小麦HMW谷蛋白亚基基因克隆研究进展

高分子量麦谷蛋白亚基 (HMW GS)作为小麦胚乳中的重要贮藏蛋白 ,其组成及含量对小麦面粉的烘烤品质具有重要的决定作用。因此 ,改变小麦中HMW 谷蛋白的组成及含量是小麦品质改良的主要内容。而定向克隆小麦HMW GS基因则为利用基因工程方法改良小麦品质提供新的基因资源 ,从而为优质小麦的发展起到积极的推动作用。综述了近 2 0年来国内外小麦HMW GS基因克隆的研究进展 ,并讨论了近年来发展起来的一些新的基因克隆方法及其在小麦HMW GS基因克隆上的应用前景。     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

HMW-GS的SDS-PAGE图谱在小麦品质评价中的应用

采用SDS—PAGE技术对陕西关中地区各时期大面积推广小麦品种、品种资源和新品系的HMW—GS组成进行了分析。在该地区50多年大面积推广的33个品种中,检测出9种HMW亚基(对)及其组成;品种HMW—GS的评分在5～10分之间,平均6．9分;4个时期品种HMW-GS的平均评分有升有降,优质亚基出现的频率普遍偏低;向小麦品种中聚合多种优质HMW—GS将成为陕西关中未来小麦育种的主要目标之一。53种小麦品种资源的亚基或亚基对组合类型比较丰富,具有一批携带优质亚基5 10、1、2*、7 8、14 15或17 18等资源。8个新选品系中,有4个品系携带了多种优质亚基,其中3个品系HMW—GS的评分为10分;Q1043已被审定通过,目前正在陕西关中推广种植。实践证明,采用SDS—PAGE方法对生产上推广品种、品种资源和新选品系的HMW—GS变异研究,有助于在短时间内了解生产上推广小麦品质生产现状、制订育种目标、选配亲本和品系品质性状筛选,是一种非常实用的品质快速检测方法。     

小麦HMW-GSIDx5基因在小麦不同发育阶段表达的研究(英文)

小麦面粉的烘烤品质与其高分子量谷蛋白5亚基（HMW－GSIDx5）基因的表达有关。本文从该基因的组织、器官特异性和特定发育阶段的表达特性等方面,对其在小麦发育过程中表达的变化规律进行了研究,从分子水平为小麦的栽培和育种提供依据。显微切割普通小麦钢82－122的ID染色体长臂为模板,扩增该基因400bp的片段为探针（Fig．1）,并经重组后测序（Fig．2）确认扩增无误。小麦苗期、生长期、开花期及成熟期分别取样、分离总RNA、做斑点（Fig．3,4＆5）或Northern杂交（Fig．6）、并扫描足量（Fig．7）。结果表明：该基因只在籽粒中表达（Fig．3,4＆5）,从开花15天起（Fig．4）,逐渐增加,至腊熟期达最高,以后渐次降低（Fig．6＆7）,有趣的是：在干种子和萌发种子亦有少量表达（Fig．1）。Cressey和陈和等方曾报告在开花15天可检测到HMW－GS蛋白。     

60Co-γ 诱变皖麦50HMW—GS变异的筛选及其M3代品质分析

为了探讨60Co-γ 射线对小麦HMW—GS组成的变异,以及变异后代的稳定性和品质变异效应,获得优质的种质资源,本试验用200Gy 60Co-γ射线辐照皖麦50于种子,SDS—PAGE法对M2代HMW—GS组成进行变异筛选,并对M3代HMW—GS组成变异稳定性进行鉴定和GMP含量、GMP粒度、部分营养及加工品质进行测定。结果表明：655株M2群体中,有1个单株HMW—GS组成发生变异,变异率0．15％;HMW—GS组分由亲本的7＋9／2＋12变为1／7＋9／2＋12,并在M3代保持稳定;M3代5个变异穗行,蛋白质含量、GMP含量、GMP／Pr均比皖麦50提高,GMP粒度分布范围扩大,粒径大于10．28μm的比例增加。     

Tα1小麦轮选群体高分子量谷蛋白亚基组成分析

利用了Tα1小麦(Ms2)创建改良小麦面包品质的优质群体,采用SDS—PAGE法对其2次互交轮回群C2的HMW—GS组成进行了分析。结果表明：在供试的193个样品中各HMW—GS及其组成模式的频率不尽相同,Glu—A1、Glu—Bl和Glu—Dl位点上产生频率最高的亚基分别是1、14 15和2 12,各为54．40％、35．75％和60．10％,优质亚基5 10的频率为17．6％;“null、14 15、2 12”模式产生频率最高,为13．47％,并有“14 15,5 10”的优质亚基聚合体出现,占5．2％;该群体也产生了亲本不具有的13、16、22亚基及19种新的HMW—GS组成模式。说明利用Tα1小麦轮回选择技术是创造新亚基类型的一个有效途径。     

