小麦种子萌发时期胚差异蛋白表达分析

为了解小麦种子在萌发时期胚蛋白质表达情况,通过双向电泳技术,对"晋麦-47"小麦种子未萌发和萌发3 h、16 h和25 h的胚进行蛋白质分离。结果发现,"晋麦-47"小麦种子未萌发和萌发3 h、16 h和15 h的胚在PDQuest图像分析软件可识别的蛋白点分别有127、131、135和141个,其中表达量变化2.5倍以上的蛋白点有17个。选取表达量在2.5倍以上的9个差异蛋白点进行质谱分析,初步鉴定出4个蛋白质,分别是核苷二磷酸激酶Ⅰ、17.5 kD热休克蛋白、ATP合酶和LEA蛋白27。     

转反义硫氧还蛋白基因小麦萌发种子中蛋白质的变化

硫氧还蛋白h（thioredoxin h,Trx h）是一类广泛存在于生物体内的多功能活性蛋白,分子量约为12kD,它通过还原靶蛋白中的二硫键参与酶活性调节、抗胁迫、信号传导等许多重要的生命活动。硫氧还蛋白h能促进谷物类种子萌发过程,主要表现在以下2个方面：（1）在籽粒萌发期间,硫氧还蛋白可通过还原储存蛋白的分子内二硫键使其更易于被降解;（2）硫氧还蛋白也可以直接地通过将酶还原或者间接地通过使酶抑制蛋白失活而激活酶。源于Phalaris coerulescens的trxs基因（thioredoxin s,trxs）与小麦硫氧还蛋白h基因（thioredoxin h,trx h）同属于硫氧还蛋白基因家族,它们的cDNA有94％的同源性,表达产物也有相似的生物功能。我们采用基因枪法将反义trxs基因导入小麦,获得了可稳定遗传的小麦,并检测出转基因种子中硫氧还蛋白h表达量、水溶蛋白和醇溶蛋白的还原状态以及α-淀粉酶活性均低于对照小麦;另外,通过模拟降雨抗穗发芽试验证实转基因株系具有很强的抗穗发芽能力。以转反义trxs基因抗穗发芽小麦为材料,检测反义trxs基因小麦籽粒萌发过程中蛋白质的变化,探讨转反义trxs基因小麦的抗穗发芽机理。研究表明反义trxs基因能够减缓KCl可溶性蛋白中Chloroform-methanol（CM）蛋白向代谢类蛋白的转化进程,在萌发初期降低籽粒代谢类蛋白的含量,使籽粒代谢速度下降,而CM蛋白主要包含一些分子量小于20kD的蛋白质。在籽粒成熟过程中,硫氧还蛋白能够阻止麦谷蛋白亚基形成谷蛋白聚合体的过程,在转基因小麦中麦谷蛋白更易于形成大分子量的谷蛋白大聚合体,使得转基因小麦中的谷蛋白在萌发初期更难于被水解,因此转基因小麦籽粒会因谷蛋白难于降解而萌发较慢。另外,反义trxs基因减慢了麦胚中10kD蛋白的降解过程。     

NaCl胁迫对小麦种子胚在萌发时期蛋白含量的影响

为比较不同浓度盐胁迫下小麦种子萌发前期胚蛋白表达情况,以"晋麦-47"为试验材料。选取经0、0.1和0.2mol/L处理过的种子,采用2-DE技术对3个浓度下胚蛋白含量变化进行研究。通过PDQuest图像分析软件分析盐胁迫下蛋白含量的变化,检测到正常的种子胚有121个点,0.1 mol/L NaCl胁迫下有32个蛋白点丰度下调,21个蛋白点丰度上调;在0.2 mol/LNaCl胁迫下,检测到46个蛋白质点丰度下调,16个蛋白点的丰度上调。研究结果表明,盐胁迫对小麦种子萌发时期胚中部分蛋白含量和表达产生不同程度的抑制与激活。     

