Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed by quantitative 2-dimensional gel electrophoresis (2-DE). The precise effects of the genetic modifications on the proteome were assessed in four homozygous lines generated with the same RNAi construct. Two of the lines showed >80% decreases in omega-5 gliadins with only small changes in the levels of other gluten proteins. In the other two lines, omega-5 gliadins were not detectable by 2-DE. However, there were notable reductions in all omega-1,2 gliadins. Small decreases in several other gluten proteins were also detected in one of the lines. The other line showed notable decreases in three HMW-GS and an s-type LMW-GS, increases in two m-type LMW-GS and several alpha gliadins, as well as increases in both serpins and triticin. The study demonstrates that the same RNAi construct can have differential effects on the wheat grain proteome and highlights the importance of detailed proteomic analyses of transgenic grain prior to selecting lines for further assessment of flour quality and allergenic potential.     

高温逆境对黄金葛叶片中渗透调节物质积累的影响

以3~5个叶片的黄金葛扦插苗为试材,采用单因素随机区组设计,设4个温度处理,3次重复,测定不同温度、不同时间处理下黄金葛叶片内渗透调节物质含量的变化。结果表明,在同一时间高温胁迫下,随温度的不断升高,黄金葛叶片的可溶性蛋白含量整体呈下降趋势,可溶性糖含量呈上升趋势;同一温度处理下,在35℃,不同时间处理可溶性蛋白含量和可溶性糖含量变化不大,在45℃处理40 h,可溶性蛋白含量比胁迫初期降低了35.02%,而可溶性糖含量比胁迫初期增加了97.06%。     

梅花鹿茸可溶性蛋白提取工艺及免疫活性

以梅花鹿茸为原料,采用超声波辅助法提取可溶性蛋白。以鹿茸可溶性蛋白得率为评价指标,通过单因素试验研究提取溶剂浓度、超声提取时间、超声提取功率和液料比对鹿茸可溶性蛋白得率的影响,在单因素试验的基础上进行正交试验,优化提取工艺条件。结果表明：鹿茸可溶性蛋白的最优提取工艺条件为,以pH＝10．00、0．05 mol· L-1的碳酸盐缓冲溶液为提取溶剂,液料比10∶1,超声提取功率200 W,超声提取时间20 min。在此条件下,鹿茸可溶性蛋白得率为36．849 mg· g-1。体内免疫活性试验表明,鹿茸蛋白提取物能够提高免疫功能低下小鼠的单核巨噬细胞吞噬指数、血清溶菌酶质量浓度、血清及肝组织中酸性磷酸酶活力,具有良好的免疫活性。     

苏云金芽胞杆菌Bt9875杀虫晶体蛋白可诱导Caspase和Bcl-2家族参与调控HL-60细胞凋亡

研究Caspase家族与Bcl-2家族参与调控苏云金芽胞杆菌(Bacillus thuringiensis,Bt)Bt9875杀虫晶体蛋白对人急性髓细胞性白血病细胞HL-60的影响.本实验采用MTT法检测了杀虫晶体蛋白诱导HL-60细胞凋亡后的Caspase家族的活性和Caspase凋亡酶抑制剂对杀虫晶体蛋白诱导HL-60细胞凋亡的影响;采用Western blot检测了杀虫晶体蛋白诱导多聚ADP-核糖聚合酶(PARP)降解、Bcl-2/Bax调控和细胞色素C的释放.研究结果表明,杀虫晶体蛋白作用HL-60细胞后,激活了Caspase-3、Caspase-8和Caspase-9,在48 h内Caspase家族抑制剂(Z-VAD-FMK)、Caspase-3抑制剂(z-DEVD-FMK)和Caspase-9抑制剂(Z-LEHD-FMK)均可显著抑制杀虫晶体蛋白诱导的细胞凋亡;杀虫晶体蛋白可明显上调促凋亡蛋白Bax的表达,同时下调抗凋亡蛋白BCl-2的表达,并观察到胞浆中细胞色素C的释放.初步证明了Bt9875杀虫晶体蛋白诱导的HL-60细胞凋亡是由Caspase家族和Bcl-2家族共同调控的,线粒体途径在诱导细胞凋亡过程中起着重要作用.     

Effects of Iron Deficiency on Thylakoid Membrane Structure and Composition in the Alga Dunaliella salina

Abstract

The unicellular green alga Dunaliella was previously proposed as a model photosynthetic organism for adaptation to iron deficiency. The purpose of this study was to find out how iron limitation affects the structure and composition of the photosynthetic system of Dunaliella salina. Iron deprivation did not retard proliferation of D. salina cells, but was associated with a decrease in cell volume and chlorophyll content, and with a four‐fold reduction in iron content and a two‐fold increase in Cu content. Electron microscopic analysis revealed shrinkage of the chloroplast and decrease in stacked thylakoid membranes. Measurements of chlorophyll fluorescence induction in the presence of DCMU and of 77 K chlorophyll fluorescence emission spectra indicated gross changes in the photosynthetic efficiency of reaction centers and in the organization of their associated light harvesting antenna. Differential analysis of protein composition led to the identification of a major thylakoid membrane protein (Tidi), that was specifically induced under iron deprivation. Partial sequencing suggests that Tidi is a novel type of a chlorophyll a/b binding protein. These results clearly show that iron limitation is associated with extensive reorganization of the photosynthetic system in Dunaliella.     

