长穗偃麦草DNA导入小麦后代变异系醇溶蛋白的研究

应用花粉管通道技术,将抗逆性强的长穗偃麦草总ＤＮＡ导入普通小麦甘麦８号,后代中出现了广泛的变异,并筛选出两个高产、分蘖力强、抗条锈病的新品系。以这两个品系为材料,以供体和受体作为对照,研究了外源ＤＮＡ导入后籽粒醇溶蛋白的变化,发现醇溶蛋白的增加、缺失和电泳迁移率的变化,新增加的组分与供体长穗偃麦草某些组分相对应。由此推测外源ＤＮＡ导入受体后有可能某些ＤＮＡ片段插入受体基因组从而导致受体基因表达改变或基因突变。     

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。     

不同提取剂对麦醇溶蛋白提取效果的电泳比较

以大麦、小麦品种为材料,分别采用醇溶蛋白提取剂乙醇、乙二醇、2-氯乙醇、2-巯基乙醇、尿素等配成不同浓度的单一组分提取剂和复合提取剂,在同一条件下提取大麦和小麦去胚种子的醇溶蛋白,通过A-PAGE分离样品液发现,单一组分提取剂中25％的2-氯乙醇效果较好,5％的2-巯基乙醇效果较差;复合提取剂中由2-氯乙醇和2-巯基乙醇组成的提取剂效果最好。这种复合提取剂制备的样品液经电泳后,在图谱中条带丰富,带型清晰,无论对大麦还是小麦种子,都是最佳的醇溶蛋白提取剂。     

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     

黑麦碱基因（Sec–1）表达缺失的1RS／1BL易位系的鉴定

用改良的Giemsa C－带技术、DNA原位杂交和酸性聚丙烯酰胺凝胶电泳（A-PAGE）对来源于小麦品种绵阳11与不同黑麦自交系远缘杂交获得的高代株系（BC1F7）的染色体结构和醇溶蛋白进行了研究。结果发现,在鉴定的200个株系中,有45个株系经C-带和A-PAGE检测均一致地发现它们含有一对1RS /1BL易位染色体,而一个株系843-1-1,C-带鉴定、原位杂交结果均证明它含有一对1RS/1BL易位染色体,但A-PAGE醇溶蛋白图谱却不具有黑麦1RS染色体臂的黑麦碱特征带,而表达出既不同于黑麦碱又不同于亲本绵阳11的醇溶蛋白带型。这一结果表明,利用不同的黑麦亲本资源,可以获得黑麦碱基因Sec-1表达缺失的新的1RS/1BL易位系。这种新的1RS/1BL易位系缺失了影响小麦品质的黑麦碱蛋白,因此是进一步研究1RS/1BL 易位对小麦品质影响的珍贵材料。研究指出,在利用外源基因的植物育种中,外源种供体材料的遗传多样性是值得重视的基因资源。     

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。     

农业其它

小麦种子蛋白主要是麦醇溶蛋白和麦谷蛋白。贮存蛋白富含谷氨酰胺和脯氨酸但缺少赖氨酸和某些其它必需氨基酸。这些蛋白质是小麦粉烘面包质量的主要决定因素。大量具有相似结构和特性的麦醇溶蛋白使对各个品种的纯化和分析发生困难,限制了通过蛋白质分析直接可获得结构数据的数量。从研究约小麦胚乳 mRNA 制备的 cDNA 克隆已用于在小麦基因文库中鉴定编码麦醇溶蛋白和麦谷蛋白的 DNA 克隆。基因组克隆提     

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.     

土壤肥力对小麦籽粒蛋白质组分含量及加工品质的影响

研究了基础土壤肥力对小麦籽粒蛋白质组分含量变化及加工品质的影响。结果表明,高肥力土壤栽培的小麦比低肥力土壤栽培的小麦,其籽粒蛋白质及各组分含量都显著提高,但清蛋白和球蛋白占总蛋白含量的比例为低肥力土壤高于高肥力土壤,而醇溶蛋白和麦谷蛋白为高肥力土壤高低肥力土壤;低肥力土壤栽培的小麦籽粒醇溶蛋白开始形成的时间比高肥力的早,高肥力土壤栽培小麦的籽粒醇溶蛋白和麦谷蛋白含量的提高主要源于灌浆后期合成的快,高肥力土壤垢小麦湿面筋含量,沉降值,吸水率,面团形成时间,稳定时间,断裂时间和评价值均显著或极显著高于或长于低肥力的。     

小麦醇溶蛋白的研究

根据胚乳蛋白在不同溶荆中的溶解能力,可将小 麦胚乳蛋白分为四个组份臼”,其中可溶于70%酒精 的贮藏蛋白,称为小麦醇溶蛋白（gliadin),醇溶蛋白只 存在于禾本科植物种子中,它们构成了大部分具有重 要经济价值的谷物籽粒的主要贮藏蛋白〔,．〕。在小麦 中,它的含量约占籽粒总蛋白的一半。醇溶蛋白位于 胚乳的蛋白体内,主要存在于亚糊粉层,其富含疏水性 氨基酸,如亮氨酸, 氨酸、脯氨酸和谷酞胺等,缺乏亲 水性氨基酸,如赖氨酸、色氨酸和蛋氨酸等。氨基酸谱 的不平衡是使醇溶蛋白含量较高的小麦在实际应用上 受限制的一个重要原因。     

Study of a collection of Triticum spelta L. wheat species using gliadin polymorphism

Using gliadins, endosperm storage proteins of kernels, as markers, the genetic diversity of 170 samples from the Triticum spelta L. collection of the Vavilov Institute of Plant Industry was studied. High intraspecific polymorphism of the gliadin electrophoretic patterns was revealed. On the basis of similarity of the gliadin electrophoretic patterns, groups of samples were isolated, and the genetic structurization of the collection was performed.     

