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1.
The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 ≈ 3 > 1 > 6 ≈ 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SDS-PAGE. The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order.  相似文献   

2.
Cloning and functional analysis of high molecular weight wheat glutenin subunit (HMW-GS) 1By8 from Italy durum cultivar Simeto was carried out in this study. All HMW-GS from Simeto were separated and characterized by appropriate electrophoresis methods, reversed-phased high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS). The complete gene encoding 1By8 subunit was amplified by allele-specific PCR primers, including an upstream sequence of 857 bp and an open reading frame (ORF) of 2166 bp encoding a mature protein of 720 amino acid residues. The promoter sequence, containing -300 element (cereal glutenin gene control element) and enhancer was highly conserved among HMW-GS genes. Comparison with the sequence of subunit 1By9 from bread wheat demonstrated 99% identity with the main difference being that the 1By8 subunit possesses an additional insertion of 15 amino acid residues (QYPASQQQPA QGQQG) at position 342 and two residue substitutions at position 78 (leucine/proline) and 442 (arginine/glutamine). The molecular weight differences between MALDI-TOF-MS and deduced amino acid sequence of the coding gene revealed the possibility of some kinds of post-translational modifications present in 1By8 subunit. The protein subunit expressed in Escherichia coli showed a very similar mobility to the endogenous 1By8 of Simeto on SDS-PAGE. The function of the isolated protein on wheat processing quality was determined by 10 g Mixgraph analysis. Results demonstrated that addition of y-type HMW glutenin subunits into the base flour had significant positive effects on main mixing parameters and significant difference in effects were observed among different y-type subunits.  相似文献   

3.
Cloning and functional analysis of high molecular weight wheat glutenin subunit (HMW-GS) 1By8 from Italy durum cultivar Simeto was carried out in this study. All HMW-GS from Simeto were separated and characterized by appropriate electrophoresis methods, reversed-phased high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS). The complete gene encoding 1By8 subunit was amplified by allele-specific PCR primers, including an upstream sequence of 857 bp and an open reading frame (ORF) of 2166 bp encoding a mature protein of 720 amino acid residues. The promoter sequence, containing -300 element (cereal glutenin gene control element) and enhancer was highly conserved among HMW-GS genes. Comparison with the sequence of subunit 1By9 from bread wheat demonstrated 99% identity with the main difference being that the 1By8 subunit possesses an additional insertion of 15 amino acid residues (QYPASQQQPA QGQQG) at position 342 and two residue substitutions at position 78 (leucine/proline) and 442 (arginine/glutamine). The molecular weight differences between MALDI-TOF-MS and deduced amino acid sequence of the coding gene revealed the possibility of some kinds of post-translational modifications present in 1By8 subunit. The protein subunit expressed in Escherichia coli showed a very similar mobility to the endogenous 1By8 of Simeto on SDS-PAGE. The function of the isolated protein on wheat processing quality was determined by 10 g Mixgraph analysis. Results demonstrated that addition of y-type HMW glutenin subunits into the base flour had significant positive effects on main mixing parameters and significant difference in effects were observed among different y-type subunits.  相似文献   

4.
为了解我国黄淮麦区主栽品种的主体HMW-GS基因的动态表达规律,以该麦区的35个主栽小麦品种（系）为材料,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）技术对参试材料高分子量麦谷蛋白亚基（HMW-GS）进行分离和鉴定,并对HMW-GS的积累动态进行了研究。结果表明,籽粒灌浆过程中,7亚基大约在花后10 d开始表达,25 d左右所有亚基均已表达,但不同亚基开始表达的时期略有差异。20 d之后,各亚基表达量开始快速增加,约在35 d左右表达量达最高,之后略有下降,在40 d左右趋于稳定。就其相对表达量而言,5亚基和10亚基明显高于其他亚基类型。  相似文献   

5.
The degradation effects of wheat bug protease(s) on glutenin proteins of durum wheat cultivars were investigated by electrophoresis and modified rapid visco analyser (RVA) test. Glutenin patterns of the bug damaged durum wheats changed substantially due to bug protease(s). Although high molecular weight glutenin subunits (HMW-GS) of three cultivars (Ege, Svevo, and Zenith) disappeared after 60 min of incubation, the HMW-GS of other two cultivars (Diyarbakir and Firat) were still visible even after the longest incubation period at medium damage level. It shows that there was an intercultivar variation in susceptibility to hydrolysis by bug proteolytic enzymes. Low molecular weight glutenin subunits of all cultivars decreased substantially after 30 min of incubation. The RVA curves indicated a clear reduction in viscosity in semolina samples with both medium and high damage levels as compared to their respective undamaged (control) samples. There were significant correlations (p < 0.001) between bug damage level and viscosities at 3 min (r = −0.765), at 4.5 min (r = −0.549) and at 10 min (r = −0.835), breakdown value (r = −0.534) and decay rate (r = 0.600). Consequently, hydrolysis rate of wheat bug protease(s) can be determined by modified RVA technique without much more chemicals, procedures and expensive equipments.  相似文献   