小麦幼芽水分胁迫诱导蛋白的特征

水分胁迫（－1．2MPaPEG-6000）处理萌动的小麦种子,24h后诱导小麦幼芽产生41．5kD蛋白,其含量随着胁迫时间延长明显增加,48h时含量最高,到72h后不再变化。复水后,该蛋白消失;再胁迫48h时则又出现,其含量与处理24h时相当。41．5kD诱导蛋白主要位于细胞器膜上,细胞质中几乎不存在。41．5kD蛋白主要溶于10％NaCl提取液中,其等电点为pl5．65。该蛋白的氨基酸组成中,脯氨酸含量最高,其次为丙氨酸、天冬氨酸、谷氨酸、甘氨酸,没有发现半胱氨酸和组氨酸。     

假槟榔种子脱水耐性的发育变化

对假槟榔（Archontophoenix alexandrae）种子和胚发育过程中脱水耐性的变化、不同脱水速率对脱水耐性的影响及种子的萌发和贮藏特性进行了研究。种子含水量在花后55～70d逐步降低,随后不再变化,并保持在较高水平（37％）;花后90d的种子获得最大干重。花后60d后种子获得萌发能力,花后70d达到最大值。在交替光照下（14h光照,10h黑暗,12μnmol m^-2s^-1）,种子在15℃～40℃下均能萌发,其萌发的适宜温度范围为30℃～35℃;但光照对种子的萌发有较大的抑制作用。种子和胚在花后55～90d,脱水耐性逐渐增强;花后90d种子和胚的脱水耐性最强,此时种子和胚的半致死含水量分别为0．18g／g和0．3g／g。脱水至相同含水量,快速脱水的种子的存活率明显高于慢速脱水。无论是否进行脱水处理,-18℃下贮藏1个月后,种子均丧失萌发能力;在4℃,10℃和15℃下,适度脱水能延长种子的贮藏寿命。假槟榔种子不耐脱水,不适合在低温、低含水量条件下长期贮藏,属顽拗性种子。     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

大豆中L34基因的克隆与表达分析

目的:采用分子手段试图分离与大豆种子低温吸胀相关的基因.方法:利用cDNA-AFLP(cDNA-amplified fragment length polymorphism)和RACE技术从大豆种子胚轴中克隆到一个编码132 kD的全长核糖体蛋白基因,命名为SOL34.结果:序列分析表明,SOL34和苜蓿(gi | 113205273 |)、茄科植物(gi | 48057670 |)及拟南芥(gi | 2500376 |)中L34蛋白基因的同源性分别为95%、95%和90%.半定量RT-PCR结果表明:大豆种子在4℃下吸胀24h内,胚轴中的SOL34被诱导表达,其中当种子低温吸胀6h,SOL34表达量升高非常明显,18h的表达量最大,24h表达量和18h相似;SOL34在大豆不同部位的表达不同,4叶期的大豆幼苗经过低温处理后,根尖中SOL34的表达比非处理材料增强约5倍,同时比胚轴中高约2倍;但是在叶片中,SOL34表达量并没有受到温度的影响,表达量也很弱,说明SOL34在叶片中是组成型表达.结论:结果分析表明,SOL34可能和大豆根的代谢有关.     

不同发育时期小鼠胚泡表面Lewis寡糖抗原的表达

在胚泡表面表达的Lewis寡糖抗原 (LewisX ,LewisY)在胚胎发育以及着床过程中起重要作用 .应用免疫印迹和免疫荧光等方法对着床前小鼠胚泡表面的Lewis寡糖抗原进行分析 .结果发现 :小鼠胚泡LewisX寡糖蛋白有 2 7kD、2 9kD、6 8kD和 80kD 4种 ,LewisY寡糖蛋白有 70kD和 90kD 2种 ;2种寡糖抗原均在 8细胞时期开始表达 ,其中 ,LewisY寡糖抗原在胚泡表面的表达持续升高 ,直至胚泡着床 ;而LewisX寡糖抗原的表达则在桑椹期后逐渐降低 ,但仍在胚胎期的囊胚腔侧的顶端可见有部分表达 ;应用RT PCR的分析结果显示 ,LewisX合成的关键糖基转移酶FUT9基因在 4细胞及桑椹期高表达 ,到胚泡期虽然强度明显减弱 ,但仍有表达 ;而LewisY合成关键酶FUT1基因在 4细胞未见表达 ,在桑椹和胚泡阶段均有表达并逐渐升高 ,表达趋势与相应寡糖的表达趋势基本一致 .结果说明 ,在小鼠胚泡表面表达的Lewis寡糖抗原的表达受到相应糖基转移酶基因转录的调控     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

