类大麦材料y型高分子量谷蛋白基因不表达的鉴定

利用SDS-PAGE检测了2份类大麦属植物的高分子量谷蛋白亚基组成,在小麦的高分子量区域仅检测到1条蛋白带,因此怀疑其y型亚基没有表达.根据其它高分子量谷蛋白提取方法的结果以及基因编码区部分序列测定,确认其y型高分子量谷蛋白基因是沉默的.     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS-PAGE检测了2份类大麦属(Crithopsis delileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2052bp．编码长度为661个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C．delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

小麦HMW-G12亚基基因启动子克隆及序列分析

为了研究高分子量谷蛋白基因启动子在种子中的特异性表达,以小麦品种“东农7742”的基因组DNA为模板,根据已发表序列设计并合成引物,用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为424bp与Thomspon报道的序列比较,同源性为97.9%,有9个核苷酸发生了改变。推测的TATA box位于-27— -30bp,Prolamin-box位于-175— -181bp,认为该元件可能与转录速率的调控有关。     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS_PAGE检测了2份类大麦属(Crithopsisdelileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2 0 5 2bp ,编码长度为6 6 1个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D ,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C .delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

小麦新品种“川麦42”低分子量谷蛋白亚基新基因的分子克隆

采用PCR方法从小麦（Triticum aestivum L.）新品种“川麦42”中克隆得到一个低分子量谷蛋白亚基（LMW-GS）新基因,暂命名为LMWCM42-1。该基因编码区全长846 bp,编码281个氨基酸,具有LMW-GS基因的典型结构特征。推导氨基酸序列比较显示,尽管LMWCM42-1与已知LMW-GS高度相似,但在N-末端重复区部分重复单元和C-末端区中仍存在明显差异。聚类分析表明,LMWCM42-1可能是由Glu-D3位点编码的。     

小麦HMW-GS基因及其AS-PCR分子标记研究进展

小麦高分子量谷蛋白亚基(HMW-GS)与小麦品质性状,尤其是沉降值性状显著相关。利用其做分子标记选育聚合优质亚基的品种,具有快速、简单、实用、有效的特点。目前,普通小麦基因组中已有15个已命名Glu-1基因被克隆和测序,这使设计引物序列、进行等位基因的特异性扩增成为可能。对普通小麦高分子量谷蛋白亚基基因组成特点及分子标记现状进行了分析,并针对国内利用高分子量谷蛋白亚基进行分子标记辅助育种做了展望。     

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。     

四川小麦地方品种AS1643中低分子量谷蛋白基因的克隆及其蛋白质的二级结构预测

根据小麦低分子量谷蛋白基因保守区序列设计引物P1/P2, 采用PCR法对四川小麦地方品种AS1643的基因组DNA进行扩增, 获得1条约900 bp的片段, 分离、纯化后连接到载体pMD18-T上, 对筛选阳性克隆测序, 获得1个低分子量谷蛋白基因LMW-AS1643(GenBank登录号: EF190322), 其编码区长度为909 bp, 可编码302个氨基酸残基组成的成熟蛋白。序列分析结果表明, LMW-AS1643具有典型的低分子量谷蛋白基因的基本结构, 其推导氨基酸序列与其它已知的LMW-GS相比, 最高相似性为93.40%。生物信息学分析表明, 在LMW-AS1643低分子量谷蛋白中, 无规则卷曲含量最高, 为67.90 %, 其次是a-螺旋, 占30.46 %, b-折叠含量最少, 为1.64 %。     

西藏半野生小麦高分子量麦谷蛋白亚基组成分析

应用SDS-PAGE分析了50份西藏半野生小麦(Triticum aestivum ssp.tibetanum Shao)的高分子量麦谷蛋白亚基等位基因组成。结果表明,43份材料的HMW-GS组成是同质的,7份材料为异质。供试材料共有7种HMW GS组合,以Null、7 8、2 12为主要类型,占所分析材料的68．4％。在Glu-1位点共检测到10种等位基因,Glu- A1位点2种,Glu～B1位点4种,Glu～D1位点4种。Null(96％)、7 8(80．4％)和2 12(94．9％)分别是Glu-A1、 Glu-B1和Glu～D1位点上主要的等位基因。在Glu-B1位点还新发现2个亚基,暂时分别命名为8*和7**。说明西藏半野生小麦中存在着较广泛的HMW-GS等位基因变异,是小麦品质育种潜在的可利用的遗传资源。     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.     

