【作者】 祁鹏飞；

【导师】 郑有良； 魏育明； Thérèse Ouellet；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 小麦种子储藏蛋白主要包括谷蛋白和醇溶蛋白,它们决定小麦独特的加工品质。醇溶蛋白有四种类型,即α-、γ-、ω-和LMW-醇溶蛋白,占小麦种子储藏蛋白总量的40-50%,是人类日常摄取所需蛋白营养的主要来源之一。因此醇溶蛋白影响面粉的加工品质和营养品质。小麦近缘属种中含有丰富的醇溶蛋白基因资源。赤霉病是世界小麦三大主要病害之一,其主要致病菌是禾谷镰刀菌(Fusarium graminearum)。小麦感染赤霉病后不仅产量降低、种子储藏蛋白的合成受到抑制,而且会被赤霉菌分泌的真菌毒素污染,对食品加工和饲料行业造成严重威胁。因此世界各主要小麦生产国对赤霉菌毒素含量都有限制性标准。本文对醇溶蛋白进行了基因克隆、结构分析、二硫键形成方式和进化等方面的研究,并探索了植物激素与小麦赤霉病抗性之间的关系。主要结果如下1.从野生二粒小麦中克隆了4个α-醇溶蛋白基因(DQ140349-DQ140351)的编码序列。这些序列全长约800 bp,没有内含子。推测DQ140351和DQ140352在胚乳中可以正常表达,而DQ140349和DQ140350是假基因。忽略提前终止密码子和移码突变,得到这4条基因的推导氨基酸序列：它们均含有6个保守的半胱氨酸：重复区的重复单元与已报导的结果有区别；乳糜泻抗原主要分布于成熟蛋白N末端；脯氨酸密码子的使用有明显偏好；谷氨酰胺在整个蛋白中含量丰富,但在特定区域呈现集中分布的特点。2.从小麦中克隆了10条新型α-醇溶蛋白基因(Gli-ta、Gli-turgl-6和Gli-cs1-3)其中Gli-csl、Gli-cs2、Gli-cs3和Gli-turg6是假基因。这些新型α-醇溶蛋白具有显著不同于典型α-醇溶蛋白的特征。推测这些新型α-醇溶蛋白基因起源于发生在典型α-醇溶蛋白基因两个多聚谷氨酰胺区之间的非常规重组。与典型α-醇溶蛋白相比,Gli-cs3的分子量更大且有10个半胱氨酸残基,它的一级结构多一个特征区I和一个多聚谷氨酰胺区；其余9条序列缺失一个特征区1和一个多聚谷氨酰胺区,其推导蛋白的分子量较小且仅有2个半胱氨酸残基。Gli-talGli-ta-like a-醇溶蛋白基因在种子发育过程中能正常表达。SDS-PAGE分析表明Gli-ta的2个半胱氨酸都能参与形成分子间二硫键；回收种子提取物中与Gli-ta具有相似迁移率的条带并进行质谱分析,结果表明该条带中包含Gli-ta/Gli-ta-like a-醇溶蛋白,而且它们在种子内参与形成分子间二硫键。3.从Genbank中下载了79条节节麦α-醇溶蛋白基因序列和40条已知染色体来源的α-醇溶蛋白基因序列用于开发6D染色体特异的α-醇溶蛋白基因的SNP标记。总共设计了85条SNP标记引物用于检测68个潜在的6D染色体特异的α-醇溶蛋白基因的SNP位点。以中国春第6部分同源群染色体的缺体-四体材料和六倍体小麦的二倍体和四倍体祖先种的基因组DNA为PCR模板,验证得到11个6D染色体特异的SNP标记。以1份人工合成六倍体小麦及其亲本DNA为PCR模板,证明在节节麦和圆锥小麦杂交形成六倍体小麦的过程中六倍体小麦基因组会丢失部分α-醇溶蛋白基因。以节节麦、普通小麦及其4个亚种的DNA为PCR模板,证明至少两种遗传背景不同的节节麦参与了普通小麦D基因组的形成。4.从普通小麦及其近缘物种中克隆了170条γ-醇溶蛋白基因序列,其中138条是潜在的功能基因。这些基因开放阅读框的长度介于678-1089 bp。基因之间长度的差异主要是因为它们重复区的长度不一致,即重复单元(P(Q/L/S/T/I/V/WA)F(S/Y/V/Q/I/C/L)P (R/L/S/T/H/C/Y)Q1-2(P(S/L/T/A/F/H)QQ)1-2)的数目多少不一。序列多样性和连锁不平衡分析表明Y-醇溶蛋白基因家族具有非常高的遗传多样性。系统进化分析表明节节麦和斯卑尔脱山羊草组(Siptopsis)物种的γ-醇溶蛋白基因差异不显著(即相比于A基因组,B(S)和D基因组的γ-醇溶蛋白基因的亲缘关系更近)。根据半胱氨酸数目和相对位置的不同,可将Y-醇溶蛋白基因分为9种类型(C1-9)和17(SG-1至SG-17)个亚类；根据重复区长度的差异,γ-醇溶蛋白可被分为两类。氨基酸组成分析表明人体所需的8种必需氨基酸含量在γ-醇溶蛋白中差异巨大。SG-10.SG-12和重复区较短的γ-醇溶蛋白必需氨基酸含量较高。C9类和重复区较短的γ-醇溶蛋白几乎不含有乳糜泻抗原表位。不同γ-醇溶蛋白类群的乳糜泻抗原含量和必需氨基酸含量不同。因此下一步研究应当更重视重复区较短的γ-醇溶蛋白,其营养品质更高且几乎不含乳糜泻抗原。这为培育品质优良、无乳糜泻毒性的小麦品种提供了可能。5.从小麦和山羊草属物种中克隆了4条新型Y-醇溶蛋白基因(Gli-ngl-Gli-ng4),其中Gli-ng1、Gli-ng2和Gli-ng3的核酸序列完全一致。与典型Y-醇溶蛋白相比,新型Y-醇溶蛋白的分子量相对较小,其一级结构缺失非重复区、谷氨酰胺富集区、重复区的3’端和C-末端区的5’端。依据核酸序列特征,我们推测这些新型γ-醇溶蛋白基因起源于发生在典型Y-醇溶蛋白基因重复区和C-末端区之间的非常规重组,并找到了其可能的祖先基因。典型γ-醇溶蛋白具有8个或9个半胱氨酸,而Gli-ng1和Gli-ng4仅分别含有2个和3个半胱氨酸。cDNA克隆表明,Gli-ng1能在中国春种子发育过程中正常转录。SDS-PAGE分析表明Gli-ng1的2个半胱氨酸在体外均可参与形成分子间二硫键。系统进化分析表明这4条新型γ-醇溶蛋白基因与来源于小麦B基因组或山羊草S基因组的Y-醇溶蛋白基因亲缘关系最近。6.首次克隆得到了大麦和黑麦α-醇溶蛋白的基因序列。结构特征分析表明它们与Gli-ta/Gli-ta-like新型α-醇溶蛋白基因具有相似的结构,这说明非常规重组可能导致α-醇溶蛋白基因从黑麦和大麦基因组中丢失了。首次得到黑麦40Kγ-secalin基因序列,结构分析表明它们与典型γ-醇溶蛋白基因具有高度同源性,而且由于75Kγ-secalin的重复区比40Kγ-secalin长,因此分子量较大。系统分析了小麦族二倍体物种707个α-醇溶蛋白基因和280个γ-醇溶蛋白基因的多样性。根据一级结构特点,可将α-和γ-醇溶蛋白分别分为4和4类。不同类群醇溶蛋白的一级结构差异很大。