Comparison of isoenzymes in Prunus avium separated by two different electrophoretic techniques

Isoenzyme variation for seven systems revealed by two different electrophoretic procedures was compared in Prunus avium. Fourteen cultivars and 14 wild selections were analysed for acid phosphatase (ACP), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), phosphoglucomutase (PGM), shikimate dehydrogenase (SKD) and superoxide dismutase (SOD). Extracts were separated by isoelectric focusing (IEF) and by polyacrylamide gel electrophoresis (PAGE). For the eight loci that had been described previously in these enzyme systems on the basis of IEF analysis, we compared the variation revealed with IEF and PAGE. Similar variation was revealed for Acp‐1 and Pgm‐1, and the alleles revealed by PAGE could be identified directly with those reported for IEF. For Lap‐1, Mdh‐1 and Skd‐1, variation was seen with IEF but not with PAGE. For Mdh‐2, PAGE revealed additional variation not revealed by IEF. For Idh‐1, different patterns of variation were revealed by PAGE and IEF, and both procedures would be needed to genotype cherry accessions. We were unable to detect variation corresponding to that reported previously for Sod‐1 with either technique. The implications of these findings for allele labelling, for studies of genetic diversity and for linkage analysis are discussed.     

The electrofocusing composition of the Large and Small Sub-unit polypeptides of Fraction-1-Protein in rice x wheat and rice x sorghum hybrids

Summary The polypeptide composition of Fraction-1-Protein (F1P) from rice × sorghum, rice × wheat hybrids and their respective parents have been analyzed by a microelectrofocusing method. The large sub-unit (LSU) is composed of three polypeptides and the small sub-unit (SSU) of two polypeptides in rice and sorghum parents and rice × sorghum hybrids. Similarly, LSU is composed of three polypeptides in the rice and wheat parents and rice × wheat hybrids. Two polypeptides occur in the SSU of rice parent and rice × wheat hybrids where as only one polypeptide in the wheat parent. These polypeptides also differ in their isoelectric points. Based on the previous reports of F1P inheritance in hybrids in other crops, F1P analysis of rice × sorghum and rice × wheat hybrids does not seem to be an important marker to identify such intergeneric hybrids. Since this is first such report of F1P inheritance in hybrids between distantly related plants, its implication in different modes of inheritance are discussed.Abbreviations F1P Fraction-1-Protein - IEF Isoelectric focusing - pI Isoelectric points - LSU Large sub-unit - RuBPCase Ribulose 1,5-Bisphosphate Carboxylase-oxygenase - SSU Small sub-unit     

Identification of novel alleles from wild barley for the improvement of alpha‐amylase and related malt quality traits

Germplasms consisting of 64 wild barley accessions (Hordeum vulgare ssp. spontaneum) were examined for polymorphisms in α‐amylase using both isoelectric focusing (IEF) and thermostability assays. Wide variation was found for the high pI α‐amylase with 20 IEF band patterns identified. Enzyme activity and thermostability assays showed large differences among α‐amylase isoenzymes. Two wild accessions Tel‐Shoket CPI 77146‐32 and Afiq CPI 77128‐41 showed superior enzyme activity and thermostability compared with commercial varieties such as ‘Baudin’, ‘Flagship’ or ‘Navigator’. The functionality of the Tel‐Shoket allele was validated in backcross lines with ‘Flagship’ as the recurrent parent. The Tel‐Shoket allele at the amy1 locus increased α‐amylase thermostability at 75°C by 8.4% and α‐amylase activity in kilned malt by 18.7%. The introgression of the wild allele also led to significant improvements in fermentability, hot water extract and viscosity. Gene sequencing showed that there are three single nucleotide polymorphisms in the Tel‐Shoket amy1 sequence, which can be used as diagnostic markers for the selection of this allele in breeding programmes.     

