trxS基因对大麦发芽籽粒中蛋白质降解的影响

对转trxS基因大麦籽粒发芽过程中蛋白酶活性、不同蛋白组分含量和贮藏蛋白SDS-PAGE图谱的变化进行了研究。结果表明：与对照相比,转基因籽粒中的蛋白酶活性提高;清蛋白、球蛋白、醇溶蛋白和谷蛋白含量低于对照。SDS-PAGE图谱也表明,转基因籽粒中贮藏蛋白降解快于对照。     

甜荞不同品种不同海拔地区种子蛋白组分含量研究

该研究通过分析甜荞10个品种在4个不同海拔栽培的种子蛋白质组分(清蛋白、球蛋白、醇溶蛋白和谷蛋白)的含量变异,以揭示不同荞麦品种之间以及不同栽培地点甜荞种子蛋白组分的变异规律。结果表明:在所有甜荞品种种子蛋白组分含量中清蛋白谷蛋白球蛋白醇溶蛋白。其中,种植于海拔最低的内蒙古通辽的甜荞种子平均球蛋白含量最高(1.081%),而种植于海拔1 450 m的河北甜荞谷蛋白平均含量最高(2.805%);海拔2 620 m的青海甜荞清蛋白平均含量为4.750%,而在海拔最高的西藏日喀则收获的甜荞种子的醇溶蛋白最高(平均为0.393%)。另外,蒙0530在4个地区的平均种子清蛋白和谷蛋白含量都最高,而球蛋白含量最高的品种是赤甜荞1号,定甜荞2号的种子醇溶蛋白含量最高。双因素方差分析表明,种子清蛋白含量品种间变异达极显著水平,不同地点间的种子醇溶蛋白含量达极显著水平,而地点和品种两个因素对种子球蛋白含量和谷蛋白含量的变异都有极显著影响。相关性分析表明,赤甜荞1号的醇溶蛋白含量与海拔呈显著正相关,蒙0530的球蛋白含量与海拔呈显著负相关,其他品种蛋白组分与海拔的相关性不显著。该研究结果对于甜荞优质品种培育和栽培以及推广都有一定的指导意义。     

Changeability of protein fractions and their amino acid composition during maturation of barley grain

The formation of the protein complex during barley grain maturation is characterized by unequal synthesis of different protein fractions. In ten day-old grain the dominating protein is glutelin, followed by albumin and globulin. The content of prolamin is at this stage negligible. Particularly after the eighteenth day of maturation intensive synthesis of prolamins begins, which continues during the entire maturing period in almost the same ratio as total N accumulation in the grain. From the twenty-fourth day the amount of prolamins exceeds that of glutelins, and at full maturity 50% of the proteins are prolamins. The glutelin content increases absolutely, but relatively it decreases from 41 to 26%. The amino acid composition of flour from total grain as well as the amino acid composition of the individual proteins is significantly changed during maturation. Generally the changes are manifested in an increasing content of Glu and Pro and a decreasing content of Asp, Ala and Lys. These changes seem to occur as a result of an increasing representation of prolamin, which is characterized by large content of Glu and Pro and low content of Asp, Ala and Lys compared with other protein fractions.     

The Extraction and Separation of Barley Glutelins and their Relationship to Other Endosperm Proteins

Procedures are described for the extraction of unmodified andalkylated barley glutelin fractions and their subsequent separationby sodium dodecyl sulphate-polyacrylamide gel electrophoresis.The resulting separations are considerably clearer and sharperthan those previously published. One major polypeptide is presentin the salt-soluble and glutelin fractions in varying proportionsdepending on whether or not the salt-soluble fraction is extractedin the presence of a reducing agent. There is also variationin the amount of contaminating hordein polypeptides in the glutelinsand this appears to depend not only on the conditions used toextract hordein but also those used to extract the salt-solublefraction. Finally, variation in the glutelin pattern also occurswhen different denaturing agents or reducing agents are used. ¹ Visiting Scientist, permanent address: U.S. Department ofAgriculture, Science and Education Administration, AgriculturalResearch, Dept. of Agronomy Univ. of illinois, 1102 S. GoodwinAve., Urbana, IL 61801, USA.     

