小麦高分子量谷蛋白亚基对加工品质影响的效应分析

分析了 2 50份小麦材料的高分子量谷蛋白亚基 (HMW- GS)组成以及其中 66份材料的加工品质及面条制作品质。回归分析表明 :HMW- GS与 1 0种加工品质性状均有显著的线性关系。不同亚基对综合品质效应的得分大小依次为 :Glu- Al,1 >2 * >null;Glu- Bl,1 4 +1 5>7+8>1 7+1 8>>7+9;Glu- Dl,5+1 0 >>2 +1 2 >4+1 2。不同基因位点对品质的贡献大小顺序为 :Glu- Dl>Glu- Al>Glu- Bl。首次提出了 HMW- GS综合品质评分系统     

烟草花粉原生质体与叶肉原生质体的融合及培养早期变化

用ＰＥＧ—高Ｃａ高ＰＨ法诱导抗卡那霉素的烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ）品系Ｎ３６４＋Ｋｍ＋花粉原生质体和黄花烟草（Ｎｉｃｏｔｉａｒｕｓｔｉｃａ）叶肉原生质体融合。幼嫩花粉原生质体和叶肉原生质体之间的融合体培养启动胚胎发生分裂,经卡那霉素筛选后,少数多细胞团存活并形成小愈伤组织。成熟花粉原生质体与叶肉原生质体之间的融合体则仅产生管状结构。这一结果表明,作为融合一方的花粉原生质体的发育时期对融合产物的发育途径有重要影响。     

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis. Received: 13 June 2000 / Accepted: 3 July 2000     

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.     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin band of an apparent M_r lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably transmitted to the progenies in all the events except one where in some of the segregating T₂ seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to a low copy number. The revertant T₂ seeds expressed the five endogenous subunits plus the 1Ax1 transgene. Received: 16 June 1999 / Accepted: 29 July 1999     

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。     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.     

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。     

Field evaluation and agronomic performance of transgenic wheat

Seven transgenic lines of wheat have been evaluated under field conditions during 2 agonomic years. Four lines contained the transgenes for beta-glucuronidase ( uidA), herbicide resistance ( bar) and for one high-molecular-weight (HMW) subunit, and three lines contained only one transgene for one HMW glutenin subunit and no marker genes. Agronomic traits and yield components were studied in transgenic lines and compared with the non-transgenic parent and null segregant lines. Although phenotypic differences for many traits have been found, only heading date and the number of spikelets per spike showed clear genotypic differences for both field trials. All transgenic lines had a longer heading date than parent lines whereas the number of spikelets per spike in transgenic lines was around that for L88-31 and higher for L88-6 than the corresponding parent lines. No differences were found between lines constitutively expressing the uidA and bar genes from those which only expressed the HMW genes. We conclude that differences between transgenic lines and their parents are small, and could be eliminated by backcrossing transgenic lines with their parents and selecting for the wanted genotype.