转trxS基因大麦发芽种子水解酶活性的变化

利用转基因技术是改良大麦品种品质的有效途径.研究了转trxS基因对大麦种子发芽过程中水解酶活性的影响,结果表明转基因种子中α-淀粉酶、自由态β-淀粉酶和极限糊精酶的活性比未转基因种子高;转基因种子醇溶蛋白和谷蛋白中巯基的含量提高,说明该基因能够表达,为大麦育种和品质改良提供新的途径.     

野生大麦与栽培大麦醇溶蛋白遗传多样性比较

利用A-PAGE(acid-polyacrylamide gel electrophoresis)法对采自以色列的野生大麦的一个野生自然群体的15个系和来自世界不同国家的14份栽培大麦品种醇溶蛋白的遗传多样性进行了分析.结果表明:在所有的29份供试材料中,共发现52条相对迁移率不同的谱带.52条谱带的出现频率为3.44%～93.1%,多样性指数为0.066～0.368;以中国春醇溶蛋白为标准,ω区大麦醇溶蛋白的谱带数最多,其次是β区;野生大麦Shannon多样性指数依次为β区>ω区>α区>γ区,而栽培大麦Shannon多样性指数依次为ω区β>区>γ区>a区;野生大麦自然群体和栽培大麦品种间的遗传相似系数变幅相当,且聚类分析结果显示,野生大麦自然群体和来自全球不同区域栽培大麦品种间的醇溶蛋白遗传多样性同样丰富.以上结果说明,野生大麦中保存了较栽培大麦更为丰富的基因资源,今后栽培大麦的品质改良应该重视野生大麦资源的合理利用.     

普通小麦×大麦杂交后代中间材料的GISH及PAGE鉴定

利用基因组荧光原位杂交 (GISH)及种子贮藏蛋白聚丙烯酰胺凝胶电泳 (PAGE)对普通小麦×大麦杂交后代中间材料进行了鉴定分析。GISH结果表明 ,WBA984和WBA9812为二体小大麦异附加系 ,WBS0 2 15和WBS0 2 6 4为小大麦二体异代换系 ,WBT0 2 12 5和WBT0 2 183为端部易位系 ;种子贮藏蛋白PAGE分析表明 ,WBA9812和WBS0 2 6 4含有大麦特有的高分子量麦谷蛋白亚基和在γ区含有大麦特有的醇溶蛋白带型 ,WBA9812为大麦 5H附加系 ,WBS0 2 6 4为 1B/ 5H代换系 ,WBT0 2 12 5为 1BL/ 5HL端部易位系。     

僵蚕药材蛋白质分级指纹图谱建立及产地鉴定相关分子研究

建立云南与四川僵蚕药材的总蛋白质指纹图谱及分级指纹图谱,对比分析以发现其中的差异表达蛋白质分子并进行鉴定及生物信息学分析,探索其用于产地鉴定的可行性。提取两种僵蚕的酸溶、碱溶、水溶及醇溶性总蛋白质,对其进行丙酮分级沉淀,对所得样品进行SDS-PAGE指纹图谱对比分析,对所得差异条带进行LC-MS/MS分析,数据库检索后进行生物信息学分析。两种僵蚕的总蛋白质指纹图谱相似度很高,难以据此进行产地鉴定;两种僵蚕的分级指纹图谱则有较显著差异,有望用于产地鉴定;依据分级指纹图谱差异共鉴定出β-葡萄糖苷酶、抗胰凝乳蛋白酶、抗胰蛋白酶、含脂肪酶结构域蛋白等273种蛋白质。云南与四川僵蚕的蛋白质组成及其分级指纹图谱存在显著差异,有望用作产地鉴定的分子依据;醇溶蛋白质构建分级指纹图谱可以提供更加丰富的信息及更好的分辨率;分级指纹图谱技术具有简便易行、分辨率较高的优点,为其他中药材的产地鉴定研究提供了方法学思路。     

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。     

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．     

不同品种水稻种子贮藏蛋白质组分的差异

水稻是我国的主要粮食作物,它的蛋白质含量为6—14%。在水稻种子蛋白质中,谷蛋白占80%,球蛋白占10%,醇溶蛋白占5%,清蛋白占5%。据研究,水稻种子清蛋自主要由分子量16800的亚基组成,球蛋白由10种不同分子量的亚基通过疏水交互作用相结合,谷蛋白由分子量38000、25000和16000三种亚基通过双硫键相结合。但是,不同品种水稻种子贮藏蛋白质组分有何差异?研究报道的很少。本文简要报道不同品种水稻种子(籼稻和粳稻)贮藏蛋白质组分的差异,为     

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.     