根据半胱氨酸数目和相对位置的差异,可将具有潜在功能和完整开放阅读框的225条α-醇溶蛋白序列和168条γ-醇溶蛋白基因分别分为15和17种类型。一些α-和γ-醇溶蛋白包含奇数个半胱氨酸,这表明它们有可能形成分子间二硫键,因此有可能在种子内参与谷蛋白聚合物的形成。7.水杨酸在固体和液体培养基中能显著抑制F. graminearum菌丝的生长及孢子的萌发,并随着水杨酸浓度的提高最终杀死病菌；水杨酸在抑制菌丝体生长的同时也抑制F. graminearum合成真菌毒素DON；酸性条件下水杨酸对F. graminearum的抑制作用较强,在碱性条件下F. graminearum能使用水杨酸作为唯一碳源。高效液相色谱分析进一步证明F. graminearum具有代谢水杨酸的能力。为深入了解水杨酸对F. graminearum菌丝生长的抑制作用,我们比较了在液体培养基中添加水杨酸8h和24 h后F. graminearum基因在菌丝中的表达情况,结果表明F. graminearum能通过儿茶酚和/或龙胆酸途径代谢水杨酸。因此,虽然高浓度的水杨酸可直接影响孢子萌发率和菌丝生长速率,但F.graminearum具有代谢水杨酸的能力。将水杨酸与F. graminearum孢子混合后接种极易感染赤霉菌的小麦品种’Roblin’能显著减轻其赤霉病的症状；但若先接种水杨酸后接种孢子或者先接种孢子再接水杨酸,赤霉病的症状与对照没有显著差异。通过检测PR1、NPR1、Pdf1.2和PR4等与植物防御反应相关基因的表达情况,证明水杨酸诱导的抗病反应对提高小麦赤霉病抗性无显著作用。8.小麦穗子被F. graminearum侵染会累积水杨酸(SA)、茉莉酸(JA)、脱落酸(ABA)及其代谢物和生长素(IAA)及其缀合物。F. graminearum能合成IAA、少许ABA及其代谢物是这些激素在穗子中含量增加的原因之一。小麦穗子的激素反应网络显示SA刺激JA的累积,但不影响ABA和IAA；JA能增加ABA含量,降低SA水平,但对IAA无影响：施加ABA导致JA和IAA水平的提高及SA水平的降低；施加IAA引起ABA浓度的增加,但不影响SA和JA；12种受检赤霉素的含量均低于检测水平。JA和IAA可显著抑制F. graminearum菌丝的生长和孢子的萌发,而ABA只抑制菌丝的生长。在改良SNA平板上,IAA在抑制菌丝蔓延的同时能显著增加菌丝体的厚度。将JA或IAA与孢子混合后接种能显著减轻赤霉病症状。若不将JA/IAA与孢子在接种前混合,它们对赤霉病的抑制效果明显减弱。接种ABA显著降低小麦对赤霉病的抵抗力。除去植物激素对F. graminearum的直接影响,我们发现SA不影响,甚至对赤霉病有轻微的促进作用；JA和IAA显著抑制赤霉病发病水平；ABA促进赤霉病的发生和发展,但不影响F. graminearum的生长,同时意外的发现ABA显著抑制染病穗子积累真菌毒素DON。降低小麦穗子JA和ABA的水平证明它们分别能增强和减弱小麦对F. graminearum的抗性水平。通过检测ABT2、(3-expansin、LEA、PR1、NPR1、Pdfl.2和PR4等与植物激素信号途径相关基因的表达,证明SA与JA相互拮抗控制PRl基因的表达,而JA与ABA在拮抗调控小麦赤霉病抗性的同时,相互协作控制一些抗性相关基因的表达。更多还原

【Abstract】 The unique properties of wheat flour primarily depend on seed storage proteins, one of the most important sources of protein for human being, which mainly consist of glutenins and gliadins. Gliadins can be classified as four groups, i.e.α-,γ-,ω-and LMW-gliadins, and account for about 40-50% of the total seed storage proteins. Gliadins significantly affect the processing and nutritional qualities of flour. There is a great diversity of gliadin genes in wheat and its closely related species. Fusarium head blight （FHB） is one of the most common diseases on wheat worldwide. FHB can cause significant yield losses and quality reductions. Fusarium graminearum is the principal causal agent of FHB, and produces the mycotoxin deoxynivalenol （DON）. DON is a potent inhibitor of protein synthesis, and thereby presents hazards for both humans and animal health. Therefore, many countries have adopted maximum allowable limits for the mycotoxin DON in grain. This research focused on gene cloning, structural analysis, disulphide bond formation and gene evolution of gliadins, and investigated the role of salicylic acid on wheat FHB resistance. The main results are as follows:1. The coding regions of four a-gliadin genes of wild emmer wheat （GenBank No. DQ140349-DQ140352） were obtained. Each was about 800 bp long, and intronless. DQ140351 and DQ140352 were potentially functional in