibitors in cultivated wheat were encoded by multi-gene.Forty-five cSNPs,including 35 transitions and 10 transversions,were found, and resulted in a total of ten amino acid changes.The cSNPs at the first site of a codon cause much more nonsynonymous(92.9%)than synonymous mutations,while nonsynonymous and synonymous mutations were almost equal when the cSNPs were at the third site.It was observed that there was Ile105 instead of Val105 at the active region Vai104-Val105-Asp106-Ala107 of theα-amylases inhibitor by cSNPs in some inhibitors from Aegilops speltoides,diploid and hexaploid wheats.Thirty genes encoding dimericα-amylases inhibitors were isolated from Triticum aestivum L.'Chinese Spring' and characterized by nucleotide and amino acid sequence analysis.Eleven representativeα-amylases inhibitor genes were identified and the deduced amino acid sequences of these genes were of high coherence(95.1%).These inhibitors and others obtained from the wheat EST database were clustered into three groups,the genes from 'Chinese Spring' were present in each group.Specific primer sets were designed for each group,based on the SNPs of these genes,and the chromosome locations of each group of inhibitor genes investigated by amplification of the 'Chinese Spring' ditelosomic lines.There were two and one groups of inhibitor genes on chromosomes 3BS and 3DS, respectively,whereas no group of inhibitor genes was found on chromosome 3AS.Thus, the primer set for each group of inhibitor genes was genome allele-specific.The two known inhibitors,0.53 and 0.19,were located on chromosomes 3BS and 3DS,respectively. The validity of the three genome allele-specific primer sets was confirmed by amplifications in 15 accessions of Triticum urartu,Triticum monococcum,Aegilops tauschii and Triticum dicoccoides.These results gave further support at the molecular level, that the 24 kDa dimericα-amylases inhibitors in cultivated wheat are encoded by a multigene family.Seventy-three gene sequences encoding monomericα-amylase inhibitors were characterized from cultivated wheat "Chinese Spring",group 6 nullisomic-tetrasomic lines of "Chinese Spring" and diploid putative progenitors of common wheat.The monomericα-amylase inhibitors from the different sources shared very high homology(99.54%).The differentα-amylase inhibitors,which were determined by the 24 single nucleotide polymorphisms(SN-Ps)of their gene sequences,were investigated.A total of 15 haplotypes were defined by sequence alignment,among which 9 haplotypes were found with only one single sequence sample.Haplotype H02 was found to be the main haplotype occurring in 83 WMAI sequence samples,followed by haplotype H11.The median-joining network for the 15 haplotypes of monomericα-amylase inhibitor gene sequences from hexaploid wheats was star like,and at least two subclusters emerged.Furthermore evidence of homologous recombination was found between the haplotypes.The relationship between nucleotide substitutions and the amino acid changes in WMAI of hexaploid wheats was summarized.It was clear that only 5 polymorphic sites in the nucleotide sequence of WMAI resulted in amino acid variations,and that should be the reason for different structure and function of inhibitors.However,little evidence could be found that there were WMAI genes in the A genome of hexaploid wheat,whereas it could conclude from our results that the A genome diploid wheat had WMAI genes.The overall information on the monomericα-amylase inhibitors from wheat and Aegilops strongly support the view that these inhibitors have evolved from a common ancestral gene through duplication and mutation.3.Eighty dimericα-amylases inhibitor genes were characterized from 68 accessions of the einkorn wheats Triticum urartu,T.boeoticum,and T.monococcum.The mature protein coding sequences of WDAI genes were analyzed.Nucleotide sequence variations in these regions resulted from base substitution and/or indel mutations.Most of the WDAI gene sequences from T.boeoticum and all sequences from T.monococcum had one nucleotide insertion in the coding region,such that theseα-amylases inhibitor sequences could not encode the correct mature proteins.We identified 21 distinct haplotypes from the diploid wheat WDAI gene sequences.A main haplotype was found in 15 gene samples from the Au genome and 35 gene samples from the Am genome.The T.monococcum and T. boeoticum accessions shared the same main haplotype,with 25 samples from T. monococcum and 10 from T.boeoticum.The WDAI gene sequences from the Au and Am genomes could be obviously clustered into two clades,but the sequences from the Am genome of T.boeoticum and T.monococcum could not be clearly distinguished.The phylogenetic analysis revealed that the WDAI gene sequences from the Am genome had accumulated fewer variations and evolved at a slower rate than the sequences from the Au genome.Although some accessions from only one or two areas had unique mutations at the same position,the diversity of WDAI gene sequences in diploid wheat showed little relationship to the origin of the accessions.One hundred and fifty-nine sequences encoding dimericα-amylases inhibitors were characterized from Triticum and Aegilops.These sequences had 375 nucleotides in length, among which there were 255 conserved sites,50 singleton variable sites(the nucleotide polymorphism only observed in a single accession)and 70 polymorphic sites(the polymorphisms found in two or more accessions).The frequency of SNPs in the B(a.k.a.S) genomes codingα-amylases inhibitor genes was 1.7 out of 10 bases.A total of 59 haplotypes were defined,among which 4 main haplotypes occurring in more than 10 genes and 36 haplotypes with single gene were found,indicating that the dimericα-amylases inhibitors might derive from a very limited number of ancestral genes.The phylogenetic median-joining network of the 59 haplotypes