种子醇溶蛋白提取及检测条件探索

以大麦、小麦、玉米、高粱和苏丹草的种子为材料提取种子的醇溶蛋白,分析了不同提取剂及不同固液比[种子粉末样品质量(g)与提取剂的体积(mL)的比例]对种子醇溶蛋白提取效果的影响,并对SDS-PAGE检测醇溶蛋白中的不同胶浓度、厚度以及样品上样量等的影响进行了研究.结果表明,60%的正丙醇、乙二醇、异丙醇和叔丁醇分别是小麦、大麦、玉米以及高粱和苏丹草的最佳提取剂,将1∶6比例提取的种子醇溶蛋白以15μL上样,0.5mm厚度的15%分离胶电泳可以得到清晰的电泳检测图谱.     

特优系列杂交稻种子纯度的鉴定

用等电聚焦技术(IEF)对特优系列品种及其亲本种子蛋白进行了分析,2-氯乙醇不能提取F1代的亲本互补蛋白带,而用40%的2-氯乙醇与15%的巯基乙醇混合液则能有效提取F1代亲本互补带.采用此种互补谱带,可鉴定"特优524"和"特优721"品种的种子样品,其结果与田间小区种植鉴定的差异较小.     

萌发大麦种子水溶性蛋白组分提取

采用二次回归正交旋转组合试验方法,对大麦种子中水溶性蛋白组分的提取工艺进行了研究,得出了大麦种子水溶蛋白组分提取量与料液浓度,浸提温度和浸提时间的数学模型,确定了大麦籽粒水溶蛋白组分最佳提取条件为料液浓度9．1％,浸提温度20℃,浸提时间3h;通过连续检测萌发大麦种子水溶蛋白含量发现：随萌发时间增长,蛋白含量逐渐升高,麦芽中水溶蛋白含量约为大麦的4倍．     

外源DNA导入小麦引起遗传变异的验证

对由波兰小麦ＤＮＡ注射到普通小麦鄂恩１号子房获得的Ｄ５代稳定遗传变异２个转化株系的种子醇溶蛋白,进行单向和双向聚丙烯酰胺凝胶电泳分析,结果表明由外源ＤＮＡ导入获得转化株系的变异,与其种子醇溶蛋白电泳图谱出现供体的某些组分和缺少受体的某些组分相印证。     

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。     

鸡冠花种子蛋白质的提纯及分析

鸡冠花种子干燥后用石油醚脱酯,用凯氏定氮法测定蛋白质含量。按Osbern系统分别提取白蛋白、球蛋白、醇溶蛋白、谷蛋白;用Folin一酚试剂法测定各自相对含量,并用单向和双向SDS—PAGE方法分析种子总蛋白和4类不同溶性蛋白质的组成成分及这些组份的热稳定性。实验发现:鸡冠花种子蛋白质含量达26.04％,白蛋白、球蛋白、醇溶蛋白、谷蛋白的相对含量分别为11.5％、72％、4.6％、12％。SDS—PAGE表明;其种子蛋白中20KD球蛋白成份含量最高,其它一些次要组分为63KD、61KD、15KD、13KD白蛋白成份,58KD、37KD、23KD球蛋白成份,39KD、34KD、25KD谷蛋白成份及26KD醇溶蛋白成份,其中58KD由37KD和20KD两亚基经二硫键连接而成,39KD组份由24.5KD和18KD两亚基通过二硫键连接而成,23KD球蛋白组份遇热沉淀。     

小麦体细胞无性系醇溶蛋白电泳图谱初步分析

本试验对小麦品种的休细胞无性系及其对照品种的种子醇溶蛋白进行电泳图谱比较分析。结论是：(1)同一小麦品种存在着不同的醇溶蛋白生物型;(2）小麦醇溶蛋白与外部性状在总体变异程度上有一定的正相关;(3)利用小麦醇溶蛋白电泳图谱,可以鉴别混杂株系,确定体细胞无性系的来源,但需注意选择合适的对照图谱。     