蓖麻毒蛋白的分离纯化和毒理作用研究

经（NH4）2SO4沉淀, 分子筛层析和亲和层析等处理,从去壳蓖麻籽中分离纯化得到蓖麻毒蛋白, 在SDS-聚丙烯酰胺凝胶电泳中呈32kDa和34kDa两条蛋白带, 证明该蓖麻毒蛋白已被纯化至均一。 用等电聚焦法测出该蓖麻毒蛋白的等电点为pI 6.1和pI6.7。 高效液相色谱测出该蓖麻毒蛋白在非变性条件下有五个峰, 表明蓖麻毒蛋白可能存在五个不同的多聚态。 动物实验表明该蓖麻毒蛋白对小白鼠有较强的毒性,但对斜纹夜蛾幼虫无毒杀作用。     

聚丙烯酰胺凝胶电泳鉴定烟草品种

在本实验条件下，得到的8个烟草品种种子的可溶性蛋白质的SOD-聚丙烯酚胺凝胺电泳（SDS-PAGE）和聚丙烯酚胺等电聚焦电泳（IEF-PAGE）图谱，分辩率高，重复性强，其多态性条带分别占61.5%和52.6%,各品种的电泳图谱可以相互区别,表明种子的可溶性蛋白的SDS-PAGE和IFF-PAGE图谱可作为烟草品种鉴定的蛋白质指纹图谱.     

Allelic variants of granule-bound starch synthase proteins in European bread wheat varieties

The composition of 324 European wheat cultivars were analysed at the three granule‐bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE. A specific two‐dimensional (2D) electrophoresis (immobilized pH gradient × SDS‐PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx‐A1 null allele (Wx‐A1b) and none was found to have the Wx‐2D null allele. As described in the literature the Wx‐B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx‐D1 protein in SDS‐PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx‐B1 null allele. Indeed this band probably corresponded to the Wx‐B1? or Wx‐B1e allele overlapping with the Wx‐D1a allele in SDS‐PAGE.     

The use of reversed-phase high performance liquid chromatography as an aid to the identification of European barley cultivars

Summary A reversed-phase high performance liquid chromatography (RP-HPLC) system was used to separate the storage proteins (hordeins) extracted from European barley cultivars. From a total of 38 barleys tested, 26 types of hordein patterns could be distinguished after RP-HPLC. This appears to be a marked improvement in resolution over that achieved in a similar survey of European barley cultivars using SDS polyacrylamide electrophoresis (32 hordein patterns resolved by SDS PAGE from a total of 160 spring and winter barleys tested).Different hordein patterns were resolved by RP-HPLC within each of two groups of barley previously classified by SDS PAGE as indistinguishable within groups (three distinct patterns identified in a total of five cultivars tested from group 1A and five patterns observed among eight cultivars from group 3B). Thus RP-HPLC achieves a higher resolution than undirectional electrophoresis and promises to be a valuable aid in the identification of European barley cultivars.     

Variation in seed proteins from mutagen-treated cultivars and selected lines of Vigna unguiculata L. Walp

The storage protein profiles of the seeds of two IITA cowpea cultivars (‘IT84E–124’ and ‘Vita 7’) exposed to three mutagens—sodium azide (NaN₃), ethyl methane sulphonate (EMS) and ⁶⁰Co gamma rays—and those of 18 selected M₃ lines were investigated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). The total seed protein, globulin and albumin fractions showed differences in number and intensity of subunit bands, but the least differences were found in the albumin fraction. Both high and low molecular weight bands were observed, the highest being 94.5 kDa and the lowest 12.0 kDa.‘Vita T showed less variability compared with ‘IT84E–124’, as indicated by the relative similarity indices (S.I.) of the two cultivars. The lowest S.I. of 0.200 was found among ‘IT84E–124’ lines while the lowest S.I. among ‘Vita T lines was 0.4706. A number of lines with particular traits were found to be characterized by the presence of specific polypeptide bands. This study demonstrates that induced mutation could create additional variability to supplement existing germplasm and that SDS-PAGE is a useful tool for discriminating and estimating genetic similarities among selections.     

Phaseolus coccineus Storage Proteins II. Electrophoretic Analysis and Erythroagglutinating Activity in Various Cultivars

Biochemical analyses of seed storage proteins of Phaseolus coccineus have been carried out to identify seventeen different cultivars. The electrophoretic patterns in native polyacrylamide gel electrophoresis (PAGE) and denaturing conditions (SDS-PAGE); evidenced qualitative and quantitative differences for the three major protein components: legumin, vicilin and phytohaemagglutinin (PHA). The results were confirmed by isoelectrofocusing (IEF) analyses. Erythroagglutination tests showed the presence of high agglutinating activity particularly in cultivars with low vicilin content. The experimental results allow one to distinguish all the cultivars by their electrophoretic spectra and agglutinating activities.     

