小伞山羊草高分子量麦谷蛋白亚基及其基因的鉴定

运用SDS-PAGE和分子克隆技术,对小伞山羊草(Aegilops umbellulata,UU, 2n = 2x = 14)的高分子量麦谷蛋白亚基(1Ux, 1Uy)及其编码基因进行了鉴定.SDS-PAGE分析表明小伞山羊草不同基因型中的1Ux的电泳迁移率接近或慢于普通小麦1Dx2.2亚基的电泳迁移率,1Uy亚基的电泳迁移率一般接近或慢于普通小麦的1Dy类亚基.采用PCR扩增技术获得了1Ux和1Uy亚基编码基因的全长编码区,并对一个1Uy基因的全长编码区进行了全序列测定.对推导的氨基酸序列进行比较发现1Ux和1Uy亚基具有与来自于其他物种的高分子量麦谷蛋白亚基一致的一级结构,聚类分析显示1Ux和1Uy亚基与D基因组编码的高分子量麦谷蛋白亚基在起源和进化上具有较高的相似性.     

高冰草中高分子量麦谷蛋白亚基的编码基因

通过SDS-PAGE法分析了高冰草(Agropyron elongatum (Host) Nevski)种子麦谷蛋白亚基,发现高冰草的麦谷蛋白亚基种类比普通小麦更加丰富。通过基因组PCR法用高分子量麦谷蛋白亚基基因的特异引物从高冰草核基因组中分离出了7条麦谷蛋白亚基的全编码序列,分别命名为AgeloG1~AgeloG7。其中的5条已进行全序列测定,对AgeloG1和AgeloG4进行了末端测序。尽管其中的4条基因的编码序列(AgeloG4, AgeloG5, AgeloG6和AgeloG7)小于1.8 kb,但是对从克隆到的序列推导出的氨基酸序列与已经发表的小麦高分子量麦谷蛋白亚基序列进行对比分析发现,这些亚基与来自小麦的高分子量麦谷蛋白亚基具有很高的同源性。并且对信号肽、N-、C-末端的氨基酸序列分析显示,这7条序列编码的亚基皆为y-型亚基。用5条全部测序的编码序列与普通小麦的A、B、D、粗山羊草的D、圆柱山羊草的C、伞穗山羊草的U、黑麦的R染色体的编码高分子量麦谷蛋白的序列进行了聚类分析。表明,AgeloG2与小麦1Dy, AgeloG3与小麦1By, AgeloG5、AgeloG6和AgeloG7与小麦1Ay在起源和进化上有较高的相似性。     

高冰草中高分子量麦谷蛋白亚基的编码基因

通过SDS-PAGE法分析了高冰草(Agropyron elongatum(Host)Nevski)种子麦谷蛋白亚基,发现高冰草的麦谷蛋白亚基种类比普通小麦更加丰富.通过基因组PCR法用高分子量麦谷蛋白亚基基因的特异引物从高冰草核基因组中分离出了7条麦谷蛋白亚基的全编码序列,分别命名为AgeloGl～AgeloG7.其中的5条已进行全序列测定,对AgeloGl和AgeloG4进行了末端测序.尽管其中的4条基因的编码序列(AgeloG4,AgeloG5,AgeloG6和AgeloG7)小于1.8kb,但是对从克隆到的序列推导出的氨基酸序列与已经发表的小麦高分子量麦谷蛋白亚基序列进行对比分析发现,这些亚基与来自小麦的高分子量麦谷蛋白亚基具有很高的同源性.并且对信号肽、N-、C-末端的氨基酸序列分析显示,这7条序列编码的亚基皆为y-型亚基.用5条全部测序的编码序列与普通小麦的A、B、D、粗山羊草的D、圆柱山羊草的C、伞穗山羊草的U、黑麦的R染色体的编码高分子量麦谷蛋白的序列进行了聚类分析.表明,AgeloG2与小麦lDy,AgeloG3与小麦1By,AgeloG5、AgeloG6和AgeloG7与小麦1Ay在起源和进化上有较高的相似性.     

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。     

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

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1Av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lAy基因沉默的机制.     

乌拉尔图小麦（Triticumurartu）lAy高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticumurartu)不同种质材料中获得并测定了表达型和沉默型lAy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lAy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lAy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lAy基因编码区的克隆序列可经诱导而产生lAy蛋白,该蛋白与种子中lAy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lAy基因的全长编码区。但是,本研究所克隆的沉默型lAy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lAy蛋白。讨论了表达型lAy基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制。     

高冰草中一种新型高分子量麦谷蛋白亚基编码序列的研究

高冰草(Agropyron elongatun)是普通小麦(Triticum aestivum)的近缘禾草,SDS-PAGE显示其所编码的麦谷蛋白亚基的类型较普通小麦更加丰富,是普通小麦品质改良的重要亲本之一。利用基因组PCR的方法从高冰草中克隆到一个新的高分子量麦谷蛋白亚基(HMW-GS)基因(AgeloG2)全编码序列,同源性分析表明：与普通小麦的1Dy12基因比较在少数位点发生了碱基替换和一处6碱基序列的缺失,同源性为99％;与普通小麦的1Dy10基因比较,该基因亦只有少数碱基的替换和两处18碱基序列的增加及一处6碱基序列的缺失,同源性为98％。从推导的编码序列分析,AgeloG2编码y型HMW—GS。综上分析,AgeloG2是一个新的高分子量麦谷蛋白y-型亚基基因。聚类分析结果显示,无论在基因序列还是推导的氨基酸序列上,小麦1Dy亚基与AgeloG2的同源性都高于与粗山羊来源的y型亚基的同源性。     

