大豆质核互作雄性不育系NJCMS1A和其保持系的不同器官蛋白质组比较

质核互作雄性不育在杂种优势利用中起着重要作用,探讨质核互作雄性不育发生的机理具有重要的理论和实践意义.本研究开展大豆质核互作雄性不育系NJCMS1A与其保持系NJCMS1B的不同器官蛋白质组比较分析.以大豆质核互作雄性不育系NJCMS1A和其保持系NJCMS1B的种子、叶片和花药为材料,采用双向凝胶电泳技术对其蛋白质进行分离,考马斯亮蓝染色,获得重复性好的蛋白质双向电泳图谱,利用PDQuest软件处理分析,寻找差异表达蛋白.结果发现不育系NJCMS1A与其保持系NJCMS1B的种子2-DE图谱间存在7个差异表达蛋白点,其中3个在NJCMS1A中表达而在NJCMS1B中缺失,3个在NJCMS1A中缺失而在NJCMS1B中表达,1个在NJCMS1A中表达量明显增强;苗期叶片2-DE图谱间基本一致,没有差异表达蛋白点;单核小孢子期花药2-DE图谱间有9个差异表达蛋白点,其中3个在NJCMS1A中表达而在NJCMS1B中缺失,6个在NJCMS1A中缺失而在NJCMS1B中表达;二胞花粉期花药2-DE图谱间有24个差异表达蛋白点,其中10个在NJCMS1A中表达而在NJCMS1B中缺失,12个在NJCMS1A中缺失而在NJCMS1B中表达,2个在NJCMS1B中表达量明显增强.两系花药间存在较多差异表达蛋白点,种子间仅有少量差异表达蛋白点,而苗期叶片间基本没有差异表达蛋白点,说明不育基因表达具有时空性和器官特异性,与育性有关的蛋白主要在花药中表达.     

大豆种子发育过程中差异表达蛋白的蛋白质组分析

采用蛋白质组学技术研究了大豆N2899种子发育过程中蛋白质的差异表达。运用PDQuest软件比较分析不同发育时期（15,20,30,40,50 DAF和成熟种子）大豆种子蛋白的双向电泳图谱,在考染的2-D胶上共检测到337个蛋白点。有些蛋白质在整个发育过程中都出现,而另外一些只出现在发育早期或成熟的种子中。利用基质辅助-激光解吸／电离-飞行时间-质谱（MALDI-TOF-MS）技术,分析了不同发育时期30个差异表达蛋白,并用Pro-found（http：／／www．prowl．rockefeller．edu）工具,对质谱产生的肽质量指纹（PMF）数据进行NCBInr数据库检索,结果鉴定了18个蛋白质。比较发现,这些蛋白主要参与种子的成熟（如伴豆球蛋白）、逆境胁迫反应（如抗坏血酸过氧化酶）、细胞分裂（如Skp1）和蛋白运输（如钙网蛋白）等。研究表明,种子发育过程十分复杂,所鉴定的蛋白质,可为从分子水平上研究大豆种子发育进程中蛋白的积累和调控奠定基础。     

四种油料作物中的细菌型PEPC基因的鉴定及在发育种子中的表达

鉴定和获取了四种油料作物（油菜、大豆、花生和芝麻）中的细菌型PEPC基因,分析了所编码蛋白的保守结构域（BOX I-IV）和蛋白作用功能位点。基因包括甘蓝型油菜的Bna10093361、Bna1009749和Bna10093360,大豆的Glyma10g34970.1, Glyma01g22840.1和Glyma02g14500.1,芝麻的SIN1018296和花生的AhPPC5。这8个基因含有19～21个内含子,内部插入一个约350～600bp的高度变异区,编码的蛋白在C端形成R/KNTG结构域,在N端缺乏磷酸化作用位点。在种子发育的不同时期,油菜中仅Bna10093360表达,但其表达量不到油菜BnActin表达量的0.1%;大豆中Glyma10g34970.1表达量最高（接近大豆GlymaActin的2%）,Glyma02g14500.1次之;花生AhPPC5表达量为花生AhActin的32%～175%,在种子不同发育时期表达量为早期>中期>晚期;芝麻SIN1018296表达量为芝麻SINActin的3%～18%,在种子发育时期的表达趋势和花生AhPPC5相似。8个基因种子中的表达模式差异明显,说明细菌型PEPC基因可能存在着广泛的功能分化。       