水稻谷蛋白的一个新基因克隆及表达分析

与其他禾本科作物以醇溶蛋白为主不同，水稻种子含有醇溶蛋白和谷蛋白两种主要蛋白质贮藏形式。其中，谷蛋白约占胚乳蛋白总量的70%~80%。水稻谷蛋白是由多基因家族编码合成的，到目前为止至少已克隆获得了9个全长cDNA，根据这些cDNA编码的氨基酸序列同源性可将谷蛋白分为A、B两个亚家族。B亚族谷蛋白成员富含赖氨酸等人体必需氨基酸，与稻米的营养品质直接相关，因此挖掘、利用B亚族基因成员对于改良稻米蛋白品质性状至关重要。本文报道了以32P标记的谷蛋白基因GluB-2 cDNA片段为探针筛选水稻胚乳cDNA文库获得1个新的水稻谷蛋白基因全长cDNA。序列分析显示该基因核苷酸序列共1588 bp，含有1个由495个氨基酸残基组成的开放阅读框，编码蛋白分子量约为56 kD。推导的氨基酸序列与其他已知谷蛋白基因家族成员间序列相似性介于57.8%~97.8%之间，并与B亚族谷蛋白基因的同源性更高，因此命名为GluB-7（GenBank注册号AY987390）。Northern杂交显示，GluB-7具有高度的胚乳表达特性。     

Detection of HMW glutenin subunit variations among 205 cultivated emmer accessions (Triticum turgidum ssp. dicoccum)

The high molecular weight glutenin subunits (HMW‐GS) encoded by Glu‐1 loci among 205 accessions of cultivated emmer wheat (Triticum turgidum ssp. dicoccum Schrank) collected from different regions of Europe and China were separated and characterized by SDS‐PAGE in combination with two‐dimensional gel electrophoresis (A‐PAGE × SDS‐PAGE) and acidic capillary electrophoresis. High genetic polymorphisms in HMW‐GS compositions were found. A total of 40 alleles (6 for Glu‐A1 and 34 for Glu‐B1) and 62 subunit combinations (genotypes) were detected, some of which were not previously described. At Glu‐A1 locus, two novel alleles, designated Glu‐A1x coding for the subunit 1A × 1.1 and Glu‐A1y coding for the subunit 1A × 2.1′ were found while seven new subunits (1B × 17*, 1B × 6′, 1B × 13′, 1B × 20*, 1By9*, 1By14.1 and 1By8.1) and 20 novel alleles at Glu‐B1 locus were detected. In particular, some additional protein components were detected, which probably were 1Ay subunits encoded by Glu‐A1 locus. The introduction of both Ax and Ay subunits from tetraploid wheats into hexaploid wheats may increase the genetic variability of gluten genes and consequently improve flour technological properties.     

Characterization of kafirin composition and in vitro digestibility in CMS‐lines,fertility restorers and F1 hybrids with the new types of CMS‐inducing cytoplasms of sorghum

One of the reasons of poor nutritive value of sorghum grain is resistance of its seed storage proteins (kafirins) to protease digestion. To reveal sorghum entries with increased kafirin digestibility, the sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) of endosperm proteins of 10 lines [cytoplasmic male sterility (CMS)‐lines and fertility restorers] and five F₁ hybrids before and after pepsin digestion was carried out. For quantitative estimation of kafirin digestibility the SDS‐PAGE banding patterns were scanned by laser densitometer. Significant variability for both individual fractions and total kafirin digestibility was found. The line KVV‐45, fertility restorer for the Indian ‘M35‐1A’ type of CMS, had the highest level of kafirin digestibility (30% and 25% of undigested γ‐ and α₁‐kafirins, respectively), while in some entries 80–90% of kafirins remained undigested. Increased α₁‐kafirin digestibility coincided with relatively high γ‐kafirin digestibility. High‐molecular weight kafirins (HMWK) (45 kDa and 66 kDa) resistant to pepsin digestion were found in some lines, the F₁ hybrids had the same HMWK as parental lines. These data demonstrate possibility for isolation of sorghum genotypes with increased nutritive value by screening their flour for in vitro protein digestibility.     