6.
The variations of the amounts of individual high molecular weight glutenin subunits (HMW-GS), of the ratios HMW-GSy to HMW-GSx and HMW-GS to low molecular weight glutenin subunits (LMW-GS) and of protein content were evaluated for eight durum wheat cultivars in two regions using four fertilizer combinations during two successive years. All measured parameters showed significant variation with genotypes (G), environments (E) and fertilizers (F). The interaction E × G × F was highly significant for glutenin amount variation. Amongst cultivars possessing HMW-GS 20, landraces seem to better value the N-fertilizer use for the accumulation of HMW-GSy than high yielding cultivars. Both HMW-GSy to HMW-GSx and HMW-GS to LMW-GS ratios were found to be positively correlated (p < 0.05) with total protein content.  相似文献   

7.
为给小麦高产优质育种提供参考,以黄淮麦区35个主栽小麦品种（系）为材料,利用SDSPAGE电泳技术对其进行了高分子量麦谷蛋白亚基鉴定,并对各参试品种的灌浆特性进行了分析。结果表明,35个黄淮麦区小麦品种（系）中 GluA1、GluB1和 GluD1位点分别只有2、5和4种亚基类型,分别以Null、7+9和2+12亚基为主,所占比例分别为71.4%、45.7%和62.7%。依据 GluB1和 GluD1位点的亚基特点,将参试品种（系）分为三类。对这三类品种（系）的灌浆期粒重增长特性分析发现,第I类（亚基组合：7+8/2+12）品种（系）灌浆速率较快,最终粒重较高;第II类（亚基组合：7+9/2+12）品种灌浆持续时间较长,但灌浆速率不及第I类,最终粒重较高;第III类（亚基组合：7/5+10或7+8/5+10）品种（系）灌浆持续时间较短,且灌浆速率较慢,最终粒重不高。三类品种（系）相比,粒重增长速率在灌浆前期基本相同,但35 d以后,第III类品种（系）粒重增长速率明显减慢,这可能是导致其最终粒重显著低于前两类品种（系）的主要原因。  相似文献   

8.
从小麦谷蛋白大聚合体与HMW GS的关系   总被引：1，自引：0，他引：1  
为了探索小麦谷蛋白大聚合体对小麦品质的影响,采用SDSPAGE方法,分析了166个来自不同地区的小麦品种高分子量谷蛋白亚基（HMWGS）的组成,并对不同亚基与小麦谷蛋白大聚合体（GMP）的关系进行了分析。结果表明,所用小麦材料HMWGS的变异类型丰富,但亚基组合仍以N/7+9/2+12为主。HMWGS对面粉GMP含量有显著影响,GluA1位点亚基1>N,GluB1位点亚基13+16>7+9或7+8或14+15或17+18,GluD1位点亚基5+10>2+12,其他亚基差异不显著。说明含亚基1、13+16和5+10的品种能形成较多的GMP。HMWGS评分与面粉GMP含量的相关系数为0.288,达到极显著水平。  相似文献   

9.
为从一级结构水平揭示小麦高分子量麦谷蛋白亚基的结构、功能及其进化上的关系,利用生物信息学软件(DNAMAN)对已在GenBank数据库中登录的22个小麦高分子量麦谷蛋白亚基序列进行了分析.结果表明,这些高分子量谷蛋白亚基平均含氮量为16.186%;其各种氨基酸含量基本恒定,其中谷氨酰胺含量最高(33.74%),且主要以QQ形式连接在一起,这种结构特点可能影响小麦面粉的加工品质;同时在一级结构序列上还发现有232个保守的氨基酸位点(56个单个氨基酸座位和一些长短不同的氨基酸片段),较大的保守性氨基酸片段主要为AEGEAS-QLQCER/HEL,GSFY PG/SETTP-QQLQQ,YYPGQAS/FP/SQQ/RP/SGQG/RQQ,PGQGQQ/PG/A/YYPTS-QQ等;分别有6组共13个序列相似度均达到100%,推测这些高分子量谷蛋白亚基在进化上具有较强的保守性.  相似文献   