大豆叶片蛋白响应干旱胁迫的双向电泳分析

大豆是我国重要的农作物之一,蛋白质组学技术是解析植物响应逆境胁迫的重要工具之一。为进一步推进对响应干旱胁迫的大豆叶片蛋白的鉴定工作,以大豆幼苗的叶片为研究对象,用甘露醇溶液模拟干旱胁迫,处理24 h后提取叶片蛋白粉进行双向电泳分离和图谱差异分析。结果表明:采取三氯醋酸(TCA)-丙酮沉淀法提取的叶片蛋白进行双向电泳分离能够得到稳定清晰的双向电泳蛋白图谱。采取PD-Quest软件对干旱胁迫下的大豆叶片蛋白表达图谱进行差异分析,共检测和匹配425个大豆叶片蛋白点,其中差异表达的蛋白点有101个,60个表达量上调,41个表达量下调。本研究结果表明有一部分大豆叶片蛋白响应了干旱胁迫。     

利用蛋白质组学技术鉴定大豆籽粒贮藏蛋白A1aB1b亚基缺失体

利用SDS-PAGE对220份大豆种子贮藏蛋白亚基含量进行筛选,在获得大豆籽粒贮藏蛋白11S组分组I亚基变异种质的基础上,经双向电泳分辨大豆贮藏蛋白亚基正常品种(南农大黄豆)和亚基变异品种(桂阳紫金豆)成熟种子总蛋白的蛋白质组.用PDQuest软件分析双向电泳凝胶图谱,发现在大豆贮藏蛋白11S酸性亚基位置有等电点和分子量分别为5.40和37.85kDa、5.24和37.2kDa、5.15和37.05kDa的蛋白质点在正常种子中表达而在变异种质种子中未表达.对这3个蛋白质点用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定其胶内酶解后的肽质量指纹谱(PMF),对获得的PMF用Mascot软件在NCBInr数据库中查询比对,鉴定出这3个蛋白质点均为大豆球蛋白A1aB1b亚基同源三聚体,表明变异种质种子缺少贮藏蛋白A1aB1b亚基.     

适用于大豆叶片蛋白质组分析的双向电泳最佳条件研究

针对大豆叶片中蛋白含量较低,含有大量色素、酚类、醌类等次生代谢产物干扰蛋白质双向电泳分离效果的问题,以大豆叶片为材料,对样品制备、蛋白质裂解液组分、等电聚焦(IEF)参数等条件进行研究.结果表明:采用TCA/丙酮法提取大豆叶片总蛋白,裂解缓冲液为9 mol·L-1尿素,2 mol·L-1硫脲,4%CHAPS.100 mmol·L-1DTT,0.5%Bio-Lyte,IEF等电聚焦条件为24 000Vh时,2-DE图谱分离到的蛋白点效果最好.初步建立了一套适用于大豆叶片蛋白质组分析的双向电泳(2-DE)体系.     

萌发过程中不同进化类型大豆种子贮藏蛋白的电泳分析

采用SDS聚丙烯酰胺凝胶电泳,分析了室温条件下不同进化类型大豆种子贮藏蛋白在萌发过程中的代谢。结果表明:(1)在萌发过程中,大豆种子贮藏蛋白各种组分的降解时间不同,表现为7S组分早于11S组分。(2)萌发初期以大分子贮藏蛋白为主,随着萌发天数的增加,大分子组分逐渐减少,而小分子组分逐渐增加。(3)不同进化类型大豆种子贮藏蛋白的降解表现出连续性差异。     

玉米/大豆间作增强大豆种子在萌发期间的抗逆能力

玉米/大豆间作模式下,低位作物大豆受到高位作物玉米的遮荫,导致大豆种子在发育期间受到荫蔽胁迫。为研究荫蔽胁迫对大豆种子在随后的萌发期间抗逆性的影响,本研究以大豆品种菏豆19、玉米品种浚单26为试验材料,采用1∶1玉米/大豆间作(intercropping,IC)模式,对照为净作大豆(monocropping,MC),大豆种子收获后,测定百粒重、蛋白质含量以及脂肪含量等性状;并分析其在萌发期间的抗逆能力(以高温、甘露醇、葡萄糖、聚乙二醇、Na Cl以及脱落酸进行胁迫处理);进一步以qRT-PCR分析了ABA信号转导相关基因的表达情况。结果表明,玉米/大豆间作模式下收获的大豆种子的百粒重、脂肪和蛋白质含量与净作大豆无明显差异;在上述非生物胁迫条件下,与对照相比,间作大豆种子具有较快的萌发速率,表现出较强的抗逆能力;qRT-PCR数据显示,在萌发过程中,间作大豆种子中ABA信号通路上的正调控基因Gm ABI4、Gm ABI5表达量较净作有所下调。因此,本研究表明玉米/大豆间作模式不会降低大豆种子品质,但是会通过削弱ABA信号,进而增强间作大豆种子在萌发期间的抗逆能力,提高其萌发期间对非生物逆境的适应性。     