Flax Seed Proteins Comparison by Various PAGE-Techniques in Slabs

Seed proteins of a number of Egyptian, French, and British cultivars of flax have been separately extracted with buffer, and water and buffer respectively, and analyzed by slab electrophoresis: PAGE, SDS-PAGE, and poro-SDS-PAGE (22.5%–10%), and by PAGIF. The protein patterns were compared to find out the best method to differentiate between flax cultivars.
The results of the water extracts and the residue extracted with Tris/borate buffer and analyzed on SDS-PAGE indicated that the approximate MWs of native albumins and globulins ranged from 4.2 to 1.2 and 7.6 to 1.6 × 10⁴, respectively. It is also shown that SDS-PAGE of globulins extracted with Tris/borate buffer are the method recommended for differentiation of flax cultivars.
The data of the total proteins extracted with Tris/HCl buffer and analyzed on SDS-PAGE under reducing and non-reducing conditions exhibited 5 disulphide bonded bands which on reduction gave acidic subunits and basic subunits.     

烤烟与晒烟品种同工酶差异初探

对来源中国，美国等国家的烤烟品种，晒烟品种９种同工酶进行了聚丙烯胺凝胶电泳分析。结果表明：品种间表现多态性的５种同工酶中，烤烟品种成熟期腰叶乳酸脱氢酶，花蕾中的过氧化物酶分别比晒烟品种多一条带，成为烤烟区别晒烟器品种的同工酶标记，这些标记可应用于烤烟和晒烟的杂交后代单株类型鉴定。     

38–48 kDa subunits of buckwheat 13S globulins are controlled by a single locus

The inheritance of buckwheat (Fagopyrum esculentum Moench) seed storage proteins was investigated by control crosses between accessions containing different SDS‐PAGE protein patterns in the range of 38–48 kDa. Analysis of segregation of individual F₁ and F₂ seeds obtained by 17 crosses showed segregation of eight electrophoretic bands, from which 12‐banding variants were inferred. Two variants contained one band, nine contained two bands and one variant contained three bands and behaved as a single co‐dominant unit. The segregation of banding variants fits the expected ratios and thus supports the hypothesis of single Mendelian gene inheritance and that alleles are co‐dominant. The results therefore suggest that the genes controlling globulin subunits are tightly linked and inherited as a single locus. The locus was designated Glob‐1 and the twelve segregated protein variants as alleles a‐l. This is the first buckwheat globulin locus identified with multiple alleles, phenotypically expressed as groups of protein bands and thus applicable as a functional marker in buckwheat genomic studies.     

小麦抗白粉病生化标记--过氧化物酶pⅠ 6.1酶带

采用等电聚焦凝胶电泳比较了抗、感白粉病小麦品种的过氧化物酶同工酶酶谱,发现抗、感品种之间的叶片过氧化物酶同工酶pⅠ6.1酶带(位于过氧化物酶同工酶等电聚焦凝胶电泳酶谱pⅠ6.1)在拔节期至白粉病发生期表现水平有很大差别.抗病品种中编码pⅠ6.1酶带的基因终生正常表达,该酶带在小麦全生育期均有较强的活性,染色后着色深,多属一级酶带.而该基因的表达在感病品种拔节之后受到抑制,酶带变得模糊不清甚至消失踪迹,多为三级、零级酶带.感染白粉病之后酶活性才回升,再度表现为一、二级酶带.为了探索造成这一差异的原因,比较了119株抗病品种与感品种杂交F2代和65株抗病品种/感病品种//感病品种的BC1代植株叶片过氧化物酶同工酶IEF酶谱,证明感病品种成株的pⅠ6.1酶带活性下降是因另一隐性基因所至.     