10.
Two winter wheat (Triticum aestivum L.) cultivars differing in grain protein content were selected to study the effect of N application rate on changes in contents of glutenin macropolymer (GMP) and high molecular weight glutenin subunits (HMW-GS) during grain filling. Contents of GMP and HMW-GS were much higher in the high GPC cultivar, Xuzhou 26, than those in low GPC cultivar, Ningmai 9. N increased contents of GMP and HMW-GS in Xuzhou 26 with N rate between 0 and 300 kg ha−1, while at the very high N rate of 300 kg ha−1 the contents of GMP and HMW-GS in Ningmai 9 decreased. The high contents of GMP and HMW-GS at maturity were closely related to the rapid increase in contents of GMP and HMW-GS during the initial period of their synthesis. HMW-GS and GMP content were closely correlated. The total HMW-GS content was important in determining GMP content than the content of any HMW-GS pair or any individual HMW-GS present in the selected cultivars. The pattern of response of GMP content to N application rate was closely related to the regulatory effect of N on HMW-GS synthesis.  相似文献   

11.
Brachypodium distachyon, an emerging model plant system for some economically important temperate grasses such as wheat, barley and switchgrass, has recently caught wide attention in modern biological research. In the current study, the glutenin, albumin and globulin components of 13 B. distachyon accessions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) followed by peptide mass finger printing (PMF) and MS/MS protein identification. Abundant wheat low molecular weight glutenin subunit (LMW-GS) like proteins and a few high molecular weight glutenin subunits (HMW-GS) with low expression level were detected in B. distachyon. A total of 18 storage proteins and 15 albumin proteins were identified through PMF and MS/MS. The results demonstrated that the major seed storage proteins in B. distachyon are wheat LMW-GS like proteins and globulins. The identified albumins and globulins were mostly various enzymes that were classified into five groups according to their functions. The 2-DE spot distribution and MS results suggested that post-translational modifications (PTMs) such as phosphorylations and glycosylations are common phenomena in B. distachyon seed proteome.  相似文献   

12.
甘肃小麦品种(系)HMW-GS遗传变异分析   总被引：4，自引：1，他引：4  
为了了解甘肃省小麦品种HMW-GS的遗传变异和组成，为甘肃优质专用小麦品种筛选和品种改良提供依据,采用SDS-PAGE法，分析了254份甘肃省小麦材料(育成品种(系)和农家品种)Glu-1位点的HMW-GS变异，共检测到22种HMW-GS变异，Glu-A1位点3种，Glu-B1位点11种，Glu-D1位点8种。其中110个育成品种(系)的15种HMW-GS有27种亚基组合类型。Glu-A1位点有2种亚基，47．3％的品种该位点具有优质亚基；Glu-B1位点有7种亚基，76．4％的品种具优质亚基；Glu-D1位点有6种亚基，32．7％的品种具优质亚基。14．5％的品种(n：15)Glu-1位点具优质亚基。144份农家品种的18种HMW-GS共有29种亚基组合形式，Glu-A1位点有3种亚基,Glu-B1位点有9种亚基，Glu-D1位点有6种亚基，无优质亚基组合。  相似文献   

13.
The high molecular weight glutenin subunits (HMW-GS) play a key role in end-use quality of wheat. Their particular primary structure is mostly derived from DNA sequencing, which gives no information on potential post-translational modifications. This paper reveals the primary structure of HMW-GS 1Dx2 by proteomic analysis. For this purpose, HMW-GS were first isolated from wheat flour (cv. Contra). The relative molecular mass (Mr) of subunit 1Dx2 present in the HMW-GS mixture was then very accurately determined with high-performance liquid chromatography–electrospray ionization-mass spectrometry using a quadrupole-time-of-flight mass analyzer (HPLC–ESI-QTOF-MS). The obtained Mr value (87,105) differed from the value derived from its protein sequence in the NCBI database (87,007). The subunit was further purified by preparative reversed-phase HPLC and partially hydrolyzed with chymotrypsin. The resulting 1Dx2 peptides were then analyzed by HPLC–ESI-MS/MS and the MS data were compared to amino acid sequences in protein databases. The discrepancy between the calculated and the measured Mr of 1Dx2 was explained by a missing proline in the 1Dx2 amino acid sequence from the database and not by any post-translational glycosylation.  相似文献   

14.
The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H2O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.  相似文献   

15.
粗山羊草高分子量谷蛋白亚基组成分析   总被引：1，自引：0，他引：1       下载免费PDF全文
为了发现能够用于小麦品质改良的优异高分子量谷蛋白亚基(HMW-GS),应用SDS-PAGE技术分析了47份粗山羊草Glu-Dt1位点的HMW-GS组成,分别检测到5种x-型亚基(1.1t、1t、1.5t、2t、3t)和4种y-型亚基(10.1t、11t、12t、12.4t).其中,1Dx1.1t是1种新的x-型亚基,它的迁移率比普通小麦的1Ax1亚基稍慢,这是目前在粗山羊草中发现的分子量最大的HMW-GS.供试粗山羊草中有8种HMW-GS组合类型:1.1t 11t、1t 11t、1.5t 12t、2t 10.1t、2t 11t、2t 12t、2t 12.4t和3t 11t.其中,1.1t 11t和2t 12.4t未见报道.  相似文献   