冰草属(Agropyron Gaertn.)植物遗传多样性取样策略基于醇溶蛋白的研究

对冰草属(Agropyron Gaertn.)植物的5个种22个居群分8个取样梯度,分别随机取3、6、9、12、15、18、21、24粒种子的混合样进行醇溶蛋白的A-PAGE分析,结果发现:平均Simpson指数依取样量的增加表现增大,以3粒种子0.922表现最低,以21粒0.933表现最高,变异系数(CV%)为0.41.醇溶蛋白谱带数依所提取的混合样的籽粒数目发生显著变化,冰草属内5个种表现趋势相同,22个居群不同取样整体平均以3粒种子提取的醇溶蛋白谱带数18.38为最少,以18粒种子提取的醇溶蛋白谱带28.24为最多,CV%为13.21.取样量达到12粒以上醇溶蛋白谱带差异不明显(P=0.01),图谱表现基本一致.建议在利用生化指纹进行冰草属居群间及种间的遗传多样性研究中,混合取样量最低应保持在12个个体及以上方能代表居群整体,反映居群的整体遗传特性.     

一种改良的小麦醇溶蛋白电泳法

在小麦种子中,醇溶蛋白约占蛋白总量的50％,可分为40—50个组份,每种醇溶蛋白基因已定位于一定染色体上。近年来,世界各国对小麦醇溶蛋白进行电泳分析,发现不同小麦品种之间醇溶蛋白的差异很大,因此醇溶蛋白电泳法可作为小麦品种鉴     

我国大麦醇溶蛋白多肽的多态性研究

对22个省的大麦农家品种487份和3个近缘野生种的10份材料,应用SDS-PAGE、IEF及双向电泳(IEF、SDS-PAGE)进行了醇溶蛋白多肽多态性的研究,主要结果如下:(1)B醇溶蛋白多肽多态性十分显著。经SDS-PAGE可分出16种带型,归为α、β、γ、ω四组,不同的多肽带型中间也有一些多肽是共有的;(2)5种带型的地理分布表明,具有同种带型的品种分布于一定的地理区域;各区域品种所具有带型类型的数目虽然不同,但表现出一定的趋向,这种趋向似与大麦进化和生态区域有关;(3)近缘野生大麦的带型有的与农家品种带型相似,有的则相异;但一些农家品种的带型在供试近缘野生种中还未找到与之相近的带型;(4)初步讨论了醇溶蛋白多肽多态性在品种鉴定上的应用和作为栽培大麦生态区域划分辅助手段的可能性。     

D hordeins of <Emphasis Type="Italic">Hordeum chilense</Emphasis>: a novel source of variation for improvement of wheat

The high molecular weight subunits of wheat (Triticum aestivum L.) glutenin (HMW-GS) are important in determining the bread-making quality of flour and dough. There is therefore interest in transferring orthologous HMW-GS present in other grass species into wheat by wide crossing in order to extend the range of end use properties. In this work, we have isolated and characterized two genes encoding D hordeins from Hordeum chilense (Roem. et Schult.) lines H1 and H7, representing two ecotypes. The fragments were 4,305 bp for line H1 and 4,227 for line H7 and contained the promoter, coding and terminator regions. Both sequences differ in the presence of single base changes (SNPs) and insertions/deletions in the open reading frame (ORF). The encoded proteins comprise 870 and 896 amino acids for lines H1 and H7, respectively. The primary structure is similar to those of D hordeins of cultivated barley (H. vulgare L.) and HMW-GS from wheat. However, the D hordeins from H. chilense are significantly larger than those from cultivated barley due to the presence of longer repetitive regions. The H. chilense D hordeins also differ from those of cultivated barley in the distribution of the cysteine residues: whereas the D hordeins of cultivated barley contain ten cysteines with four in the repetitive domain, only nine are present in the H. chilense proteins with two in the repetitive domain. As in the HMW-GS, the central part of the D hordein proteins comprises repeated sequences based on short peptide motifs. The repetitive domain is divided in three regions named as R1 (N-terminal repeats), R2 (central degenerate repeats) and R3 (C-terminal repeats). Hexapeptide motifs are present throughout the repetitive domains of D hordeins with a consensus motif of PFQGQQ in R1 and R2 and PHQGQQ in R3. In addition, the tetrapeptide motif TTVS, which is characteristic of D hordeins of cultivated barley is present in the repetitive domain close to the protein C-terminus.     