不同倍性水稻胚乳蛋白的差异表达研究

以4份同源四倍体水稻和4份相应的二倍体水稻为研究材料, 对其种子内清、球蛋白(清蛋白和球蛋白)、醇溶蛋白和谷蛋白的含量进行了测定, 利用聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了其特异性。结果表明: 同源四倍体水稻胚乳蛋白各组分含量与相应的二倍体相比, 大部分呈增加趋势; 同源四倍体水稻胚乳蛋白的亚基类型与相应的二倍体水稻基本一致, 仅在全蛋白电泳和清、球蛋白电泳中各发现1条差异条带。研究结果认为: 二倍体水稻经过染色体组加倍之后, 同源四倍体水稻重复基因组在蛋白质水平上的表达结果趋于“二倍化”, 即与二倍体水稻表现出相似的特点, 但在蛋白质表达量上前者往往高于后者。基因组多倍化对同源四倍体水稻重复基因组进化最主要的影响并不是体现在蛋白质结构上的差异, 而是体现在蛋白质表达量上的差异。     

Changes in protein fractions and leucine-[14C] incorporation during Sorghum grain development

Incorporation of leucine and changes in different protein fractions have been studied during Sorghum grain development. Most of the label from the injected leucine-[¹⁴C] was found in glutelin and residue fraction towards later stages of maturity. The label in albumin, globulin and prolamin decreased with a concomitant increase in label in glutelin and residue proteins. The concentration of lysine, aspartic acid and glycine decreased while that of leucine, proline, alanine, tyrosine, phenylalanine, and cystine increased during grain development. Increase in serine, methionine, valine and isoleucine was only marginal. The proportion of glutamic acid was high at all stages of grain development. Glutelin fraction resolved into two peaks on gel chromatography, only one of which with higher MW was labelled, while in albumin both the peaks were found to be labelled. Tannin content also increased during grain development.     

Effect of high-lysine mutations on the protein fractions of barley grain

We have studied the effects of six high-lysine barley mutations (Risø mutants 1508 and 56, Notch 1 and 2, lys 95 and 449) on the protein fractions of the grain. All mutants had a decreased relative and total amount of the lysine-poor hordein fraction, but only in Risø 1508 and 56 was the polypeptide composition of this fraction greatly affected. In all the mutants there were increases in the lysine-rich glutelin proteins and in nonprotein N while in Risø 1508 and the Notch mutants the total amount of salt-soluble proteins was also increased and their relative polypeptide composition substantially altered.This work was supported by EEC Contract No. 473 under the common program on plant protein improvement.     

Incorporation of 15N labelled urea and ammonium into proteins and amino acids of Sorghum

Protein fractionation studies in developing Sorghum kernel indicated a considerable decrease in the proportion of albumin and increase in prolamin, glutelin and residue proteins during grain development. The globulin fraction remained more or less constant. ¹⁵N analysis indicated a turnover of albumin and globulin fractions. The nitrogen present in these fractions appeared into glutelin and residue proteins. At an early maturation stage ¹⁵N from ammonium was detected in the residue fraction while that from urea was incorporated in both albumin and residue fractions. However, this difference disappeared as the grains matured. Incorporation of ¹⁵N into basic amino acids was lower when compared to that in neutral and acidic amino acids at all stages of grain development.     

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.     

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.     

Differential Protein Accumulation during Barley Grain Development

Barley grains were analysed during development for the presenceof salt-soluble proteins, hordeins and glutelins. Characteristictemporal differences between the fractions were observed withhordeins being produced relatively late during maturation. Analysisof this fraction by gel electrophoresis revealed differentialaccumulation of its component polypeptides. The C hordeins madeup a relatively higher percentage of total hordein at the earlystages, decreasing from 20% of the total at 33% final dry weightto 15% at maturity. The relative amount of the lowest molecularweight B hordein band (Bl) increased throughout developmentfrom 30% of the total at 33% final dry weight to about 45% atmaturity. Electrophoresis of the salt-soluble fraction showedthat a group of low molecular weight polypeptides appeared atthe same stage of development as did the hordeins. There wereonly relatively minor changes in the polypeptide compositionof the glutelin fraction.     

Development of wild barley-derived DArT markers and their integration into a barley consensus map

Wild barley-specific genomic libraries were developed for the purpose of creating a ‘comprehensive’ genomic representation of the primary Hordeum genepool capable of more robust genotyping of barley. In order to enrich for wild barley-specific sequences in the DArT libraries, suppression subtraction hybridization (SSH) was performed using cultivated barley as the subtraction driver and wild barley as the tester. Four doubled-haploid populations were genotyped with the comprehensive barley DArT array, including two from wild × cultivated crosses (Damon/Harrington and Shechem/Harrington) and two from cultivated × cultivated crosses (Albacete/Barbarrouse and TX9425/Naso Nijo). Analysis of genotyping data revealed that the SSH process was somewhat ineffective at enriching for unique sequences in this application of DArT marker development. However, the addition of markers derived from wild barley proved to be an effective means for increasing the number of polymorphic markers obtainable from a single DArT assay. Genetic maps of the four component populations were developed and 607 newly developed DArT markers were integrated with a barley consensus map to create a new synthetic map of the barley genome containing 3542 markers. This significantly increased the resolution of the consensus map and improved the power of the map to provide a reference for profiling genetic diversity within the primary Hordeum genepool. The improvement in the genotyping capability of the comprehensive DArT genomic representation and the higher resolution of the synthetic map facilitates an even greater flexibility of DArT markers to be utilized as a fast, high-throughput platform for molecular marker-based barley breeding.     