云南小麦高分子量麦谷蛋白亚基基因1Dy12*的分离

通过SDS-PAGE分析,从云南小麦中鉴定出一个电泳迁移率比高分子量麦谷蛋白亚基1Dy12稍快的亚基1Dy12*.利用Glu-Dy位点特异引物对1Dy12*基因编码区进行了克隆和序列测定.1Dy12*基因全长为1980 bp,编码658个氨基酸.氨基酸序列比较结果表明:与亚基1Dy12相比有3个氨基酸的差异和1个二肽(GQ)的缺失,与亚基1Dy10相比有15个氨基酸的差异、2个六肽(IGQGQQ)的插入以及1个二肽(GQ)的缺失.这表明1Dy12*亚基是一个新型高分子麦谷蛋白亚基,其对小麦加工品质的影响正在评价中.     

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。     

云南小麦高分子量麦谷蛋白亚基基因1Dy12*的分离

通过SDS-PAGE分析,从云南小麦中鉴定出一个电泳迁移率比高分子量麦谷蛋白亚基1Dy12稍快的亚基1Dy12^*。利用Glu-Dy位点特异引物对1Dy12^*基因编码区进行了克隆和序列测定。1Dy12^*基因全长为1980bp,编码658个氨基酸。氨基酸序列比较结果表明:与亚基1Dy12相比有3个氨基酸的差异和1个二肽(GQ)的缺失,与亚基1Dy10相比有15个氨基酸的差异、2个六肽(IGQGQQ)的插入以及1个二肽(GQ)的缺失。这表明1Dy12^*亚基是一个新型高分子麦谷蛋白亚基,其对小麦加工品质的影响正在评价中。     

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

High-Molecular-Weight Glutenin Subunit Genesin in Decaploid Agropyron elongatum

Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences suggested that the primary structure of the subunits encoded by the seven genes was very similar to that of y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6 and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L. (CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most closely related to 1Dy; the AgeloG3 was to 1By; the AgeloG5, AgeloG6 and AgeloG7 were to 1Ay.     

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D^tx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1D^tx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat. Received: 5 November 1999 / Accepted: 12 February 2000     

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D ^t 1 and Glu-D ^t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5^t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D ^t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties. Received: 3 March 2001 / Accepted: 23 March 2001     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F₁ of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.     

Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin,a major protein of wheat endosperm

Summary The electrophoretic mobilities of the high-molecular-weight (HMW) subunits of glutenin from 7 varieties were compared by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). In total, 12 subunits were clearly resolved and they had nominal molecular weights of between 95,000 and 140,000. The chromosomes which control their synthesis were determined using monosomic lines and inter-varietal substitution lines. All subunits were shown to be controlled by the homoeologous group 1 chromosomes. Each variety contains between 3 and 5 HMW subunits; two are under the control of the 1D chromosome, 1 or 2 are controlled by chromosome 1B and 0 or 1 by chromosome 1A. The segregation of two 1D-controlled subunits of similar electrophoretic mobilities were analysed in the F₂ progeny of crosses between Chinese Spring and Holdfast. The results suggest that the genes which code for the two proteins are allelic.     

Molecular characterization of the HMW glutenin genes Dt x1.5?+?Dt y10 from Aegilops tauschii and their PCR-mediated recombinants

The high molecular weight (HMW) glutenin subunits, D^tx1.5 + D^ty10, are special types of storage proteins found in Aegilops tauschii that are never found in common wheat (Triticum aestivum). This study reports the characterization of the complete open reading frames (ORFs) of the HMW glutenin genes, D^tx1.5 and D^ty10, using a restrict-enzyme based method named the restricted deletion method (RDM). The D^tx1.5 and D^ty10 were found to have an identical structure compared with the other published HMW glutenin genes. Comparison of the deduced protein sequences also indicated that the D^ty10 in Ae. tauschii differed from its counterpart Dy10 in common wheat, by having insertions and deletions in the central repetitive domain. This result confirms the two subunits with same mobility in SDS-PAGE are different types of HMW glutenin subunits. In addition, four PCR-mediated recombinants of the D^tx1.5 and D^ty10 genes were amplified using a PCR program with shorter extension time. The recombinants had a similar structure to their corresponding natural genes, but a significantly different central repetitive domain. Western blot analysis exhibited a normal expression of the recombinants in E. coli. In addition to its usefulness for studying structure and function of the HMW glutenin subunits, the PCR-mediated recombination may provide an efficient method to generate novel HMW glutenin genes for wheat breeding.     

带芒草属物种新型高分子量谷蛋白亚基的鉴定

采用SDSPAGE方法对牧草带芒草属3个种8份材料的高分子量谷蛋白进行了检测和鉴定。结果显示,带芒草物种具有的高分子量谷蛋白亚基与普通小麦中发现的不一样,其迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率大。8份材料中共发现了4种x型亚基新类型(Tax1,Tax2,Tax3和Tax4),5种y型亚基新类型(Tay1,Tay2,Tay3,Tay4和Tay5)和6种亚基组合类型(Tax1+Tay3,Tax3+Tay2,Tax4+Tay1,Tax1+Tay1,Tax2+Tay5,Tax4+Tay2),该项研究结果揭示了带芒草属植物可能具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。