农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究

 谷氨酰胺合成酶（Glutamine synthetase,GS,E.C. 6.3.1.2）是植物氨同化过程中的关键酶,对植物的氮素吸收和代谢起着至关重要的作用。谷氨酰胺合成酶还是除草剂草胺膦(Phosphinothricin　(PPT)或Basta)的靶标酶。前期工作已从我国特有的豌豆(Pisum satium)品种中克隆了细胞质型谷氨酰胺合成酶（GS1）cDNA和叶绿体型谷氨酰胺合成酶（GS2）cDNA。为了验证谷氨酰胺合成酶的功能,构建了同时含有GS1 cDNA和GS2 cDNA的植物表达载体p2GS。以该表达载体通过农杆菌介导法,转化小麦(Triticum aestivum)的未成熟胚愈伤组织,经PPT筛选及分化再生培养,获得了抗PPT的转基因小麦植株41株。PCR和基因组Southern 杂交分析证实了GS1 和GS2基因已经整合到转基因小麦的基因组。用除草剂草胺膦Basta溶液涂抹转p2GS小麦叶片,结果证明GS转基因植株可以抗高达0.3%的 Basta溶液,而对照植株叶片逐渐变黄直至枯死。转基因小麦植株能正常结实。上述实验结果表明：1) GS基因在小麦植株中获得了有效表达,从而赋予小麦植株抗PPT特性;2) GS基因能够作为研究小麦遗传转化的筛选标记基因。     

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。     

粗秆高产小麦茎结构特性分析

以高产小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）新品种“兰考９０６－４”与北京大面积种植的小麦品种“京４１１”为实验材料,运用植物解剖学、化学和力学的理论与方法,对粗秆的高产小麦茎结构特性做了详细的比较研究。结果表明：粗秆的高产小麦品种茎秆在物理力学特性、维管束结构特征以及木质素含量等方面,均明显优于一般小麦品种。因此,在超高产小麦品种的育种中,重视外源基因的引进,改进茎秆的结构特性及提高木     

远缘杂交在转移有益基因创造小麦新种质中的潜力

本文概述了小麦远缘杂交技术的发展以及这些技术的应用对以染色体易位方式转移有益基因到普通小麦中的影响。通过对小麦远缘杂交技术的总结得出,普通小麦由于本身的多倍性,对导入的外源基因具有较强的调节能力,是适宜外源有益基因导入的良好受体。而以染色体易位方式转移有益基因是创造小麦新种质的有效方法之一,许多研究也表明以染色体易位导入的外源有益基因更利于表达。近几年,随着细胞遗传学以及其它生物技术的发展,对小麦族进化途径和染色体间的亲缘关系进一步明确,从而更便于进行易位导入的技术选择,也使得染色体易位鉴定方法更趋完善。现在已有更良好的外源导入的工具和方法,使多基因控制的外源优良性状导入成为可能。在小麦远缘杂交中染色体易位所具有的上述优势,在育种实践中逐步显示出来,为开拓小麦种质资源开创了一条新的途径。     

Multiplex PCR identification of wheat HMW glutenin subunit genes by allele-specific markers

Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Molecular Characterization of Glutenins in Wheat Varieties Differing in Chapati Quality Characteristics

Five wheat (Triticum aestivum) varieties differing in chapati quality characteristics viz. C-306, K-68, HD-2745 and HD-2735 with good and Sonalika with poor chapati quality characteristics, were selected for the characterization or distribution of glutenin genes. Polymorphism was observed when genomic DNA of wheat varieties was hybridized with a HMW glutenin probe [glutenin subunit 10 (Dy10)]. No hybridization was observed in Sonalika. PCR amplification of genomic DNA with the LMW glutenin gene-specific primers did not show any polymorphism. However, with HMW glutenin gene-specific primers a single band of ～ 650 by was obtained in all the good chapati characteristic wheat varieties.The amplified fragment was sequenced and found to have sequence homology with HMW glutenin subunit Dx5.The deduced protein structure analysis showed that the peptide was made up of N-terminally placed (x-helices and centrally placed repetitive β-turns.     

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.