Physiological basis of UV-C induced resistance to Botrytis cinerea in tomato fruit. V. Constitutive defence enzymes and inducible pathogenesis-related proteins

Changes in the protein content and profile of postharvest tomato fruit treated with the hormetic dose (3.7 kJ m^?2) of ultraviolet light C (UV-C) at the mature green stage was investigated. In UV-C treated fruits, the total protein content increased until 10 d after treatment and decreased thereafter during a 30 d storage period; whereas in control fruit, protein content decreased constantly throughout the storage period. Using polyacrylamide gel electrophoresis (PAGE) it was shown that UV-C treatment affected the protein profile of tomato fruit in several manners: (1) UV-C repressed the expression of some proteins presumably associated with ripening; (2) it enhanced the expression of several constitutive proteins, of which one was an acidic β-1,3-glucanase, three acidic chitinases and three basic chitinases; and (3) it induced the synthesis of at least 5 new proteins of which four were basic proteins. Among the proteins induced by UV-C, three (a basic β-1,3-glucanase and two acidic chitinases) were apparently pathogenesis-related proteins as they were also induced by inoculation with Botrytis cinerea. The molecular mass (MM) of five of the UV-C induced proteins was determined using SDS-PAGE. Their molecular masses were 45, 39.4, 34.6, 10 and 8.9 kDa. The UV-C induced β-1,3-glucanase had a MM of 33.1 kDa. The MM of two constitutive chitinases were 48.3 and 30.5 kDa, and those of the two UV-C and pathogenesis-induced chitinases were 37.1 and 20.6 kDa. Furthermore, the glucanohydrolase activities induced by UV-C were maintained until the end of the storage period. It is likely that the PR-proteins with glucanohydrolase activities induced by UV-C are an integral part of the long-term resistance observed in UV-C treated tomato fruit.     

Comparative analysis of different methods revealing the involvement of proteins during ageing of rice seeds

Seed longevity is a very important characteristic controlling seed quality. However, the mechanism underlying this characteristic is poorly understood. In this study, 'FH7185', a storable rice variety, the germination rate of which was 66.63%, much higher than the control after artificial ageing under relative humidity 88% and 42°C for 21 days. Different methods were applied to reveal the involvement of proteins during ageing of rice seeds. In total, 35 differential proteins were identified from 2D‐PAGE, and 3,719 proteins from iTRAQ analysis. A comparison of these two methods showed that not all protein types could be detected on the 2D‐PAGE gels, and the dynamic range was somewhat limited. So the comprehensive proteome map from the iTRAQ analysis was used to identify the quantitatively regulated proteins that played roles in seed longevity, and found that the most marked change was the increased abundance of many metabolic enzymes, especially the ones involved in α‐linolenic acid metabolism in the embryos during ageing. OsLOX2 and OsLOX3 had a negative effect on seed longevity, which were lower in FH7185 than the control. The dehydrogenase (LOC_Os07g44430) which played a major catalytic role in lipid peroxidation, also changed significantly during ageing. Therefore, OsLOX2, OsLOX3 and Loc_OS 07G44430 may be involved in the regulation of seed longevity with a synergistic effect.     

高压处理对肌球蛋白质的影响

从牛骨骼肌中提取肌球蛋白质后进行纯化,并用不同程度的压力(100,200,300,400 MPa)进行处理,再与未处理样品进行比较。采用SDS—PAGE分析了解压力对肌球蛋白的影响。从SDS—PAGE可观察到,随着压力的升高,肌球蛋白质条带逐渐变淡,压力为400 MPa时,13.8 kDa的肌球蛋白轻链几乎已经消失,而200 kDa的肌球蛋白重链下方出现了一条比较模糊的其他蛋白质条带。     

Stability of esterases and total proteins during seed storage of perennial ryegrass (Lolium perenne L.) and their use for cultivar discrimination

Polymorphisms in electrophoretic patterns obtained by isoelectric focusing (IEF) were examined to evaluate their suitability for cultivar identification in perennial ryegrass (Lolium perenne L.). It was possible to discriminate 64 (94%) of 68 cultivars by combining results from esterase and total protein analysis. Discrimination was based on quantitative differences (relative band intensity) rather than on qualitative differences (presence or absence of bands). Esterase patterns from different recent (fresh to seven years old) seed lots of the same perennial ryegrass cultivars were very stable. Occasionally, minor differences in band intensity were observed between recent and old (up to 30 years old) seed lots of a cultivar. Storage of meal samples up to two years at −20 ^°C had no effect on the total protein patterns. No correlation was found between esterase patterns and ploidy level, cultivar type (pasture or turf), heading date or breeding company. Esterase patterns appeared to be unsuitable markers for the selection of reference cultivars for distinctiveness, uniformity, and stability (DUS) testing, because no correlation was found between cultivars on the basis of esterase banding patterns and morphological characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.