两对高分子量麦谷蛋白亚基等位基因及其相互作用对小麦面筋品质的影响

小麦谷蛋白赋于面筋弹性,其亚基组成类型对加工品质有着重要的决定作用。采用一对杂交组合(烟农19×安农9914)的后代,随机选择至F3,种植871个F4穗系,分别检测了高分子量麦谷蛋白亚基(high molecular weight glutenin subunits,HMW-GS)、低分子量麦谷蛋白亚基(low molecular weight glutenin subunits,LMW-GS)组成、SDS沉降值和和面图指标,分析了麦谷蛋白等位亚基及其相互作用对品质的影响。结果表明:该群体麦谷蛋白组成仅在Glu-A1(1亚基或N亚基)与Glu-B1(14 15亚基或17 18亚基)位点有差异。Glu-A1位点1亚基的SDS沉降值显著高于N亚基,1亚基的峰高、7 min尾高显著大于N亚基,而在和面时间、7 min带宽以及衰落角(耐揉性),两亚基间差异不显著。Glu-B1位点亚基间SDS沉降值17 18>14 15/17 18>14 15,和面时间、7 min带宽两个指标17 18亚基显著高于14 15亚基,衰落角显著小于14 15亚基,峰高和7 min尾高差异不显著。对Glu-A1和Glu-B1两位点互作,除7 min带宽互作达5%显著水平外,其它四个指标均未达显著水平。1亚基相对于N亚基,17 18亚基相对于14 15亚基,虽以SDS沉降值为标准其效应相当,但两者却作用于面团的不同性能。     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定(英文)

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列。表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似。在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区。但是,本研究所克隆的沉默型1Ay基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白。讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及1Ay基因沉默的机制。     

小麦高分子量麦谷蛋白亚基1Dx5启动子驱动外源基因的表达

将小麦高分子量麦谷蛋白亚基(HMW-GS)基因的胚乳组织特异性表达启动子驱动的外源突变型1Dx5基因和gus基因导入小麦中.对其转基因植株连续3代的跟踪研究表明,突变型1Dx5基因的重复序列导致其表达蛋白分子量增大,并影响其它1Bx17 1By18亚基基因的表达.组织化学分析观察到gus基因在1Dx5基因启动子驱动下的表达表现出胚乳组织特异性,在开花2周后开始表达,表达量呈持续上升,至腊熟期达到最高,其次为籽粒成熟期.     

小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

 Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively. Received : 30 May 1997 / Accepted : 29 July 1997     

外源1Dx5基因导致小麦高分子量麦谷蛋白亚基表达变异

目的:为了结合基因枪转化和传统杂交方法培育优质小麦品种,对转基因小麦和国内主栽小麦品种杂交后代外源基因遗传表达行为进行了研究。方法:采用SDS-PAGE对2个小麦杂交组合川89-107×B72-8-11b和鄂麦18×B72-8-11b的杂交及回交后代籽粒进行高分子量麦谷蛋白亚基遗传表达分析。结果:在亲本中能够稳定超量表达的外源基因1Dx5在杂交后代中出现了不同的表达量,而且在外源基因的影响下,杂交后代出现了新的、杂交亲本并不表达的高分子量麦谷蛋白亚基。结论:多拷贝的外源基因在不同于受体环境的细胞质中的表达发生了变化,且由于外源基因的插入引起了内源高分子量麦谷蛋白亚基组成的变异。     

小麦Na^＋／H^＋反转运蛋白基因的克隆和特性

以水稻 (OryzasativaL .)Na /H 反转运蛋白cDNA片段为探针 ,从小麦盐胁迫cDNA文库中筛选和克隆了 2个小麦Na /H 反转运蛋白基因 ,分别命名为TaNHX1和TaNHX2。序列分析表明TaNHX1为 2 0 2 9bp ,包含一个完整的 16 38bp的ORF ,编码 5 4 6个氨基酸 ,其中含有DIFFIYLLPPI跨膜区。TaNHX2为 16 93bp ,包含部分ORF及 80 8bp的 3′_UTR。这 2个基因与已知的水稻、拟南芥 (Arabidopsisthialiana)和滨藜 (Atriplexgmelini)中的同类基因NHX的相似性约为 70 %。RT_PCR分析表明小麦苗经 4 0 0mmol/LNaCl处理 1h后 ,TaNHX1的转录水平有所提高。     

Modification of the Low Molecular Weight (LMW) Glutenin Composition of Transgenic Durum Wheat: Effects on Glutenin Polymer Size and Gluten Functionality

