The identification of a barley haze active protein that influences beer haze stability: The genetic basis of a barley malt haze active protein

In bright beers, the formation of haze is a serious quality problem, which places limitations on the storage life of the product. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot analysis using an antiserum that was raised against a silica eluate (SE) protein fraction (obtained from silica gel, used for the colloidal stabilisation of beer), detected a range of protein bands in barley, malt, beer and haze. A polymorphism was observed in which some barley varieties contained a molecular weight (MW) 12,000 band (SE +ve) while in other varieties this band was absent (SE −ve). A survey of 219 Australian and international barley varieties, including a comprehensive selection of current and past malting varieties, identified 181 varieties as SE +ve, and 38 varieties as SE −ve. Previous pilot brewing trials demonstrated that SE −ve varieties are desirable as the beer brewed from the malt of these varieties formed less haze after accelerated ageing than beers brewed using SE +ve malt varieties. The genetic basis for the absence of the SE protein was conferred by a recessive allele at a single locus. Interval mapping analysis showed that the MW 12,000 band mapped to the short arm of chromosome 3H.     

The identification of a barley haze active protein that influences beer haze stability: Cloning and characterisation of the barley SE protein as a barley trypsin inhibitor of the chloroform/methanol type

Previous pilot brewing trials have demonstrated that in the absence of a molecular weight (MW) 12,000 (barley silica eluate (SE) protein), the beer brewed from the malt of these SE −ve varieties formed less haze after accelerated ageing than beers brewed using SE +ve malt varieties. The previously described SE protein was characterised using comparative two-dimensional (2-D) gel electrophoresis immunoblots of barley seed extracts from both SE +ve and SE −ve varieties. The SE protein spot identified was excised and its partial sequence determined, after in-gel cleavage using trypsin and separation of the resulting fragments by reversed-phase high performance liquid chromatography (HPLC). N-terminal sequence analysis of the tryptic peptides from SE +ve and SE −ve varieties identified the SE protein as the barley trypsin inhibitor-CMe precursor (BTI-CMe). The mature BTI-CMe protein is 13.3 kDa and its functional gene is located on chromosome 3H. Cloning of the BTI-CMe protein demonstrated that both SE −ve and SE +ve barley varieties contain a BTI-CMe protein family member that is similar but has a consistently different sequence, primarily in the last 30 amino acid residues of their C-termini.     

氮肥运筹对大麦啤酒混浊性状的影响

啤酒混浊是一个严重的品质问题,它显著缩短啤酒的贮藏与货架时间。为给生产低混浊啤酒大麦提供科学依据,本研究选用混浊特性不同的4个大麦品种,设置不同的氮肥用量与施用时期,探讨氮肥运筹对啤酒混浊性状的影响。结果发现,供试大麦品种之间和氮肥施用时期之间混浊性状差异极显著;澳大利亚大麦品种Franklin和Yerong的酒精冷混浊值(ACHD)显著低于国产大麦品种浙大9号和花30。Franklin的混浊敏感蛋白最低,花30次之,浙大9号和Yerong的混浊敏感蛋白含量较高。增施抽穗期氮肥,显著提高大麦的混浊度。不同氮肥用量之间,混浊敏感蛋白HAP1和HAP3差异极显著,但酒精冷混浊值和HAP2无显著差异;品种与氮肥用量及施用时期之间的互作效应对部分混浊性状表现显著。因此,为生产低混浊的啤酒大麦,生产上首先应考虑选用低混浊品种,并在氮肥施用上适当减少穗肥用量。     

Analysis of Differential Proteins in Two Wing-Type Females of Sogatella furcifera (Hemiptera: Delphacidae)

Sogatella furcifera (Horvath) is an important rice pest with the wing dimorphism, including macropterous and brachypterous morphs. The protein expression profiles in two wing-type adults and two wing-type disc fifth-instar nymphs were analyzed using two-dimensional gel protein electrophoresis and mass spectrometry. In adults and fifth-instar nymphs, 127 and 162 protein spots were detected, respectively. Fifty-five differentially expressed protein spots were identified between the long-winged adults and the short-winged adults, and 62 differentially expressed protein spots were found between the long-winged disc fifth-instar nymphs and short-winged disc fifth-instar nymphs. In long-winged and short-winged adults, six and seven specific protein spots were identified, respectively, with five and seven protein spots having more than threefold increased level, respectively. In long-winged and short-winged disc morph nymphs, 8 and 12 specific protein spots were identified, respectively, with 11 and 17 spots containing more than threefold increased level, respectively. Among the 16 identified proteins, five proteins are associated with muscle function, suggesting that muscle is a main tissue where the genes were differentially expressed between the two wing types. In addition, the content of a peptidase with an insulinase domain was higher (by 3.02 ± 0.59 fold) in the short-winged fifth-instar nymphs than in the long-winged fifth-instar nymphs, which suggests that this peptidase may be involved in wing differentiation by regulating insulin receptors. The results of this study provide some genetic clues for the wing differential development in S. furcifera and provide more references for future studies.     