反相高效液相色谱分析种子醇溶贮藏蛋白质鉴定大麦品种(英文)

本文采用反相高效液相色谱(reversedphase high-performance liquid chromatography,RP-HPLC)技术分析了中国种植的24个不同大麦品种的种子醇溶贮藏蛋白质。首先,根据所获得的色谱图的相似性可以将供试品种分成10组,每组各有自己的共同特征色谱峰;其次,再依据各组内不同品种间色谱图的定性或定量上的差异可以将它们分别区别和鉴定;这表明大麦种子醇溶贮藏蛋白质的异质性较强,其组成随基因型的不同而有所变异。因此,应用RP-HPLC技术分析大麦种子醇溶贮藏蛋白质可以准确、快速地对大麦品种进行鉴定。     

In vitro synthesis of barley storage proteins

Membrane-bound polysomes were isolated from developing endosperms of barley (Hordeum vulgare L.) and shown to support the synthesis of trichloroacetic acid-insoluble material by an in vitro wheat germ protein synthesis system. The mRNA associated with the polysomes was separated from the ribosomes by affinity chromatography on oligo-dT cellulose and was also shown to support in vitro protein synthesis. The poly-A⁺ RNA isolated contained material of between 0.55 and 2.55 kilobases in length with about 6% poly A. The products of in vitro protein synthesis resembled hordeins (the prolamin storage proteins of the barley endosperm) in that they were predominantly soluble in 55% propan-2-ol, contained a low proportion of lysine as compared with leucine and had similar, but not identical, electrophoretic properties. The differences in the electrophoretic behaviour between the products of poly-A⁺ RNA translation and authentic hordeins is suggested to be due to the presence of an extra (leader?) sequence on the former.     

Genetic diversity among and within Iranian and non-Iranian barely (Hordeum vulgare L.) genotypes using SSR and storage proteins markers

The objectives of this study were evaluation of genetic diversity and marker–trait association of 64 barley (Hordeum vulgare L.) genotypes using hordeins and simple sequence repeats (SSRs) markers under optimal moisture and drought stress conditions. Moreover, to evaluate the response of barley genotypes to drought stress, five drought tolerance indices were calculated. SSRs and hordeins generated clear patterns with high polymorphism. SSRs and hordeins analysis provided us with useful information on the level of polymorphism and diversity in barley. Marker–trait associations were studied for 22 agronomic traits using 122 SSR markers (obtained from 14 primer pairs) and 51 hordeins bands in 64 barley genotypes under both normal and stress conditions. Phenotypic traits strongly associated with SSRs were also strongly associated with hordeins. Generally, we believed that at least some of these markers would be informative and validated and can be used in marker-assisted selection (MAS) under drought stress.     

Uptake and Distribution of Stable Strontium in 26 Cultivars of Three Crop Species: Oats,Wheat, and Barley for Their Potential Use in Phytoremediation

The main objective of this study was to investigate the accumulation and distribution of strontium (Sr) in 26 cultivars of wheat (Triticum aestivum L.), husk oat (Avena sativa L) and naked oat (Avena nuda), and barley (Hordeum vulgare L.) for their potential use in phytoremediation.Sr levels had no effect on the accumulation of shoot biomass at tillering or at maturity. Mean shoot Sr concentration of naked oat and barley at tillering was significantly (P < 0.05) higher than that of wheat; Neimengkeyimai-1, a naked oat cultivar, had the highest Sr concentrations. At maturity, of four naked oat cultivars, Neimengkeyimai-1 had the highest Sr content at all measured Sr levels. Leaves had the highest Sr concentrations, followed by roots and straw, and then grain with the lowest. Mean enrichment coefficients from soil to shoots ranged from 0.521 to 1.343; the percentage of stable Sr removed from the soil to the shoots at harvest time was more than 1.4% after 120 days. Neimengkeyimai-1 could be used as a model for further research to find more effective cultivars; and naked oat plants could be selected for phytoremediation to clean up contaminated soil.     

