【作者】 王际睿；

【导师】 郑有良； 魏育明； George Fedak； Thérèse Ouellet； 颜泽洪；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 依据功能不同可将种子蛋白划分为贮藏功能的谷醇溶蛋白和维持正常代谢功能的非醇溶性蛋白两类。高分子量谷蛋白亚基（HMW-GS）属于种子醇溶性蛋白,优质HMW-GS可明显改善小麦加工品质。小麦近缘属种中广泛存在的HMW-GS等位变异基因可能成为小麦品质改良的重要基因资源。麦类作物种子中还富含属于种子非醇溶性蛋白中清蛋白的α-淀粉酶抑制因子（AAI）,能有效抑制内源或外源α-淀粉酶活性,控制种子穗发芽、抑制昆虫繁殖。赤霉病是小麦主要病害之一,易感染开花期的小穗和发育早期的籽粒。赤霉菌会扰乱种子贮藏蛋白的合成或者消耗种子中的蛋白、糖来供自己繁殖,从而破坏发育中的籽粒。本文对小麦种子蛋白HMW-GS、AAI进行了基因克隆、与进化分析,并对抗赤霉菌材料的基因表达谱进行了分子鉴定,主要结果如下:1.从二倍体长穗偃麦草中分离并鉴定了4个新型HMW-GS编码基因,分别编码x-型亚基（Ee2.1、Ee1.9）与y-型亚基（Ee1.8、Ee1.5）。其中,Ee2.1和Ee1.5各存在1个与普通小麦HMW-GS相比特殊的Cys,推测这两个亚基在小麦品质育种中具有潜在利用价值。对小麦族不同物种HMW-GS的N-端序列进行比对分析发现,x-型和y-型HMW-GS均具有很高的同源性,但y-型HMW-GS变异程度比x-型亚基小。依据是否存在15bp或24bp的碱基插入将x-型HMW-GS及其编码基因划分为3种类型。系统进化分析表明,HMW-GS编码基因在漫长的基因复制过程中相对独立,且一直保持着其生物学功能。来自普通小麦A、B、D基因组的HMW-GS存在明显差异,具有染色体组特异性。2.从普通小麦及近缘物种中克隆获得与昆虫抗性有关的635个二聚α-淀粉酶抑制因子编码基因（WDAI）,377个单聚α-淀粉酶抑制因子（WMAI）和20个四聚α-淀粉酶抑制因子（WTAI）编码基因序列。对α-淀粉酶抑制因子序列与功能分析,探讨了该家族成员间的理化、功能（如等电点、迁移率、对蛋白酶的抑制范围和活性）差异的分子基础。建立了一种利用序列中SNP位点开发出特异标记引物将功能基因定位于普通六倍体小麦具体基因组的方法,由此将来自普通小麦的WDAI基因定位在3BS和3DS上。虽然从A基因组一粒系小麦中得到了WDAI、WMAI编码基因序列,但分子证据揭示普通小麦A基因组不含有WDAI编码基因,而是否含有WMAI编码基因则需要进一步研究。依据序列特征,推测小麦及其近缘物种中WDAI源于共同的祖先基因,而小麦及山羊草的WMAI也可能由一共同的祖先基因通过复制与突变进化而来,但是WMAI相似性明显比WDAI高,变异程度更低。3.依据序列分析发现二倍体一粒系小麦由于碱基插入导致无α-淀粉酶抑制因子活性的分子基础。一粒系小麦中WDAI编码基因差异与基因组来源有关,与地理来源关系不明显。A^m基因组的WDAI基因与A^u基因组的基因相比进化速率更慢、变异程度更低。依据普通小麦B基因组和拟斯卑尔托山羊草组S(S、S^l、S^s、S^sh和S^b)基因组159个WDAI基因序列中SNP位点差异,共发现59种呈“星”形分布的单倍型,并可归为5个主要的单倍型组。普通小麦和野生二粒小麦B基因组上的WDAI基因与来自拟斯卑尔脱山羊草S基因组上的WDAI基因最相似,但是推测多个拟斯卑尔托山羊草组物种可能通过种间杂交时的基因渐渗参与了多倍体小麦B基因组WDAI基因位点的形成。小麦族11个属20个基因组的WDAI基因的分子系统进化分析表明,WDAI编码基因差异与基因组系统进化有关,且在基因组分化以前该位点就已经存在。这些结果为深入了解WDAI基因位点上基因重组、整合事件及发生过程提供了重要信息,有助于加深对WDAI功能的了解。4.对以色列野生二粒小麦α-淀粉酶抑制因子基因的遗传结构、分化程度及其与生态因子间的关系进行了系统研究。WMAI序列所包含遗传信息较低;WTAI序列同样非常一致。无论是在居群内还是居群间,野生二粒小麦WDAI基因位点都存在变异,居群间遗传距离与地理来源关系密切。野生二粒小麦WDAI基因序列ω值（Ka/Ks）大于1,表明该基因位点为正选择位点,存在明显的选择压力导致WDAI基因序列产生多样性及遗传和功能差异。WDAI编码基因中SNP标记所揭示的16个居群遗传多样性指数（P）、Nei’s基因多样性（He）和Shannon指数（I）分别为0.887、0.404和0.589。SNP服从于自然选择,生态因子单独或联合作用导致WDAI基因序列中SNP位点的多样性,特别是几个水分因子对SNP位点有极显著影响,推测水分作为主要选择压力通过影响昆虫数量间接影响α-淀粉酶抑制因子遗传结构变异、分化程度。5.获得与过敏性哮喘相关的小麦种子潜在变应原编码基因。利用生物信息学手段对小麦种子蛋白质变应原与公认主要变应原α-淀粉酶抑制因子进行了比较,并预测编码蛋白质高级结构。由于过敏哮喘与由IgE介导的超敏反应有关,推测这些蛋白质具有能与IgE结合相似的结构域。6.对一套中国春-长穗偃麦草附加系、代换系、端体异附加系材料的赤霉病抗性进行了评价,发现7E附加系（CS-7E）和7ES端体异附加系（CS-YES）抗性最强,推测7E染色体上存在潜在的赤霉病抗性基因或能诱导/抑制普通小麦CS上抗性/易感基因表达的调控基因。利用基因芯片对CS、CS-7E、CS-7ES在赤霉菌诱导0-96小时后的表达谱进行了分析,初步筛选出70个赤霉病菌诱导差异表达的抗性相关基因;生物信息学比对和功能注解发现,候选基因所编码蛋白质或是属于某些特定的生理生化代谢途径关键酶、或是已知功能真菌诱导抗性应答蛋白、或与生物或非生物胁迫应答有关。利用实时定量PCR对16个候选基因的表达水平进行了验证,与芯片数据相比基因表达水平变化的方向（上调或下调）一致,但Q-PCR所检测到的差异要大于芯片所显示的表达差异。更多还原

【Abstract】 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.更多还原