大豆2S清蛋白基因生物信息学分析

2S清蛋白含有动物胰蛋白酶抑制剂及过敏原等多种具有生理活性的多肽,可引起特殊人群尤其是儿童的过敏反应,其编码基因为LTP基因家族。为系统地分析2S清蛋白编码基因,利用生物信息学分析方法获得2S清蛋白基因的全序列、定位以及转录本等信息,鉴定了30个大豆LTP家族基因。基因定位结果表明:23. 33%基因在染色体上串联排布,反映了该家族基因的串联重复进化。通过基因功能注释得到22个非特异性脂质转运蛋白,1个类伸展素蛋白,4个脯氨酸蛋白,2个种子储存2S清蛋白和1个雄蕊特殊蛋白。根据系统发育分析将这些蛋白分成3个类群。类群I有类伸展素蛋白、脯氨酸蛋白、雄蕊特殊蛋白和种子储存2S清蛋白,类群II有16个非特异性脂质转运蛋白,类群III有6个非特异性脂肪转移蛋白。种子储存2S清蛋白基因在种子发育中后期特异表达,表达量显著高于其它同源基因。本研究为进一步研究其功能提供指导,为该类基因应用于大豆品质改良提供理论支撑。     

大豆Glyma.05G222700.2基因生物信息学与逆境表达分析

为研究Glyma.05G222700.2基因编码的丝氨酸/苏氨酸蛋白激酶在抗非生物胁迫过程的功能和原理,促进大豆抗逆候选基因的开发利用,本研究通过生物信息学方法对大豆Glyma.05G222700.2基因进行同源序列、蛋白结构、进化树和转录组分析,通过qRT-PCR分析盐胁迫下大豆不同组织中该基因的表达情况。结果表明:该基因编码区长2 040 bp,编码697个氨基酸,预测分子量为656.54 kD,pI8.026。多序列比对发现Glyma.05G222700.2蛋白包含1个Pkinase结构域。进化树分析表明该蛋白与野大豆、刺毛黧豆、赤豆一致性较高。转录组数据表明Glyma.05G222700.2基因在大豆各组织中均有表达,其中在种子中表达量最高,在根中表达量最低。qRT-PCR结果发现Glyma.05G222700.2基因在毛状根、茎、叶中均有表达,在茎中表达量最高,在叶中表达量最低;在毛状根中盐胁迫12 h表达量达到极值,盐胁迫24 h表达量下降;在茎中表达量呈上升趋势,在24 h表达量达到极值;在叶中表达量不稳定,盐胁迫2 h该基因不表达,盐胁迫6 h该基因表达量达到极值并高于...     

复硝酚钠及其组分对大豆种子萌发的影响

采用复硝酚钠及其有效组分邻硝基苯酚钠、对硝基苯酚钠和5-硝基愈创木酚钠对大豆浸种,通过研究发芽第1、3、5、7 天的大豆种子蛋白质含量、脂肪含量、可溶性糖含量和种子发芽情况,探讨复硝酚钠及其组分对大豆种子萌发的影响.结果表明:5 mg·L-1的5-硝基愈创木酚钠和10 mg·L-1复硝酚钠浸种均使大豆的芽重、发芽势、发芽率、发芽指数和种子活力增加,达到了5%的显著水平.同时使种子中的储藏蛋白质从第3天分解加速,脂肪的转化速率从第5天明显加快,可溶性糖含量始终低于其它处理.以上结果表明,5-硝基愈创木酚钠在大豆种子萌发中起主要作用.     

Feasibility study of a tissue-specific approach to barley proteome analysis: aleurone layer,endosperm, embryo and single seeds

Proteome analysis in the pI 4–7 range of aqueous extracts from whole barley seeds results in complex 2D-gel spot patterns that represent proteins from all seed tissues. Proteins were extracted from dissected aleurone layer, embryo and endosperm for separate analysis of these components and comparison with single seed extracts. The analysis showed that while the starchy endosperm comprises approximately 85% of the mature seed dry weight, it contributes less than 50% of the soluble protein to seed extracts. Although the aleurone layer and embryo are minor components of the seed in terms of dry weight, their proteins contribute significantly to the 2D-gel pattern. Distinct but overlapping patterns were observed for the dissected tissues. While whole seed gels contained about 850 spots, endosperm, aleurone layer and embryo gels had about 575, 850 and 1000 spots, respectively, representing a total increase of at least 15%. Protein patterns were also reduced in complexity and this enrichment of subsets of proteins facilitated identification by mass spectrometry of proteins of interest in mature seed tissues and developing endosperm. These techniques can therefore be used with advantage to describe the proteomes of dissected seed tissues.     