The low-molecular weight (LMW) glutenin subunits are major determinants of the viscoelasticity of durum wheat gluten, and therefore of its technological quality, with both quantitative effects and qualitative effects. We have modified the LMW glutenin subunit composition of the durum wheat cultivar Ofanto by expression of a transgene encoding a B-type LMW glutenin subunit and have carried out detailed analyses of two independent transformed lines in order to assess the effect of the transgene on the size distribution of the glutenin polymers and on their functional properties. In one line the expression of the transgene led to an increase in the amount of large glutenin polymers resulting in stronger and more stable dough. In the second line, however, the expression of the transgenic subunit was accompanied by decreased expression of endogenous LMW subunits with consequent detrimental effects on glutenin polymers and dough viscoelasticity. These results demonstrate that the LMW glutenin subunits contribute to the functional properties of wheat by influencing the amount and the distribution of glutenin polymers and indicate that either plant breeding or GM technology can be used to 'fine tune' the properties of durum wheat for different end uses by manipulating the amount and structures of individual LMW subunit proteins.     

小麦HMW-GS 12基因克隆及植物表达载体构建

目的:为了利用基因遗传转化改良小麦品质,采用聚合酶链式反应(PCR)技术。方法:从小麦品种东农7742基因组DNA中扩增并克隆了小麦高分子量谷蛋白12亚基基因(HMW-GS 12)。结果:序列分析结果表明,该基因全长1 980bp,其核苷酸顺序和推导的氨基酸顺序与已发表的序列相比,同源性分别为99.5%和99.7%。经过基因拼接,分别构建了胚乳特异性表达和组成型表达的高分子量谷蛋白12亚基基因的两个植物表达载体pDNPPBIHG和pUbPBIHG。     

The Ph1 and Ph2 loci play different roles in the synaptic behaviour of hexaploid wheat Triticum aestivum

Triticum aestivum is an allohexaploid wheat (AABBDD) that shows diploid-like behaviour at metaphase-I. This behaviour is influenced by the action of several loci, Ph1 and Ph2 being the main loci involved. To study the effect of these two loci on chromosome pairing in T. aestivum we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late-zygotene, and at pachytene, of three different genotypes of cv Chinese Spring: standard line, ph1b and ph2b mutants. The analysis of the synaptic progression showed that only a few nuclei accomplish synapsis in the ph2b genotype, whereas most nuclei completed synapsis in the standard and ph1b genotypes. This result indicates that the Ph2 locus affects synaptic progression. The number of synaptonemal complex (SC) bivalents and of the different SC multivalent associations were determined in each nucleus. The mean number of lateral elements involved in SC multivalent associations (LEm) at mid- zygotene was relatively high and showed similar values in the three genotypes. These values decreased progressively between mid-zygotene and pachytene in the genotypes with the Ph1 locus because of the transformation of multivalents into bivalents. In the ph1b genotype, this value only decreased between late-zygotene and pachytene. Therefore, multivalent correction was more efficient in the presence than in the absence of the Ph1 locus.It is concluded that the Ph1 and Ph2 loci bring about diploidization of allohexaploid wheat via a different mechanism. Received: 31 July 2000 / Accepted: 15 November 2000     

PCR analysis of x- and y-type genes present at the complex Glu-A1 locus in durum and bread wheat

Genes (x-type) corresponding to different high-molecular-weight glutenin subunits encoded at the Glu-A1 locus present in bread- and durum-wheat cultivars have been selectively amplified by the polymerase chain reaction (PCR). DNA fragments corresponding to an unexpressed x-type gene were also amplified. As unexpressed y-type genes may or may not contain an 8-kb transposon-like insertion, two different sets of primers were designed to obtain amplification of DNA fragments corresponding to these genes. Amplified DNA fragments were also digested with restriction enzymes. The digestion patterns of amplified fragments corresponding to unusual x-type subunits showed similarities with genes encoding the most common subunits 2^* and 1. The unexpressed amplified x-type gene showed a restriction pattern similar to the one obtained with the allelic gene encoding high-molecular-weight glutenin subunit 1; homologies were also found within the repetitive region of the linked y-type genes. On the basis of these observations it is postulated that an ancestral active x-type gene, most likely corresponding to subunit 1, was silenced following the insertion of the 8-kb transposon-like fragment into the linked y-type gene. Received: 8 April 1996 / Accepted: 30 August 1996     

青海省小麦品种中Yr10和Yr15基因及其1BL/1RS易位的分子检测

利用抗条锈病基因Yr10和Yr15的SCAR和Barc8标记以及1BL/1RS易位的复合标记,对青海省育成和引进的137份小麦品种进行检测,以明确Yr10和Yr15基因以及1BL/1RS易位在青海小麦品种资源中的分布.结果显示:在137份材料中,有4份检测到Yr10基因,19份检测到Yr15基因,分别占参试材料的2.9%和13.9%,没有检测到同时携带Yr10和Yr15基因的材料;有22份材料为1BL/1RS易位,占参试材料的16.1%.研究表明,青海省大部分小麦抗锈品种及1BL/1RS易位品种为外引种品种.     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.     