小麦籽粒清蛋白和球蛋白表达对干旱胁迫的响应

为了解干旱胁迫对小麦籽粒灌浆期蛋白表达的影响,以强筋优质小麦品种藁城8901为材料,采用双向电泳以及质谱鉴定分析了干旱胁迫和正常生长条件下成熟籽粒清蛋白和球蛋白表达谱的差异。结果表明,与正常生长条件相比,干旱胁迫后,小麦籽粒出现130个差异蛋白质点。通过MALDI TOF-TOF质谱鉴定和数据库搜索,最终成功鉴定出57个差异蛋白,其中上调表达29个,下调表达19个,特异表达7个,沉默表达2个;涉及ADP-葡萄糖焦磷酸化酶、α-淀粉酶抑制剂、过氧化还原酶、β-淀粉酶、二硫键异构酶、丝氨酸蛋白酶抑制剂、超氧化物歧化酶等13种蛋白质,其中代谢类10个,能量类2个,蛋白质加工和储藏6个,病害防御相关17个,转录1个,功能未知和假定蛋白21个。说明干旱胁迫影响小麦籽粒灌浆期的多个代谢途径,进而影响小麦产量和品质。     

Phenotypic and allelic variation for wort protein Z in Australian and Canadian barleys

Protein Z is a major component in beer foam. Two-dimensional electrophoresis was used to analyze wort proteins of two Australian (Buloke and Commander) and two Canadian (CDC Meredith and Bentley) varieties. The Canadian barley contained more abundant proteins from MW 40–45 kDa (pI 5 to 7). These proteins were identified as either protein Z4 or protein Z7 using liquid chromatography–mass spectrometry. Full-length gene of protein Z4 and Z7 were sequenced from Canadian and Australian barleys. Sequence differences were identified in the coding region and upstream regions of the two genes, resulting in protein sequence and expression variations. Molecular markers were designed according to the indels in the upstream regions of protein Z4 and Z7 genes. These markers were highly correlated to wort protein Z content in Canadian and Australian varieties. The Canadian barleys contained ‘high level’ genotypes for protein Z4 and Z7 while most Australian barleys had ‘low level’ genotypes for protein Z4, Z7 or both. The markers identified in this study provide a valuable tool for the selection of protein Z alleles in marker-assisted breeding. Total protein Z content was assessed using different steeping conditions, and increasing air-rest time increased protein Z content in 15 varieties.     

Susceptibility to Coolness at the Young Microspore Stage under High Nitrogen Supply in Rice (Oryza Sativa L.). Proteome Analysis of Mature Anthers

Abstract

In vitro pollen germination experiment using agar plates showed that the growth under high nitrogen conditions enhanced the damage to pollen germination ability caused by the cooling at the young microspore stage. To clarify the physiological factors related to this damage to pollen germination, we performed the comparative proteome analysis of mature anthers and identified proteins that were changed by high nitrogen conditions or high nitrogen plus cooling conditions. Proteins were extracted from mature anther samples and separated by two-dimensional polyacrylamide gel electrophoresis. By comparing anther protein maps of the samples collected from the plants grown under standard nitrogen conditions, high nitrogen conditions and high nitrogen plus cooling conditions, we found 11 protein spots, which varied with the treatment. These protein spots were identified based on the rice proteome database and/or peptide mass fingerprinting (PMF) analysis after digestion with trypsin. Digested samples were analyzed by matrix-assisted laser desorption/ionization-time flight mass spectrometry to produce PMF data. Database searches using these PMF data revealed the identities of 9 proteins. Seven of these proteins were polypeptides involved in cell elongation, stress responses and sugar metabolism. The relation between the fluctuations of these proteins and the decrease in pollen germination are discussed.     