大豆种子抗老化鉴定的方法研究

选用遗传差异相对较大的皖豆24、皖豆25(杂交品种)及合豆3号的种子,在温度为40℃、湿度为85%的条件下老化处理4、8和12 d,研究加速老化过程中不同基因型大豆种子成苗率、发芽率、电导率及单株幼苗干重的变化规律。结果表明:随着加速老化时间的延长种子活力下降;加速老化处理8 d,3个品种的各项鉴定指标均发生改变:品种的成苗率、发芽率及单株幼苗干重显著降低,电导率明显升高,且品种间有显著差异;发芽率和成苗率的变化在品种间具有一致性,电导率、单株幼苗干重的变化和成苗率品种间一致性较差;成苗率和发芽率的相关性最高(r=0.972**)。利用国家大豆微核心种质中的91份资源对老化鉴定方法进行验证,结果在温度为40℃,湿度为85%条件下老化8 d的种子发芽率及老化指数与在自然条件下老化15个月的发芽率及老化指数极显著相关(r=0.943**,0.716**)。综合分析认为,温度为40℃,湿度为85%条件下老化处理8 d,以种子发芽率为活力鉴定指标可作为大豆种子抗老化性的鉴定方法。     

引发提高水稻劣变种子发芽率的蛋白质组学分析

引发处理是目前简便有效的提升种子发芽率和整齐度的方法,但引发提高水稻劣变种子发芽能力的机理并不清楚。利用蛋白质组学方法,分析清水引发和藤茶提取物二氢杨梅素引发2种处理方法与对照的差异蛋白,初步揭示引发提升种子发芽能力的机制。分析双向电泳图谱差异获得2倍以上显著差异蛋白点79个,通过MALDI TOF/TOF MS质谱分析鉴定出74个蛋白,其中2种引发处理与未引发对照相比同步上调或下调的共有蛋白,即引发相关蛋白有57个。分析引发相关蛋白可知,绝大多数的逆境防御蛋白类、能量相关蛋白类以及蛋白质合成和目标类蛋白丰度在引发处理中显著增加,推测引发提高种子发芽率的原因与逆境记忆、能量代谢活动和蛋白质合成能力增强等密切相关。     

Effect of Germinated Soybean Protein Hydrolysates on Adipogenesis and Adipolysis in 3T3-L1 Cells

Germination of soybeans increases the bioavailability of some nutrients. An evaluation was done to determine if germination increased the anti-adipogenic and lipolytic effects of soybean. Soybeans were germinated for 0 to 6 days and protein concentrates extracted from beans germinated at each period. Soy protein concentrates can retain notable amounts of phytochemicals with anti-adipogenic activity. For this reason, it was evaluated the effect of protein hydrolysates with and without phytochemicals in the adipocyte-like cells after 3T3-L1 (murine fibroblasts) cell line differentiation. Cell viability decreased with exposure to the germinated soybean protein hydrolysates during the differentiation stage, but not during the fibroblast or mature adipocyte stages. Adipogenesis and triglycerides accumulation were strongly inhibited by the hydrolysate from soybeans germinated for 2 days (with ethanol-soluble phytochemicals), when compared to ungerminated soybean. Adipolysis increased with exposure to hydrolysates from beans germinated for 2 days (with phytochemicals) and 5 days (without phytochemicals). Germinated soy protein hydrolysates had an effect on inhibition of lipid storage in adypocites and increasing lipolysis, which was improved by changes of the protein and increased phytochemical content due to germination.     

Isoflavone Contents in Germinated Soybean Seeds

The effect of germination on isoflavone contents in two soybean varieties (Hutcheson and Caviness) was investigated. Soybean seeds were soaked at 25 ^∘C for 12 h, germinated at 40 ^∘C, and freeze-dried. The isoflavone contents of dry, soaked, germinated (hypocotyl length at 0.5, 2.5, and 6.5 mm), and nongerminated seeds were determined by high performance liquid chromatography. The maximum amount of total isoflavone, genistein, and daidzein with their β-glucoside conjugates was obtained when hypocotyl length of the germinated-seed from var. Hutcheson was 0.5 mm (2.491, 1.500, and 0.671 mg/g), and from var. Caviness was 2.5 mm (2.78, 1.523, and 0.905 mg/g). A dramatic increase in malonylgenistin and malonyldaidzin (1.305 mg/g and 0.476 mg/g in Hutcheson, and 1.308 mg/g and 0.677 mg/g in Caviness, respectively) was observed at these hypocotyl lengths. A decrease was observed after this stage. Genistein and daidzein contents were highest just after soaking. Glycitein and its β-glucoside conjugates remained almost the same during germination. Controlled germination can be used to enhance isoflavone content in soybean seed.     