The Characterization and Geographical Distribution of the Genes Responsible for Vernalization Requirement in Chinese Bread Wheat

The frequency and distribution of the major vernalization requirement genes and their effects on growth habits were studied. Of the 551 bread wheat genotypes tested, seven allelic combinations of the three Vrn-1 genes were found to be responsible for the spring habit, three for the facultative habit and one for the winter habit. The three Vrn-1 genes behaved additiveiy with the dominant allele of Vrn-A1 exerting the strongest effect. The allele combinations of the facultative genotypes and the discovery of spring genotypes with ＂winter＂ allele of Vrn-1 implied the presence of as yet unidentified alleles/genes for vernalization response. The dominant alleles of the three Vrn-1 genes were found in all ten ecological regions where wheat is cultivated in China, with Vrn-D1 as the most common allele in nine and Vrn-A1 in one. The combination of vrn-A1vrn-B1Vrn-D1 was the predominant genotype in seven of the regions. Compared with landraces, improved varieties contain a higher proportion of the spring type. This was attributed by a higher frequency of the dominant Vrn-A1 and Vrn-B1 alleles in the latter, Correlations between Vrn-1 allelic constitutions and heading date, spike length, plant type as well as cold tolerance were established.     

Developmental traits affecting low-temperature tolerance response in near-isogenic lines for the Vernalization locus Vrn-A1 in wheat (Triticum aestivum L. em Thell)

Investigation of low-temperature (LT) tolerance in cereals has commonly led to the region of the vyn-A1 vernalization gene or its homologue in related genomes. Two cultivars, one a non-hardy spring wheat and one a very cold-hardy winter wheat, whose growth habits are determined by the Vrn-A1 (spring habit) and vrn-A1 (winter habit) alleles, were chosen to produce reciprocal near-isogenic lines (NILs). These lines were then used to determine the relationship between rate of phenological development and the degree and duration of LT tolerance gene expression. Each allele was isolated in the genetic backgrounds of the non-hardy spring wheat 'Manitou' and the very cold-hardy winter wheat 'Norstar'. The effects of each allele on phenological development and low-temperature tolerance (LT50) were determined at regular intervals over a 4 degrees C acclimation period of 0-98 d. The vegetative/reproductive transition, as determined by final leaf number (FLN), was found to be a major developmental factor influencing LT tolerance. Possession of a vernalization requirement increased both the length of the vegetative growth phase and LT tolerance. Similarly, increased FLN in spring Norstar and winter Manitou NILs delayed their vegetative/reproductive transition and increased their LT tolerance relative to Manitou. Although the winter Manitou NILs had a lower FLN than the spring Norstar NILs, they were able to extend their vegetative stage to a similar length by increasing the phyllochron (interval between the appearance of successive leaves). Cereal plants have four ways of increasing the length of the vegetative phase, all of which extend the time that low-temperature tolerance genes are more highly expressed: (1) vernalization; (2) photoperiod responses; (3) increased leaf number; and (4) increased length of the phyllochron.     

Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat

The present work reports new PCR markers that amplify the complete coding sequence of the specific alleles of the high molecular weight (HMW) glutenin genes. A set of AS-PCR molecular markers was designed which use primers from nucleotide sequences of the Glu-A1 and Glu-D1 genes, making use of the minor diffeences between the sequences of the x1, x2* of Glu-A1, and the x5 and y10 of Glu-D1. These primers were able to distinguish between x2* and the x1 or xNull of Glu-A1. Also x5 was distinguishable from x2, and y10 from y12. The primers amplified the complete coding regions and corresponded to the upstream and downstream flanking positions of Glu-A1 and Glu-D1. Primers designed to amplify the Glu-A1 gene amplified a single product when used with genomic DNA of common wheats and the xNull allele of this gene. This work also describes the cloning and characterisation of the nucleotide sequence of this allele. It possesses the same general structure as x2* and x1 (previously determined) and differs from these alleles in the extension of the coding sequence for a presumptive mature protein with only 384 residues. This is due to the presence of a stop codon (TAA) 1215-bp downstream from the start codon. A further stop codon (TAG), 2280-bp downstream from the starting codon is also found. The open reading frame of xNull and x1 alleles has the same size in bp. Both are larger than x2* which shows two small deletions. The reduced size of the presumptive mature protein encoded by xNull could explain the negative effect of this allele on grain quality. Received: 16 May 1999 / Accepted: 16 September 1999