Effect of high-pressure treatment on rheological,thermal and structural changes in Basmati rice flour slurry

Rheological, thermal and structural changes in high pressure (HP) treated Basmati rice flour dispersions were studied as function of pressure level (350–650 MPa), slurry concentration (with 1:5, 1:3 and 1:2 flour-to-water ratios) and holding time (7.5–15 min). Rice flour dispersions exhibited a gradual liquid–solid gel transformation as they gelatinized and/or denatured and behaved as viscoelastic fluid following HP treatment. Mechanical strength (G′) of pressurized gel increased with applied pressure and rice concentration. Differential scanning calorimeter (DSC) thermograms of rice slurry measured after pressure treatment indicated a reduction in peak enthalpy in proportion with the extent of gelatinization and/or denaturation of starch and proteins. Pressure-treated rice samples had a progressively lower gelatinization temperature. A 15 min pressure treatment at 550 MPa was found sufficient to complete gelatinization of protein free isolated rice starch while the slurry required 650 MPa. The presence of proteins might have been responsible for the slower starch gelatinization in the rice slurry during pressure treatment. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier-transform infrared (FTIR) spectroscopy results indicated some minor changes in protein subunits and secondary structure of rice protein. This study has provided complementary information on pressure-induced changes in physical (thermal stability, overall structure) and molecular level (secondary structure) of rice protein.     

Winter hardiness in faba bean: Physiology and breeding

Winter types of faba bean (Vicia faba L.) have existed for at least 200 years. Their superior use of the growing season confers strong yield advantages over spring beans. Nevertheless, yield increases have been slower than in many other crops. There are few sources of winter hardiness and efforts are in progress to combine favourable alleles from accessions such as Cote D’Or and BPL 4628 to increase the crop’s tolerance to frost. Vernalization requirements are quantitative, as vernalization hastens flowering rather than allowing it. Hardening is associated with increases in fatty acid desaturation of membrane lipids and increases in content of soluble osmoprotectants such as proline. Other osmotically active factors such as glycinebetaine, trehalose and antifreeze proteins have not yet been reported for faba bean. Frost tolerance increases after hardening and shows good heritability (h² = 0.89). Three QTLs (3.6 < LOD < 4.6) have been identified for frost tolerance (explaining 8.6% of the phenotypic variation), and further QTLs for hardening response and cell membrane fatty acid composition. Information on responsive genes and the mode of their action is increasingly available from model plant species but most remains as yet untested in faba bean. There is clear potential for increasing the winter hardiness and yield of winter faba bean so it can be grown in a wider area than at present.     

孕穗期水稻不同功能叶的发育蛋白质组学分析

 从蛋白质组学角度对杂交水稻汕优63孕穗期4个叶位功能叶片蛋白质组变化进行了研究,以揭示水稻孕穗期叶片蛋白质组的表达差异。4个不同时期功能叶片蛋白质经双向电泳得到分离,应用Imagemaster 2D Elite 5.0软件对所得到的双向电泳图谱进行分析比较,共得到差异表达蛋白质点23个,差异表达蛋白质点经质谱分析,有14个得到鉴定。对鉴定出的蛋白功能进行研究,结果表明:差异表达的蛋白质大部分(9个)为参与光合作用和呼吸作用的蛋白质;其次为与蛋白结构及功能调控相关的蛋白质(3个),表达量表现为随叶位上升而上调;其余为参与氨基酸代谢的蛋白质(2个),表达量在较低叶位叶片中无显著规律,在剑叶中的表达量显著上调。蛋白质是细胞功能执行者,受细胞功能或细胞环境变化的影响,因此,上述蛋白质发生变化的原因应与水稻不同叶位叶片生长发育特性有关。     

Alpha-amylase/subtilisin Inhibitor Levels in Australian Barleys

An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.     

野生二粒小麦根中干旱响应蛋白的分离与鉴定

为了解野生二粒小麦响应干旱胁迫的调控机制,分析了干旱胁迫下小麦根的相对含水量、丙二醛、脯氨酸和过氧化氢含量,采用双向电泳(2-DE)结合MALDI-TOF-TOF-MS方法分离和鉴定了野生二粒小麦根中干旱响应蛋白的变化。结果表明,随着干旱处理时间的延长,野生二粒小麦根的相对含水量下降,脯氨酸含量先上升后降低,过氧化氢含量和丙二醛含量上升;复水后的野生二粒小麦根的相对含水量、脯氨酸含量有所升高,但未达到对照的水平。通过对干旱处理6d后根系的总蛋白进行双向电泳分离和MALDI-TOFTOF生物质谱鉴定,成功鉴定出26个差异表达蛋白,13个上调表达,13个下调表达。26个差异表达蛋白的功能主要涉及信号传导、氧化解毒、碳代谢、能量代谢、蛋白质生物合成及细胞骨架稳定。推测野生二粒小麦为适应干旱胁迫,通过根部感应胁迫信号,并传导至细胞内,影响小麦根中蛋白质的生物合成、氨基酸代谢、碳水化合物代谢、细胞骨架形成;通过抗氧化酶系统和抗氧化物质的作用,将过多活性氧加以清除;通过增加胞内脯氨酸含量,降低根中水分损失。     

Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables

Proximate composition, energy, mineral and vitamin contentsand the effect of blanching methods and times on the trypsinand chymotrypsin inhibitor activities were studied usingcabbage, collard, turnip, peanut, and sweet potato leaves.Results of this study indicated that, crude protein, crudefat, carbohydrate and ash contents were in the range of 15.5–25.6%, 1.4–6.5%, 60.4–73.1% and 6.8–7.5%,respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%)while energy content per 100 g of vegetables was highestin sweet potato leaves (402 kcal) and lowest in cabbage(379 kcal). The mineral content per 100 g of vegetableswere in the range of 33.4–249.8 mg, 241.2–471.2 mg,12.1–75.1 mg, 14.9–98.9 mg, 0.5–3.5 mg and 0.9–3.1 mgfor Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbicacid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of45.1–112.7 mg, 0.2–0.3 mg, 0.3–0.8 mg and 2.0–7.3 mg,respectively. The trypsin inhibitory activity per gram ofthe vegetables was highest in collard (60.1 TIU/g) andlowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitoractivity was highest in the peanut (69.6 CIU/g) but lowestin the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p<0.05) reduced by most of the treatments in eitherthe conventional or microwave blanching methods. In the conventional blanching method, trypsin inhibitor activitywas reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves,respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsininhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3and 65.6 percent in cabbage, collard, turnip, sweet potatoand peanut greens, respectively. For the microwave ovenblanching, trypsin inhibitor activity was reduced by 3.8,3.3, 32.7, 5.0 and 9.5 percent in cabbage, collard, turnip,sweet potato and peanut leaves, respectively when thevegetables were blanched for 30 seconds. When blanched for60 seconds, trypsin inhibitor activity was reduced by 16.2,45.8, 46.2, 51.0 and 42.4 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively.Similar trends in the reduction of chymotrypsin inhibitoractivity were observed when the vegetables were conventionally blanched for 2.5, 5 and 10 minutes andwhen blanched by microwave oven for 30, 45 and 60 seconds.Based on the results of this study, the vegetables weregood dietary sources of minerals, vitamins, carbohydrateand proteins. Also, blanching was an effective method forreducing the trypsin and chymotripsin inhibitor activitiesin the leafy vegetables, however, further investigation onthe heating times for both conventional and microwaveblanching methods is suggested.     

乙烯刺激橡胶树胶乳黄色体蛋白差异初析

巴西橡胶树(Hevea brasiliensis)胶乳中的黄色体作为乳管细胞的一种细胞器,其稳定性受乙烯刺激影响。该试验从蛋白差异角度,选取未开割胶树,应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE),比较在乙烯刺激与未刺激条件下,黄色体蛋白的变化情况。结果表明,按蛋白表达量变化差异3倍及以上分析,胶树在乙烯刺激24h后,上调蛋白5个,下调蛋白2个;在乙烯刺激48h后,上调蛋白7个,下调蛋白1个;综合3个处理,先上调后下调的蛋白8个;先下调后上调的蛋白2个。应用质谱技术鉴定了其中部分差异表达蛋白,并进行初步功能分析,表明未开割橡胶树胶乳黄色体受乙烯刺激后,黄色体蛋白发生不同程度复杂变化,各组处理间变化明显。     

Effects of heat treatments on trypsin inhibitor and hemagglutinating activities in winged bean

The effect of different heat treatments on inactivation of trypsin inhibitor and hemagglutinin of winged bean was investigated. Trypsin inhibitor extracted from winged bean meal was stable at 60 °C for 60 min. At 80 °C, the activity of the extracted inhibitor decreased by 25% within 5 min, and continued to decline gradually to a loss of 45% of activity after 30 min. When the extracted inhibitor was incubated at 100 °C, it exhibited a triphasic pattern of inactivation. The winged bean extract incubated at 60 °C lost 60% of its hemaggluinating activity within 30 min. At 80 °C, there was a complete loss of activity within 5 min. The microwave treatment to winged bean meal had no effect on trypsin inhibitor or hemagglutinating activities in the meal. However, infrared treatment to winged bean seeds for 60 seconds was effective in destroying most of the trypsin inhibitor and hemagglutinating activities. Autoclave treatment (120 °C at 15 lb pressure) for 10 min inactivated trypsin inhibitor and hemagglutinin in winged bean meal almost completely. Cooking of presoaked beans in boiling water for 30 min was effective in destroying most of the trypsin inhibitor and hemagglutinating activities.     