endosperm, whereas DQ140349 and DQ140350 were both pseudogenes. Alignment of their deduced amino acid sequences （by removing the in-frame stop condons and frameshift mutation） indicated that six cysteine residues were not randomly distributed, but conserved in the two unique domains; The motif unit of the repetitive domain for the four newly-detected genes was different from previous reports; the N-terminal part of a-gliadins was more toxic to celiac patients; codon usage for proline was biased; codons for glutamine were clustered into specific regions.2. Ten novel a-gliadin genes （Gli-ta, Gli-turgl-6 and Gli-csl-3） with unique characteristics were isolated from wheat, among which Gli-cs1, Gli-cs2, Gli-cs3 and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical a-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly due to illegitimate recombination. Consequently, Gli-cs3 contained ten cysteine residues, while there were only two cysteine residues in the other nine sequences. Gli-ta/Gli-ta-like a-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in vitro expressed Gli-ta could form intermolecular disulphide bonds, and act as a chain extender. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular weight a-gliadins, which is a characteristic of the novel a-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo. 3. Seventy-nine a-gliadin sequences cloned from Aegilops lauschii and another 40 a-gliadin genes with known chromosome locations were downloaded from Genbank and used to design chromosome 6D-specific SNP markers for a-gliadin genes. A total of 85 SNP primers were designed to detect 68 candidate chromosome 6D-specific SNPs. Experimental tests revealed 11 chromosome 6D-specific SNP markers by using genomic DNA from homoeologous group-6 nullisomic-tetrasomic lines of Chinese Spring and putative diploid and tetraploid ancestors of hexaploid wheat as PCR templates. Detection of SNP markers in one synthetic hexaploid wheat and its parental lines indicated that some a-gliadins genes were lost from the Gli-2 loci during the formation of hexaploid wheat by amphidiploidization of the genomes of Triricum turgidum and Aegilops tauschii. Detection of these SNP markers in Ae. tauschii, Triticum aestivum and its four subspecies indicated that at least two genetically distinct sources of Ae. tauschii contributed germplasm to the D genome of T. aestivum.4. A total of 170 y-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences were putatively functional. The ORF length of these sequences ranged from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity ofγ-gliadins. The motif P（Q/L/S/T/I/V/R/A）F（S/Y/V/Q/I/C/L）P（R/L/S/ T/H/C/Y）Q,₂（P（S/L/T/A/F/H）QQ）1-2 is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that y-gliadins are highly diverse. Phylogenic