Features of recombination of nuclear genome in back crossed offspring of barley-wheat hybrids Hordeum vulgare L. (2n=14) x Triticum aestivum L. (2n+42) with the use of SSR-analysis

The backcross progenies of the barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42) and two alloplasmic lines derived from them were studied using microsatellite markers of barley and wheat. The F1 hybrids and first backcross plants BC1 contained the genetic material of both cultivated barley and the cultivars of common wheat involved in developing of these hybrid genotypes. The genomes of BC3, BC4, and alloplasmic lines contained no microsatellite markers of the cultivated barley, whereas chromosomes of each homeologous group of common wheat were identified. In chromosomes of backcross progenies BC3, BC4, and alloplasmic lines yielded by backcrosses of hybrids and various common wheat cultivars, microsatellite markers of the parental wheat cultivars were shown to undergo recombination.     

Barley allele-specific amplicons useful for identifying wheat-barley recombinant chromosomes

Barley (Hordeum vulgare L.) is potentially a new source of genes for wheat (Triticum aestivum L.) improvement. Wheat-barley chromosome recombinant lines provide a means for introgressing barley genes to wheat genome by chromosome engineering, and since these are expected to occur only rarely in special cytogenetic stocks, an efficient selection skill is necessary to identify them. To convert RFLP markers to barley allele-specific PCR markers useful for effective production of wheat-barley recombinant lines, 91 primer sets derived from RFLP clones which were previously mapped to the barley chromosomes were examined for PCR amplification using 'Chinese Spring' wheat, 'Betzes' barley and the wheat-barley chromosome addition lines. The polymorphisms were detected by an agarose gel electrophoresis of the PCR products without digestion with restriction enzymes. Out of 81 primer sets producing polymorphisms between the wheat and barley genomes, 26 amplified barley chromosome-specific DNAs which were confirmed to be located on the same chromosome as the RFLP markers by using the wheat-barley chromosome addition lines. These amplified DNAs represent barley allele-specific amplicons, which distinguish barley alleles from their wheat homoeologous counterparts. The present investigation revealed a higher probability for obtaining allele-specific amplicons from genomic DNA-derived RFLP markers than from cDNA-derived ones. The barley allele-specific amplicons developed in this study, namely, four for chromosome 2H, two for 3H, seven for 4H, eight for 5H, one for 6H and four for 7H, are suitable for identifying 'Chinese Spring' wheat- 'Betzes' barley recombinant chromosomes. However, one out of eight barley allele-specific amplicons on chromosome 5H did not detect a unique barley band in a 'New Golden' barley chromosome 5H addition line of 'Shinchunaga' wheat, indicating there may be a need to reconstruct allele-specific amplicons with different barley cultivars.     

Efficient production of wheat-barley hybrids and preferential elimination of barley chromosomes