Effect ofCallosobruchus chinensis (Bruchid) infestation on antinutritional factors in stored legumes

Effect of bruchid infestation on selected antinutritional factors, saponin, phytic acid and trypsin inhibitor activities, of three (mung bean, chickpea and pigeon pea) legumes stored for six months was studied. The three antinutritional factors, saponin, phytic acid and trypsin inhibitor activity (TIA), increased with increased storage period. Saponin, phytic acid and trypsin inhibitor activity values were significantly lower in control samples compared to stored infested samples. The difference between the contents of antinutritional factors were significant (p<0.05) during different storage periods.     

Study of faba bean (Vicia faba L.) winter-hardiness and development of screening methods

In cool-temperate regions, faba bean (Vicia faba L.) is mainly grown as a spring crop despite the higher yield potential of the winter type, because of the insufficient winter-hardiness of the present winter genotypes. The objective of this study was to assess winter-hardiness and frost tolerance, to quantify the hardening effect on physiological traits, and to identify auxiliary traits for winter-hardiness. To do so, 31 representative entries were tested in controlled frost tests for frost tolerance and in 12 European environments for winter-hardiness. Total fatty acid composition, proline content, and electrolyte leakage of leaves were analysed. Across all environments, five European winter genotypes were identified with superior winter-hardiness. Controlled frost tests indicated that frost tolerance is a significant, but not an exhaustive component of winter-hardiness (0.021 < |r| < 0.737**). These tests revealed the high frost tolerance of several poorly winter-hardy experimental lines and the limited frost tolerance of well-known winter types. Fatty acid changes due to hardening, proline content, and electrolyte leakage were more strongly correlated with frost tolerance than with field-based winter-hardiness. Although frost tolerance, fatty acid composition, proline content, and electrolyte leakage were significantly correlated with winter-hardiness, the rather low correlation values do not allow a general use of one of them alone to indirectly select for winter-hardiness.     

保水剂对水分胁迫下超甜玉米生理生化性状的影响

用聚丙烯酸钠型保水剂处理超甜玉米种子,测定室内盆栽超甜玉米苗期的一些生理生化指标。结果表明,适当浓度的保水剂处理可提高超甜玉米幼苗的株高,增加叶片叶绿素、可溶性蛋白质含量和超氧化歧化酶(SOD)活性,降低丙二醛(MDA)和脯氨酸的含量。     

水稻花粉总蛋白质双向凝胶电泳方法建立及应用

采用石英砂加液氮研磨方法破碎花粉,然后用两种不同的方法提取水稻花粉总蛋白质,之后采用固相pH梯度等电聚焦/SDS PAGE 双向凝胶电泳对红莲型细胞质雄性不育水稻单核期花粉总蛋白质进行了分离。通过银染显色, PDQuest 2DE软件可识别约1 000~1 500个蛋白质点。其中TCA 丙酮提取法更适合提取花粉总蛋白质进行双向凝胶电泳分离,并可获得分辨率和重复性较好的双向电泳图谱。对红莲型细胞质雄性不育水稻的不育系单核期和二核期花粉总蛋白进行了双向电泳分离,通过比较两个时期花粉蛋白质电泳图谱后发现,二核期的花粉蛋白质较单核期花粉蛋白质数目减少,并且部分蛋白质表达量下降。     

中国大豆种子蛋白中胰蛋白酶抑制剂等位基因的频率及分布

我国１．９万份大豆（栽、野）种质资源的胰蛋白酶抑制剂（ＳＢＴＩ）等位基因电泳分析结果指出，９８．２％的材料具有Ｔ型，１．３％的材料具有Ｔ型。在野生材料中发现有Ｔ类型。三种基因型出现的频率比例为１００：１．３：０．２。未发现不含胰蛋白酶抑制剂的ｔｉ型材料．栽培种Ｔｉ类型单一，野生种Ｔｉ类型变异较大。