萌发前后燕麦种子内酯酶的细胞化学研究

禾谷类种子萌发时,种子内的盾片和糊粉层细胞会形成和释出大量的水解酶,如酸性磷酸酶、酯酶等。水解酶对消化种子内的贮藏物质极其重要。有关这些水解酶在种子内消化贮藏物质的过程和机制,在小麦和大麦中已有很多报道,但在燕麦上还没有人报道过。燕麦和大、小麦所积聚的贮藏物质有些不同。譬如大、小麦积聚醇溶谷蛋白为主,而燕麦则积聚球蛋白为主。我们设想:既然大、小麦和燕麦种子内的贮藏蛋白不一     

3种生态型翅果油树种子贮藏蛋白分析

采用考马斯亮蓝G-250染色法和SDS-PAGE电泳技术对翅果油树3种生态类型种子贮藏蛋白含量进行测定和图谱分析.结果显示:大宫灯、长果型、小宫灯3种生态类型种子中总贮藏蛋白含量分别为33.753%、32.075%和26.633%;3种生态类型种子贮藏蛋白均以谷蛋白含量最高(65.971%～68.267%),其次为球蛋白和清蛋白,醇溶蛋白的含量最低;电泳图谱显示,3种生态类型之间清蛋白、球蛋白、醇溶蛋白和谷蛋白的蛋白质条带信息均存在差异.研究表明,翅果油树3种生态类型间种子贮藏蛋白具有多态性.     

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。     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm.

A previous study [Rahman, Shewry & Miflin (1982) J. Exp. Bot. 33, 717-728] showed differential accumulation of the major storage proteins (called B and C hordeins) in developing endosperms of barley (Hordeum vulgare). To determine how this accumulation is regulated, we have studied mRNA fractions prepared from similar endosperms. Hordein-related mRNA species were detected some days before the deposition of hordeins in vivo. The translation products in vivo directed by polyribosomes, polysomal RNA and total cellular RNA showed similar changes in the proportions of the hordein products to those observed in the accumulations of the proteins in vivo. There was a relative increase in one of the subfamilies of B hordeins (called B1 hordein) and a decrease in the second subfamily of B hordeins (B3 hordein) and in C hordeins. The populations of RNA species related to these three groups of hordeins were studied by 'dot hybridization', with specific complementary-DNA probes for B1-, B3- and C-hordein-related sequences. This showed a 10-15-fold increase in sequences related to the B1 hordein during endosperm development, but only a 4-fold increase in sequences related to B3 and C hordeins. These results indicate that the rates of synthesis of hordeins are related to the abundance of their respective mRNA species. The different results observed for the two subfamilies of B hordeins are of interest, since they indicate differential expression of two subfamilies of genes present at a single multigenic locus.     

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.     

A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.     

Placement of loci for avenins and resistance to Puccinia coronata to a common linkage group in Avena strigosa.

Alcohol-soluble seed storage proteins of oat (avenins) were extracted from two diploid accessions representing the A genome and separated by high-resolution acid polyacrylamide gel electrophoresis. Polymorphisms were detected for three clearly resolved protein bands. Linkage analysis of 88 F2:3 families mapped the three bands to a single locus. Integration of avenin segregation data with an RFLP linkage map constructed from the same population, mapped the avenin locus to a linkage group containing a locus conferring resistance to nine isolates of Puccinia coronata. Linkage between genes encoding alcohol-soluble seed proteins and genes for resistance to Puccinia species was also observed for the homoeologous group 1 chromosomes of barley (1H), rye (1R), wheat (1A, 1B, 1D), and chromosomes 4 and 10 of maize.     

On the origin of Spanish two-rowed barleys

To investigate the phylogenetic origin of Spanish two-rowed barleys, we studied 44 accessions of old land-races both morphologically and biochemically to ascertain their similarity with 51 entries of old cultivars and land-races of widespread origin across Europe. They were also compared with 20 accessions of Hordeum spontaneum from the Mediterranean basin and other regions of its distribution range, 14 accessions of Moroccan cultivated six-rowed barley land-races, and different six-rowed Spanish and two-and six-rowed European cultivars. CM-(trypsin inhibitors and subunits of the barley tetrameric -amylase inhibitor) proteins and hordeins, all of which are endosperm proteins, were used as biochemical markers. The appearance of separate clusters of the Spanish barleys in the numerical classifications for both protein systems as a result of the existence of characteristic gene combinations that do not exist in entries from other origins permitted us to postulate the existence of local ancestors for most of the Spanish two-rowed barleys studied, and, therefore, a possible in situ domestication.     

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.