was highly star like with 5 haplotype groups, and at least 2 sub clusters emerged.Evolutionary distances of the 159 genes were calculated,and subjected to the construction of neighbour-joining trees that showed that theα-amylases inhibitor genes were divided into 5 groups,and each group had at least 2 subgroups.The neighbour-joining tree of the species indicated that the genes from common wheat and Triticum dicoccoides were closely related to those from Aegilops speltoides,and it was further supported by the median-joining networks analysis of the 59 haplotypes.These results revealed the important information on genome shaping events and processes occurring at dimericα-amylases inhibitor genes loci and contributed to our understanding of functional aspects of the dimericα-amylases inhibitor genes,as well as phylogenetic relationships between species.4.In this study,we aimed to reveal the structure and diversity of dimericα-amylase inhibitor genes in wild emmer wheat from Israel and to elucidate the relationship between the emmer wheat genes and ecological factors using single nucleotide polymorphism(SNP) markers.Another objective of this study was to find out whether there were any correlations between SNPs in functional protein-coding genes and the environment.The influence of ecological factors on the genetic structure of dimericα-amylase inhibitor genes was evaluated by specific SNP markers.A total of 244 dimericα-amylase inhibitor genes were obtained from 13 accessions in 10 populations.Seventy-five polymorphic positions and 74 haplotypes were defined by sequence analysis.Sixteen out of the 75 SNP markers were designed to detect SNP variations in wild emmer wheat accessions from different populations in Israel.The proportion of polymorphic loci P(5%),the expected heterozygosity He,and Shannon's information index in the 16 populations were 0.887, 0.404,and 0.589,respectively.The populations of wild emmer wheat showed great diversity in gene loci both between and within populations.Based on the SNP marker data, the genetic distance of pair-wise comparisons of the 16 populations displayed a sharp genetic differentiation over long geographic distances.The values of P,He,and Shannon's reformation index were negatively correlated with three climatic moisture factors,whereas the same values were positively correlated by Spearman rank correlation coefficients' analysis with some of the other ecological factors.The populations of wild emmer wheat showed a wide range of diversity in dimericα-amylase inhibitors,both between and within populations.We suggested that SNP markers are useful for the estimation of genetic diversity of functional genes in wild emmer wheat.These results show significant correlations between SNPs in theα-amylase inhibitor genes and ecological factors affecting diversity.Ecological factors,singly or in combination,explained a significant proportion of the variations in the SNPs,and the SNPs could be classified into several categories as ecogeographical predictors.It was suggested that the SNPs in theα-amylase inhibitor genes have been subjected to natural selection,and ecological factors had an important evolutionary influence on gene differentiation at specific loci.5.Genes encoding five allergens(WMAI,WDAI,WTAI,Fructose bisphosphate aldolase and Glycerinaldehyde-3-phosphate dehydrogenases)associated with baker's asthma were obtained from wheat seed.Compared with other known allergens,the protein structures were calculated by bioinformatics analysis.They had different biology function in plant.Only the three amylase inhibitors had similar amino acid sequences and 3D structure,while others were different.For the baker's asthma was IgE-mediated hypersensitivity,it could be concluded that these allergens had similar domain combined with IgE. 6.Fusarium head blight(FHB)is a major disease of cereals associated with at least seventeen Fusarium species,although there is no strong evidence for race-specific resistance.Here we report the evaluation of many of wheat-Thinopyrum addition lines, substitution lines and ditelosomic addition lines in cv.Chinese Spring(CS)for resistance to the spread of Fusarium graminearum in wheat heads.The lines CS-7E and CS-7ES showed the highest resistance levels.To gain some insights about the molecular mechanism(s)of resistance in those lines,large scale RNA profiling was performed on CS-7E,CS-7ES and CS.We analyzed gene expression changes in spike of Fusarium infected and uninfected at 3 time points during disease development.829 genes(probe sets)were found to be mote than 5 fold differentially expressed in at least one time point from at least one wheat lines. It was clear that more up-regulated expression genes were found than down-regulated genes after Fusarium infection.Obvious numerical features that could be seen in the Figureure were that the numbers of up- and down-regulated genes were same in CS,but the down-regulated genes in CS-7E(S)were 2.5 fold more than up-regulated genes.To identify groups of genes with similar expression patterns,a hierarchical clustering algorithm to the 70 genes with most different expression level that induced at any of the time points in response to fungal infection were carried out.The 70 genes were clustered into 5 groups by their expression level,and belonged to many different function categories. It was found some candidate genes were belong to specific biochemical pathways or known to have functional associations and responding to fungal infection.Many changes in potential phenylpropanoid pathway genes were observed including genes associated with the salicylic acid(SA)mediated response.Genes involved in the biosynthesis of Jasmonic acid(JA)were prominently found to responsive to fungal stress treatment in CS, CS-7E and CS-7ES in this study too.
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