小麦种子萌发期抗早衰鉴定指标的初步研究

为了探索小麦抗早衰性的早期鉴定指标,采用种子萌发试验比较了不同抗早衰类型的33个小麦(Triticum aestivum L.)品种(系)萌发种子的芽长、胚芽鞘长、主胚根长、种子根数、根系活力、种子贮藏物质转运率、总鲜重、总干重、根芽干重、干物质含量等指标与抗早衰性之间的关系。结果表明,萌发种子的芽长、胚芽鞘长、主胚根长、种子根数、根系活力、总鲜重及根芽干重等7项指标均与小麦抗早衰性无明显相关性;萌发种子的总干重、干物质含量与小麦抗早衰性有部分相关性,但不能准确区分抗早衰型和典型早衰型品种(系);种子贮藏物质转运率能较好区分不同抗早衰类型的小麦品种,可作为小麦种子萌发期抗早衰性的鉴定指标。     

Characterization of Amaranthus cruentus L. seed proteins by 2-DE and LC/MS–MS: Identification and cloning of a novel late embryogenesis-abundant protein

Amaranth is taking great attention as an important cereal crop that could fulfill food requirements for the growing population, especially in developing countries. However, the protein composition of these seeds is not well known yet. We have used the proteomics tools to characterize amaranth seed proteome. About 400 proteins spots were resolved on two-dimensional gel electrophoresis (2-DE) and protein spots were analyzed by LC-MS/MS (liquid chromatography tandem mass spectrometry). Identified proteins were related to stress and defense responses, metabolic, respiratory, and oxide-reduction processes. One abundant spot was identified as a Late Embryogenesis Abundant (LEA) protein and the gene was cloned and characterized. The AcLEA cDNA contains a 418 bp open reading frame (ORF) encoding a polypeptide of 139 amino acids. Bioinformatics analysis showed that AcLEA belongs to LEAs Group 5. Proteomics is a powerful technique that could be used even in non-sequenced organisms such as amaranth. The obtained information reveals that amaranth seed, beyond the classical seed storage proteins, contains proteins related to protection against stress. The identification of these proteins opens the door to the application of new strategies to improve the quality of amaranth production.     

Contribution of low molecular weight antioxidants to the antioxidant screen of germinated soybean seeds

Soybean seed of the Mazovia variety (Glycine max) were germinated in the dark at a constant temperature of 25°C up to 168 h (24 h × 7). Changes in content of low molecular weight antioxidants (LMWA) in the course of germination were studied. The analyzed compounds were: total phenolics, soluble proteins, tocopherols (-T, -T, -T, -T), reduced and oxidized glutathione, L-ascorbic acid and L-dehydroascorbic acid. The changes in total antioxidant status (TAS) and the peroxyl radical-trapping capacity (PRTC) of the processed seeds were measured and compared to those of the raw seeds. On the basis of data obtained, the contribution of LMWA to the TAS of raw and germinated soybean seeds was calculated. The results showed a very high molar percentage contribution of phenolic compounds (49.7%) in opposition to glutathione (7.0%), tocopherols (4.0%) and soluble protein (5.7%) to the total TAS of raw soybean seeds. The germination process led to the increased contribution of phenolic compounds (67.7%), soluble proteins (9.3%) and ascorbic acid (4.4%) to the total TAS of germinated seeds and as a result, a decrease in the antioxidant gap was observed compared to the total TAS of raw soybean seeds. This study showed that the contribution of experimentally obtained total PRTC to the total TAS of raw and germinated soybean seeds after seven days ofthe process was 48% and 25.2%, respectively. The PRTC results were comparable to those obtained in TAS only when calculation was done by multiple mean values for content of investigated compounds and their relative potency with respect to Trolox. The PRTC values obtained in this manner were higher than those obtained experimentally indicating that high molecular weight antioxidants as well as are able to quench peroxyl radicals as well as low molecular weight antioxidants. Therefore, calculation of PRTC is a more accurate method that eliminates the puzzling question of unidentified antioxidants shown as the antioxidant gap in the TAS assay.