Analysis of Gliadin Polymorphism in aTriticum speltaL. Collection

Using gliadins, endorperm storage proteins of kernels, as markers, the genetic diversity of 170 samples from the Triticum speltaL. collection of the Vavilov Institute of Plant Industry was studied. High intraspecific polymorphism of the gliadin electrophoretic patterns was revealed. On the basis of similarity of the gliadin electrophoretic patterns, groups of samples were isolated, and the genetic stucturization of the collection was performed.     

Low MW gliadin-like proteins from wheat endosperm

A new group of hydrophobic endosperm proteins from Triticum aestivum has been characterized. It consists of 10 components with MWs in the range of 17 000–19 000, which have a similar range of electrophoretic mobilities at pH 3.2 as the classical gliadins. However, they have a higher proportion of sulphur amino acids and lower levels of glutamine and proline than the gliadins.     

波斯小麦醇溶蛋白遗传多样性分析

应用APAGE技术对来源于16个国家(地区)96份波斯小麦材料进行醇溶蛋白遗传多样性分析,结果表明,波斯小麦具有丰富的醇溶蛋白等位变异,共分离出55条迁移率不同的带纹。每份材料可电泳出13～26条,平均18.6条,带纹多态性为100%。96份材料共出现了75种电泳图谱类型,其中31份材料共同拥有10种图谱,剩余的65份材料的电泳图谱各不相同。供试材料间GS值变异范围为0.176～1.000,平均值为0.538。聚类分析表明,在GS值0.538水平上供试材料可聚为五大类群,且遗传聚类关系与材料的地理来源有一定的相关性。     

The Use of Artificial Neural Networks for Automatic Analysis and Genetic Identification of Gliadin Electrophoretic Spectra in Durum Wheat

Each wheat cultivar has a characteristic spectrum of gliadins. This makes it possible to use blocks of the components of reserve proteins as genetic markers when estimating seed purity and identity. However, identification of the blocks that constitute the electrophoretic spectrum is a complicated task. For this purpose artificial neural network (ANN) technology is proposed. Using experimental data, a teaching database and testing databases have been created. ANN was shown to be highly efficient (efficiency up to 100%) expert system for deciphering the electrophoretic spectra of gliadins of durum wheat cultivars.     

Use of artificial neuronal nets in automation of analysis and genetic identification of electrophoretic spectra of gliadin from durum wheat]

Each wheat cultivar has a characteristic spectrum of gliadins. This makes it possible to use blocks of the components of reserve proteins as genetic markers when estimating seed quality. However, identification of the blocks that constitute the electrophoretic spectrum is a complicated task. For this purpose artificial neural network (ANN) technology is proposed. Based on experimental data, a teaching database and testing databases have been created. ANN was shown to be highly efficient (efficiency up to 100%) expert system for deciphering the electrophoretic spectra of gliadins of durum wheat cultivars.     

Genetic marking of glume color in <Emphasis Type="Italic">Triticum spelta</Emphasis> L. var. <Emphasis Type="Italic">caeruleum</Emphasis> using gliadins

Using gliadins as genetic markers, Triticum spelta L. var. caeruleum accessions were analyzed to identify genetic control of the dark color of glumes. The research material was F₂ and BC₁ plants from crosses between spelt accessions and white-glumed common wheat varieties. The segregation for glume color fitted the monogenic control of the trait. The electrophoretic analysis of gliadins in grains from the hybrid plants has shown that the Gli-Alj* allele in the T. spelta var. caeruleum accessions is linked to the allele for the dark (black) color of glumes at the Rg-A1 locus.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Two-dimensional electrophoresis of 1D-encoded B and D glutenin subunits in common wheats with similar omega gliadins

Gli-D1-encoded omega gliadins of bread wheats show little variation; their electrophoretic patterns can be classified into two main groups which broadly resemble the patterns found in the cultivars Chinese Spring and in Cheyenne. B and D subunits of low molecular weight glutenin encoded by the chromosome 1D lociGlu-D3 andGli-D1, respectively, also showed little variation. D subunits were found only in bread wheats with Chinese Spring-type omega gliadins and they all exhibited the same electrophoretic pattern. This material also showed very similar B subunits. Cheyenne-type bread wheats displayed the same electrophoretic distribution of chromosome 1D-encoded B subunits, although they were slightly different from that found in Cheyenne itself.This work was supported by the Eclair Programme of the Commission of Europe and the Community.