Long‐Term Field Drought Affects Leaf Protein Pattern and Chloroplast Ultrastructure of Winter Wheat in a Cultivar‐Specific Manner

Recurrent drought periods of varying duration often cause extensive crop damage and affect wheat production in Southern Europe. This study compares biochemical and ultrastructural responses of four wheat (Triticum aestivum L.) cultivars to long‐term field drought, and their contribution to final grain yield. Gel electrophoresis and immunoblotting analyses combined with transmission electron microscopy and grain yield evaluation were employed to assess drought susceptibility of the wheat cultivars. Two of them behaved as drought‐tolerant, the other two presented as drought sensitive. Enhanced degradation of Rubisco large subunit (RLS), Rubisco small subunit (RSS) and Rubisco activase (RA) accompanied by an increased protease activity and reduced levels of heat shock proteins (HSP70) and dehydrins (DHNs) were associated with drought sensitivity. Drought tolerance coincided with relatively stable or increased HSP70 and DHN contents, and unchanged/higher levels of RLS, RSS and RA. Sensitive cultivars were more vulnerable to ultrastructural damages, showing obvious degradation of chloroplast membrane systems and depletion of leaf starch reserves. These drought responses affected yield potential, as tolerant cultivars gave higher yield under intense drought. Thus, our results provide additional insights into the complexity of plant drought responses, identifying multiple interacting traits that may serve as indirect selection criteria for wheat drought tolerance.     

Biochemical indices (RNA/DNA ratio and protein content) in studying the nutritional status of Ruditapes decussatus (Linnaeus 1758) juveniles

In this work, we investigated the nutritional status of Ruditapes decussatus juveniles under different rearing conditions, using biochemical indices (RNA/DNA ratio and protein content) and the linear instantaneous growth rate (IGR). Two experiments were conducted to study the effect of starvation, addition of substrate (sand) as rearing support and diet composition on somatic growth and biochemical indices. Results show that biochemical indices of fed juveniles were significantly (P<0.05) higher. Highest RNA/DNA values and protein content were recorded in fed juveniles reared with substrate (P<0.05); conversely, lowest values were recorded in starved ones reared without substrate. In the second experiment, the highest RNA/DNA ratio was recorded in juveniles fed with control algal mixture composed of species commonly used in bivalve nurseries [Isochrysis galbana (clone T-Iso), Chaetoceros calcitrans and Tetraselmis suecica] and lowest in those fed with algal species isolated from Tunisian coasts. This confirms the usefulness of the RNA/DNA ratio and protein content in the clam's nutritional status evaluation. This assay is useful to provide information about the nutritional status and health of the clam in field conditions.     

补料及开食料中不同种类蛋白质对仔猪过敏反应及腹泻程度的影响

用３周龄断奶仔猪研究了补料和开食料中不同蛋白料对断奶后的过敏反应和腹泻程度的影响、补饲和开食料中分别以玉米－豆饼、豆饼、醇处理豆饼或乳蛋白为唯一蛋白来源的补料。仔猪７日龄开始补饲，试验至３５日龄结束。结果表明，断奶后保持蛋白来源不变时仔猪的过敏和腹泻程度比改用复合开食料时低，木糖吸收和养分消化率高（Ｐ＜０．０１），但补豆饼组例外；乳蛋白引起的过敏反应最弱，腹泻程度最低，木糖吸收率和养分消化率最高，豆饼引起的腹泻最严重，腹泻开始最早，持续时间最长，木糖吸收率和养分消化率最低，玉米－豆饼和醇处理豆饼引起的腹泻程度居中（Ｐ＜０．０５）；断奶各组３５日龄木糖吸收率极显著高于２８ｍ日龄吸收率（Ｐ＜０．０１），但低于不断奶的对照组相应值（Ｐ＜０．０１）。粪中大肠杆菌计数表明，腹泻程度与微生物增殖无关。由此认为，肠道对日粮抗原过敏是仔猪断奶后腹泻的主要原因；过敏和耐受程度及补饲约影响取决于日粮蛋白质种类。     

Protein Phosphorylation and Protein Patterns in Developing Endosperms of Barley

The SDS-soluble protein-fractions of barley endosperms (2 and 3 weeks after anthesis) were compared using 1-D SDS-PAGE and 2-D IEF/SDS-PAGE. Similar procedures have been followed after in vivo phosphorylation of isolated endosperm with ³²PO₄.Protein patterns were similar when 1-D SDS-PAGE was used. On the other hand, 2-D IEF/SDS-PAGE revealed several major differences, the most important being the appearance of a major protein of Mr about 40,000 in the 3-weeks old endosperm. By contrast, the patterns of phosphorylated proteins, with both methods, were relatively simple. Only two phosphorylated bands showed up in the 3-weeks old endosperm: a highly labelled band of Mr about 65,000, and a band of about 75,000. In the case of 2-weeks old endosperm, where the phosphorylation was more active, these and several others were also labelled, but the Mr 75,000 band was labelled more intensely.