Summary Intergeneric hybridization between four common wheat cultivars, Triticum aestivum L. cultivars Chinese Spring, Norin 12, Norin 61, and Shinchunaga, and cultivated barley, Hordeum vulgare L. cultivars Betzes, Nyugoruden, Harunanijou, and Kinai 5 were carried out in a greenhouse under 15 – 20 °C and long-day (15 h) photoperiod conditions. Two days prior to pollination, a 100 mg/1 2,4-D solution was injected into wheat stems. Among wheat cultivars, Norin 12, Norin 61, and Shinchunaga showed higher crossabilities than that of Chinese Spring, suggesting the presence of crossability gene(s) other than the kr system of Chinese Spring. Variation was also found among the barley cultivars as male parents. Betzes barley showed the highest crossability with wheat. Thus, the cross Norin 12×Betzes showed the highest crossability (8.25%), followed by Norin 61 ×Betzes (6.04%), Shinchunaga×Betzes (5.00%), and Shinchunaga×Kinai 5 (5.00%). The embryos were rescued by culture at 15–20 days after pollination. Seventyfour plants were obtained from 82 embryos. The morphology of the hybrid plants resembled that of wheat parents. Among 60 seedlings observed, 28 had 28 chromosomes, 8 had 21, 23 had aneuploid numbers of chromosomes (22–27), and 1 had 29 chromosomes. About half of the aneuploid hybrids showed mosaicism for chromosome number. By analyzing five isozyme markers of barley chromosomes, the chromosome constitutions of the aneuploid hybrids were determined. Barley chromosomes 1 and 5 were found to be preferentially eliminated in the hybrids, while chromosomes 2 and 4 were eliminated infrequently. The conditions and genetic factors for high crossability and the tendency of barley chromosome elimination are discussed.     

Influence of the Russian wheat aphid <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Homoptera,Aphididae) on productive qualities of spring bread wheat and barley grown from the seeds from aphid-infested spikes

The aftereffects of the Russian wheat aphid (RWA) Diuraphis noxia on sowing and productive qualities of barley and spring bread wheat grain were assessed. Seeds of 4 cultivars of barley (Volgar, Povolzhsky 65, Kazak, and Povolzhsky 16) and 4 cultivars of spring wheat (Kinelskaya 59, Kinelskaya Otrada, Kinelskaya Niva, and Kinelskaya 2010) from spikes infested and uninfested with RWA in 2007 and in 2014 were sown in the subsequent years, using 0.5 m² experimental plots in four replications, at a seeding rate of 300 grains/m². The least significant difference (LSD_0.5) was used to compare the mean ± standard deviation (SD) values. The field germination rate of seeds from spring wheat spikes damaged by RWA was reduced by 15%. Of the components of grain yield, barley and spring wheat grown from seeds from the infested spikes showed a 23-31% smaller number of productive tillers before harvesting, a 16% smaller number of grains per spike, a 13-16% lower grain weight per spike, and a total yield loss of 33-42%. In hulless bread wheat RWA fed on the developing kernels inflicting greater damage, whereas the hulled barley seeds were practically not damaged. The mean yield loss of the barley and spring wheat spikes infested with RWA was 24-32% and 50-66%, respectively. Due to the greater tillering capacity and formation of secondary productive tillers in barley, about 52% of the productive barley tillers and 37-39% of spring wheat ones were infested with RWA, which resulted in a comparable yield loss (20-25% in barley and 19-23% in spring wheat). Resistance to RWA was higher in spring wheat and barley cultivars with a shorter vegetation period, looser spikes, and thinner culm walls. The length of productive tillers damaged by RWA was reduced by 21-28%, which determined a lower incidence of leaf diseases.     

Quantifying relationships between rooting traits and water uptake under drought in Mediterranean barley and durum wheat

In Mediterranean regions drought is the major factor limiting spring barley and durum wheat grain yields. This study aimed to compare spring barley and durum wheat root and shoot responses to drought and quantify relationships between root traits and water uptake under terminal drought.One spring barley（Hordeum vulgare L. cv. Rum） and two durum wheat Mediterranean cultivars（Triticum turgidum L. var durum cvs Hourani and Karim） were examined in soil‐column experiments under well watered and drought conditions. Root system architecture traits, water uptake, and plant growth were measured. Barley aerial biomass and grain yields were higher than for durum wheat cultivars in well watered conditions. Drought decreased grain yield more for barley（47%） than durum wheat（30%, Hourani）. Root‐to‐shoot dry matter ratio increased for durum wheat under drought but not for barley, and root weight increased for wheat in response todrought but decreased for barley. The critical root length density（RLD） and root volume density（RVD） for 90% available water capture for wheat were similar to（cv. Hourani） or lower than（cv. Karim） for barley depending on wheat cultivar. For both species, RVD accounted for a slightly higher proportion of phenotypic variation in water uptake under drought than RLD.