analyses indicated that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classifiedγ-gliadins into two types based on the length of the repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids inγ-gliadins, and thoseγ-gliadins from subgroup SG-10 and SG-12 and y-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that y-gliadins with a pattern of C9 and y-gliadins with a short repetitive domain almost lack any epitopes. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, each group/subgroup contributes differently to nutritional quality and epitope content, and it would be possible to breed wheat varieties containing the y-gliadins that are less, even non-toxic and more nutritional.5. Four novel y-gliadin genes （Gli-ngl to Gli-ng4） were cloned from wheat（Triticum aestivum） and Aegilops species. The novelγ-gliadins were much smaller in molecular size when compared to the typical y-gliadins, which was caused by deletion of the non-Repetitive domain, Glutamine-rich region,3’part of the Repetitive domain and 5’part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ngl and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ngl, as the representative of novel y-gliadin genes, has been sub-cloned into an E. coli expression system. SDS-PAGE indicated that both cysteine residues of Gli-ngl could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ng1 from seed cDNA of T. aestivum cv’Chinese Spring’ suggested that these novel y-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novelγ-gliadin genes had a closer relationship with those from the B（S） genome of wheat.6. Alpha-gliadin genes with unique structure originating from illegitimate recombination were obtained from barley and rye, indicating that the absence of a-gliadin genes in barley and rye is a result of illegitimate recombination. Analysis of the full coding region of 40K y-secalin genes indicated that they have a very close structural relationship to the typical y-gliadin genes of wheat, and that the higher molecular weight of 75K y-secalins than that of 40K y-secalins is a result of the addition of repetitive sequence. Molecular diversity ofα-and y-prolamin genes was systematically investigated. According to their distinct constitutions of protein domains, we classifyα-andγ-prolamins into 4 and 4 groups. According to the number and placement of cysteine residues, we defined 15 and 17 cysteine patterns for a-and y-prolamins, respectively. Some of theα-andγ-prolamins contain odd numbers of cysteine residues, which strongly suggests that they possibly participate in the formation of intermolecular disulphide bonds.7. The mycelial growth and spore germination of F. graminearum were significantly inhibited, and eventually halted by the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol （DON）. However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a sole carbon source. HPLC analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum spores in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cultivar Roblin. In contrast, no inhibition was observed when SA and spores were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdfl.2 and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrates that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.8. F. graminearum challenge induced the accumulation of SA, JA, ABA and its metabolites, IAA and IAA conjugates in wheat heads, partially because F. graminearum is able to produce IAA, and a little bit ABA and its metabolites. Testing of the hormone networking showed that application of SA promoted the accumulation of JA, whereas it did not affect ABA and IAA levels; application of JA increased the amount of ABA and reduced the level of SA, whereas did not affect IAA; application of ABA resulted in an increase in the JA and IAA levels and a decrease in the SA level; application of IAA induced an increased concentration of ABA, whereas it had no affect on SA and JA; GAs monitored were not quantifiable in those assays. The growth of F. graminearum mycelia and the germination of spores could be significantly inhibited by JA and IAA, whereas ABA only suppressed mycelial growth within the tested concentrations. IAA significantly increased density of mycelia on modified SNA plates, while inhibiting the spread of mycelia. Inoculation of F. graminearum spore suspensions containing either JA or IAA-containing solution led to reduced visual FHB symptoms. In contrast, less inhibition was observed when JA or IAA and spores were inoculated sequentially. Inoculation of ABA in heads enhanced susceptibility to FHB. To further understand the effect of hormones on FHB resistance, we sequentially inoculated hormones and spores into each spikelet of a spike. As a result, SA did not affect, and even slightly increased FHB severity; JA and IAA significantly reduced FHB levels; ABA promoted the visual disease symptoms, while unexpectedly suppressing the accumulation of mycotoxin without affecting fungal biomass. Down-regulation of endogenous JA and ABA levels confirmed that JA and ABA respectively functioned as a positive and a negative regulator of wheat defense response to F. graminearum infection. The expression patterns for the wheat ABT2,β-expansin, LEA, PR], NPR1, Pdfl.2 and PR4 genes, a group of indicator genes for the defence response and hormone signaling, suggested that SA antagonistically interacted with JA to control the expression of PR1, and synergistic interactions existed between JA and ABA, even though they antagonistically modulated FHB resistance in wheat.更多还原