Evaluation of some cereals, plants and tubers through protein composition

Wild and cultivated maize, sorghum, rice, amaranth, soybean, and cassava were screened for variability in seed storage proteins. Total seed proteins, albumin (Alb-1 and Alb-2), globulin, alcohol-soluble (A1 and A2), and glutelin (G1 and G2) fractions were investigated by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The comparison was done by the obtained protein patterns and their relative amounts. Using quantitative analysis of the protein composition and the electrophoretic patterns, the relationships between total proteins and amount of individual proteins were determined. Electrophoretic patterns of extracted proteins from investigated samples showed that the main protein subunits were concentrated between 10 and 45 kDa. Variation was found in major fractions and minor bands. Electrophoretic patterns of the protein fractions are directly related to the genetic background of the protein and can be identified and used to certify the genetic makeup of wild, cultivated, or newly derived cereals and plants.     

水氮互作对小麦籽粒蛋白质、淀粉含量及其组分的影响

以两个不同品质类型的小麦品种（强筋品种豫麦34、弱筋品种豫麦50）为材料,在大田条件下,研究了3个灌水处理（W₁：拔节水;W₂：拔节水+花后15 d灌浆水;W₃：拔节水+灌浆水+花后28 d麦黄水）和3个氮肥水平（0、150、270 kg·hm^-2）对籽粒蛋白质、淀粉含量及其组分的影响.结果表明：270 kg·hm^-2的施氮量有利于提高强筋小麦（豫麦34）籽粒蛋白质含量,籽粒清蛋白、醇溶蛋白和谷蛋白含量明显提高,谷/醇增大;支链淀粉和总淀粉含量提高,直/支下降;籽粒产量增加.弱筋小麦（豫麦50）在150 kg·hm^-2 的施氮量下,清蛋白和醇溶蛋白含量增加,球蛋白和谷蛋白含量下降,谷/醇降低;支链淀粉和总淀粉含量提高;不施氮肥或氮肥施用过多（270 kg·hm^-2）均影响籽粒蛋白质和淀粉的积累,使产量下降.W₂处理促进了籽粒蛋白质和淀粉积累,W₁或W₃处理均不利于籽粒蛋白质和淀粉积累,且导致籽粒产量下降.水、氮互作效应中,强筋和弱筋小麦分别以全生育期270 kg·hm^-2和150 kg·hm^-2施氮量配合拔节水+灌浆水（W₂）为比较理想的水氮运筹方式.     

Population structure and linkage disequilibrium in barley assessed by DArT markers

Diversity Array Technology (DArT) markers were used to investigate the genetic diversity, population structure, and extent of linkage disequilibrium (LD) on a genome-wide level in Canadian barley (Hordeum vulgare L.). Approximately 1,000 DArT markers were polymorphic and scored with high confidence among a collection of 170 barley lines composed mostly of Canadian cultivars and breeding lines. The reproducibility of DArT markers proved very high, as 99.9% of allele calls were identical among seven replicated samples. The polymorphism information content (PIC) of DArT markers ranged between 0.04 and 0.50 with an average of 0.38. Using principal coordinate analysis (PCoA), most lines fell into one of two major groups reflecting inflorescence type (two-row versus six-row). Within these two large groups, evidence of geographic clustering of genotypes was also observed. A cluster analysis Unweighted Pair Group Method with Algorithmic Mean suggested the existence of three subgroups within the two-row group and four subgroups within the six-row group. An analysis of molecular variance (AMOVA) revealed highly significant (P < 0.001) genetic variance within subgroups, among subgroups, and among groups. Values of LD, expressed as r ², declined with increasing genetic distance, and mean values of r ² fell below 0.2 for markers located 2.6 cM apart. Approximately 8% of marker pairs located on the same chromosome and 3.4% of pairs located on different chromosomes were in LD (r ²  > 0.2). Within both the subsets of two-row and six-row lines, LD extended slightly further (3.5 cM) than for the entire set, while 7.5% of intra-chromosomal locus pairs and <2% of inter-chromosomal pairs were in LD. We discuss the implications of these findings with regard to the prospects of association mapping of complex traits in barley.     