16.
为了克隆节节麦中的HMW-GS基因,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析了节节麦的HMW-GS,发现1种编码序列未知的y-型亚基,即Dy8.1t亚基。通过对目的片段的AS-PCR扩增、克隆、测序和氨基酸序列推导,发现这种未知序列具有典型HMW-GS的序列结构特征。通过与已知亚基序列比较发现,Dy8.1t与Dy10t、Dy10.4t、Dy10.5t和Dy12t在N-端序列有一个氨基酸差别(R-G)。与已知氨基酸序列的HMW-GS多序列比对和系统进化关系分析,证实Dy8.1t亚基是D基因组编码的高分子量谷蛋白y-型亚基家族的新成员。  相似文献   

17.
为了解应用高效毛细管电泳(HPCE)技术定量分析优质小麦高分子量谷蛋白亚基(HMW-GS)的效果,利用HPCE技术对13个小麦品种的HMW-GS进行亚基识别和定量分析,并与传统的SDS-PAGE电泳图进行了比较.结果表明,在HPCE电泳图中HMW-GS的亚基出现顺序与SDS-PAGE略有不同,亚基1Ax1早于1Bx7出峰,并且在主亚基之后还有小的亚基出现,尤其是1Dx2主亚基后面有3个小亚基.定量分析表明,优质小麦品种陕627的HMW-GS,不论单个亚基、亚基组合还是亚基总和的含量都高于其他小麦品种,且均达极显著水平.优质小麦品种陕715的HMW-GS含量除亚基1Ax1与陕627接近外,其余亚基和亚基组合都显著低于陕627.西农5211的HMW-GS因为不舍有亚基1Ax1,尽管其他亚基、亚基组合含量接近于陕627,但HMW-GS的总含量显著低于陕627.优质小麦的优质亚基含量较高.HPCE具有高效、灵敏、再现性高等特点,能够快速识别和定量分析小麦HMW-GS,可以为小麦品质育种或组合选择提供参考.  相似文献   

18.
为了建立准确有效小麦HMW-GS的检测方法,提高优质小麦品种鉴定和筛选效率,以已知HMW-GS组成的16份小麦品种为对照,优化完善SDS-PAGE结合分子标记检测小麦HMW-GS的方法,并对103份宁夏小麦品种进行了验证和分析。结果表明,8.5%的分离胶可以有效区分除了 Glu-B1位点的7*、7OE与8*亚基之外的其他亚基,分辨效果优良;SDS-PAGE结合 Bx7OE、 Bx7*/Bx7和 By8基因的分子标记可以准确鉴定小麦HMW-GS。在103份宁夏小麦品种中,发现15种HMW-GS和29种组合类型;首次在该地区小麦品种的 Glu-B1位点检测出了携带7OE、7*和8*亚基的品种;1/17+18/5+10为当地小麦HMW-GS的优势亚基组合类型,占20%。自1970年至2010年,HMW-GS的优质亚基（1、17+18和5+10）的出现频率呈明显增长趋势;宁夏小麦的HMW-GS种类和优质亚基出现频率随品种更换呈增加趋势。综上所述,分离胶浓度为8.5% 的SDS-PAGE和 Bx7OE、 Bx7*/Bx7和 By8分子标记的方法可准确有效地检测小麦HMW-GS组成,此方法可用于优质小麦品种的鉴定和筛选。  相似文献   

19.
The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.  相似文献   

20.
为探讨甲基磺酸乙酯(ethyl methane sulfonate,EMS)诱变在小麦品质育种中的价值,了解EMS诱变小麦籽粒品质变异状况,筛选优良的变异材料,对1667份西昌69的EMS诱变系M 6代籽粒的蛋白质含量、淀粉含量、面筋含量、容重、面团形成时间和稳定时间等多个籽粒性状进行了测定,初步分析了诱变系的品质变化,并通过SDS-PAGE电泳法及高效液相色谱技术分析了其籽粒高分子量麦谷蛋白亚基(HMW-GS)的组分和不溶性蛋白聚合体的含量。共筛选到101份HMW-GS变异诱变系,分别为亚基缺失、亚基增生、亚基置换和亚基缺失且置换4种变异类型。其中,有28份诱变系的蛋白质含量、面筋含量、形成时间、稳定时间等多个籽粒品质性状及不溶性蛋白聚合体含量优于亲本,且千粒重相对稳定,可以作为优质种质资源用于小麦品质育种。  相似文献   

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