Molecular characterization of high molecular weight glutenin subunit allele 1Bx23 in common wheat introduced from hexaploid triticale

High molecular weight glutenin subunit (HMW-GS) 1Bx23, an x-type subset encoded by Glu-B1p, which is only distributed in Triticum turgidum, was successfully transferred from hexaploid triticale to common wheat line SY95-71. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that subunit 1Bx23 has a faster mobility than subunit 1Bx7 and 1Bx20, but slower than 1Bx17. Primers designed from the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of SY95-71. Total nucleotide sequences of 3426 bp including an open reading frame of 2385 bp and upstream sequence of 1038 bp were obtained. Compared with the reported gene sequences of Glu-B1-1 alleles, including 1Bx7, 1Bx14, 1Bx20 and 1Bx17, the promoter region of the 1Bx23 was displayed close to 1Bx7 and 1Bx17. The deduced amino acid sequence of coding region of 1Bx23 exhibited 34, 30, 20 and 22 amino acid substitutions from that of 1Bx14, 1Bx20, 1Bx7 and 1Bx17, respectively. A phylogenetic tree based on the nucleotide sequence alignment of the Glu-1Bx alleles shows that the 1Bx23 are apparently clustered with 1Bx7 and 1Bx17, and more ancient than 1Bx14 and 1Bx20, suggesting that the evolution speeds are different among Glu-1Bx genes. Additionally, the potential use of wheat line SY95-71 to further screen the quality contribution of unique subunit 1Bx23 is also discussed.     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

 The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2^*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event. Received: 5 August 1997 / Accepted: 6 November 1997     

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 × Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.Communicated by J.W. Snape     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

高分子量麦谷蛋白1Bx14亚基AS-PCR标记

根据已发表的1Bx14亚基的基因序列在不同位点设计了10对特异引物,从中筛选出1对引物,对HMW-GS在Glu-1Bx位点已知的10个小麦品种进行了PCR扩增.结果表明,具有1Bx14亚基的4个品种都能扩增出1条1 256 bp左右的特异带.用这一特异标记对山东省种植面积较大的40个品种进行PCR扩增(即等位专一PCR,AS-PCR),发现仅有5个品种携带1Bx14亚基.该AS-PCR标记可用于检测小麦品种在该位点的亚基组成,与SDS-PAGE相比,可显著提高检测的准确性和效率,可为种质鉴定和育种工作提供参考.     

Identification and characterization of high-molecular-weight glutenin genes in Polish triticale cultivars by PCR-based DNA markers

Molecular markers were used to identify the allele/gene composition of complex loci Glu-A1 and Glu-B1 of high-molecular-weight (HMW) glutenin subunits in triticale cultivars. Forty-six Polish cultivars of both winter and spring triticale were analysed with 7 PCR-based markers. Amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis. Differences between all 3 alleles at the locus Glu-A1 [Glu-A1a (encoding Ax1), 1b (Ax2*), and 1c (AxNull)], 4 alleles at Glu-B1-1 [Glu-B1-1a (Bx7), 1b (Bx7*), 1d (Bx6), 1ac (Bx6.8)], and 5 alleles at Glu-B1-2 [Glu-B1-2a (By8), 2b (By9), 2o (By8*), 2s (By18*), and 2z (By20*)] were revealed. In total, 16 allele combinations were observed. Molecular markers are particularly helpful in distinguishing the wheat Glu-A1a and Glu-A1b alleles from the rye Glu-R1a and Glu-R1b alleles in triticale genotypes, respectively, as well as subunits Bx7 from Bx7* and By8 from By8*, which could not be distinguished by SDS-PAGE. Novel glutenin subunits By18* and By20* (unique to triticale) were identified. HMW glutenin subunit combinations of Polish triticale cultivars, earlier identified by SDS-PAGE analyses, were verified by PCR-based DNA markers. Rapid identification of wheat Glu-1 alleles by molecular markers can be an efficient alternative to the standard separation procedure for early selection of useful triticale genotypes with good bread-making quality.     

小麦HMW-GS 1Bx14基因特异标记体系的建立

比较1Bx14及其它已知HMW-GS基因的启动子和编码区,根据其不同点设计出1Bx14基因特异扩增引物。以8种已知HMW-GS组成的小麦DNA为模板进行PCR扩增。结果表明：具有1Bx14亚基的品种扩增出1条400bp左朽特异条带。结合该特异标记和已报道的1Dx5特异标记对2个F2杂交群体进行检测,从184个F2单株中筛选出111个同时含有1Bx14和1Dx5基因的单株。该研究结果可为种质鉴定和亚基整合育种提供参考。     

小麦高分子量麦谷蛋白5亚基基因的克隆及其表达载体的构建

小麦麦谷蛋白5亚基直接影响面包的烘烤品质,但我国大部分小麦品种的蛋白中缺少这种亚基。通过引物设计、PCR扩增,从Cheyenne中得到了小麦麦谷蛋白5亚基结构基因(sub5)的全长核苷酸序列(2750bp)和小麦麦谷蛋白5亚基基因启动子(Psub5)的核酸序列(630bp)。测序结果表明：得到了小麦籽粒中特异表达启动子——Psub2和用于改良小麦品质的结构基因——sub5。通过选择和改变相应的酶切位点,在构建6个中间载体的基础上,最后得到了含有目的基因的表达载体pCAM—BIAl301—Psub5—sub5—nos。酶切电泳及PCR鉴定表明：已成功地合成了sub5的表达载体。有希望通过基因工程的方法将该表达载体用于小麦的品质改良。