Effect of nitrogen nutrition on endosperm protein synthesis in wild and cultivated barley grown in spike culture

In normal growth conditions, total protein percent (salt soluble plus hordein fractions) in the endosperm at maturity in barley cultivar Hordeum vulgare L. cv `Ruth' was about 14%, whereas in an accession of wild barley, Hordeum spontaneum Koch line 297, it was about 28%. Spike culture experiments were conducted to ascertain whether there were basic differences between the two genotypes under conditions of widely different nitrogen supply. Spikes of each genotype were grown from 8 to 25 days after flowering in in vitro culture in a growth medium containing 0 to 4 grams per liter nitrogen supplied as NH<sub>4</sub>NO<sub>3</sub>. Spikes were pulse-labeled at intervals from 12 to 24 days after flowering with 3.7 megabecquerel of [<sup>3</sup>H]leucine to determine relative rates of synthesis of hordein-1 and hordein-2 polypeptides. At low nitrogen levels `Ruth' had a lower protein content than 297, but at increasing nitrogen levels its protein content increased rapidly and reached a maximum (35%) higher than 297 (30%). The relative contribution of the hordein fraction to total protein increased mainly with time, and hordein-1 to total hordein increased mainly with nitrogen level, in both genotypes. There appeared to be no fundamental limitations in the capacity of `Ruth' to accumulate protein; 297 appears to have a greater basal level of nitrogen availability under normal conditions.     

Analysis of the Genetic Structure of a Barley Collection Using DNA Diversity Array Technology (DArT)

Analysis of the extent of genetic variation within genetic resources is important for diversity preservation and also for breeders who exploit it. We investigated the recently introduced molecular marker technique of DNA diversity array technology (DArT), with the objective of characterising diversity in the likely relatively narrow genetic background of Czech malting barley cultivars. A total of 94 obsolete or registered barley cultivars and some hulless barley lines primarily of Czech origin were characterised by DArT analysis. A total of 271 polymorphic marker alleles were revealed across the analysed set of accessions, 37 of which were identified as being overrepresented; the other 234 markers were used for further analysis. The average dissimilarity value within the analysed set of accessions was 0.692. To assess how well DArT is suited for individual barley characteristic evaluation, available agronomical data from three yield field trials were used. Out of 94 barley genotypes used in the field trials that were assessed by DArTs, 41 have been grown over time as malting cultivars in the region. Similarity matrices based on Gower’s coefficient for mixed data and simple matching coefficient were used to compare DaRT and agronomical results. We demonstrate that a DArT-based similarity matrix and an agronomical data-based similarity matrix correlated well. To assess the genetic structure of the entire collection, K-means and simple matching coefficient clustering were used. Statistical analysis confirmed the power of the DArT system, in fact they efficiently grouped old genetic resources and modern cultivars in the expected way. Our results show that the level of genetic diversity has not changed substantially over time, but significant shifts in allelic frequency have occurred. In addition, a DArT-based dendrogram and principal component analysis (PCA) plots clearly demonstrated the impact of breeding practices on the diversity of Czech spring malting barley cultivars over time.     

Diversity arrays technology (DArT) for high-throughput profiling of the hexaploid wheat genome

Despite a substantial investment in the development of panels of single nucleotide polymorphism (SNP) markers, the simple sequence repeat (SSR) technology with a limited multiplexing capability remains a standard, even for applications requiring whole-genome information. Diversity arrays technology (DArT) types hundreds to thousands of genomic loci in parallel, as previously demonstrated in a number diploid plant species. Here we show that DArT performs similarly well for the hexaploid genome of bread wheat (Triticum aestivum L.). The methodology previously used to generate DArT fingerprints of barley also generated a large number of high-quality markers in wheat (99.8% allele-calling concordance and approximately 95% call rate). The genetic relationships among bread wheat cultivars revealed by DArT coincided with knowledge generated with other methods, and even closely related cultivars could be distinguished. To verify the Mendelian behaviour of DArT markers, we typed a set of 90 Cranbrook × Halberd doubled haploid lines for which a framework (FW) map comprising a total of 339 SSR, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) markers was available. We added an equal number of DArT markers to this data set and also incorporated 71 sequence tagged microsatellite (STM) markers. A comparison of logarithm of the odds (LOD) scores, call rates and the degree of genome coverage indicated that the quality and information content of the DArT data set was comparable to that of the combined SSR/RFLP/AFLP data set of the FW map.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.