小麦种子基因的表达序列标签分析(英文)

以授粉后12d的小麦(TriticumaestivumL.)种子为材料,构建起cDNA文库。从中随机挑选10000个克隆,利用Biomek2000核酸工作站制成高密度cDNA阵列。然后分别以未受精子房、胚和胚乳中提取的RNA为模板,反转录合成探针与膜杂交,进行差示筛选。根据筛选结果,选取800个在胚、胚乳或胚和胚乳中表达的克隆进行表达序列标签(EST)分析,鉴定出216个不同的基因序列。其中24个ESTs属于已知的小麦基因;122个ESTs为推测的小麦新基因,它们编码的产物与种子贮藏蛋白或与生化代谢、发育等其他的生物学过程有关;70个ESTs的序列特征尚未确定。本研究为研究种子发育和小麦品质改良等提供了基础资料。     

Protein Patterns, Characterized by Computer Image Analysis, of Lentil Embryo Axes Germinating Under Salt Stress

Following 16, 40 and 64 h exposure to 0.33 M NaCl given after 8 h water imbibition, lentil seeds showed a gradual decrease of germination upon their transfer to water. These salt related changes were accompanied by modifications in the protein patterns of embryo axes as revealed by two-dimensional electrophoresis separation and by the computer image analysis of protein spots. In comparison with 8 h water imbibed seeds, prominent proteins comprised between the 5.1 – 7.6 pH isoelectric point in the first dimension and 75 – 50 kDa molecular mass in the second dimension showed a significant increase in their abundance as salt exposure increased. On transfer to water to complete germination, the content of many of these proteins decreased at 24h in 2 – 3 cm length embryo axes in comparison with the corresponding embryo axes of seeds continuously imbibed in water for 24 h. Some groups of proteins ranging between 15.5 – 17.3 kDa, already present after 8 h water imbibition, were not detectable after 24 h but were expressed in seeds exposed to NaCl and transferred to water for 24 h. Up- and down-regulated proteins in lentil embryo axes, imbibed under non-lethal salt stress conditions, have been tentatively identified by comparison with the protein map of germinating seeds of the model plant Arabidopsis. This revised version was published online in July 2006 with corrections to the Cover Date.     

荔枝胚败育过程中内源激素与蛋白质含量的变化

连续3年(1999-2001年)对典型的荔枝焦核品种桂味、糯米糍和大核品种黑叶、怀枝花后10-40d的幼胚和胚乳内源激素、多酚含量及蛋白质动态变化进行研究。结果表明,焦核品种幼胚及胚乳中的IAA、GAs和ABA含量低于大核品种;多酚类物质含量在胚中低于大核品种,胚乳中则高于大核品种;胚和胚乳中的蛋白质含量均低于大核品种。蛋白质电泳结果显示,22．5、28．5和45kD这3类蛋白质在怀枝和黑叶的胚蛋白质代谢过程中表现出较高的稳定性,桂味和糯米糍胚蛋白质中的28．5kD蛋白质也有相似的特性。     

Differential organ-specific response to salt stress and water deficit in nodulated bean (Phaseolus vulgaris)

Nodulated bean plants were exposed to mild salt stress or water deficit in such a way that the nodule's nitrogen‐fixing activity was reduced to about 25–30% that of controls. Water‐deprived plants showed a slight decrease in the weight of the aerial part, whereas the photosynthetic parameters were not significantly affected. In contrast, salt‐stressed plants displayed a reversible decrease in the quantum yield of photosystem II photochemistry. Five water‐deficit responsive cDNA clones encoding one lipid transfer protein, two late‐embryogenesis abundant (LEA) proteins and two proline‐rich proteins (PRPs) showed different organ‐specific expression patterns depending on the kind of stress applied. PRPs and one LEA protein, PvLEA‐18, exhibited the highest expression in nodules. Anti‐PvLEA‐18 antibodies were used to immunolocalize the protein in the nodule. PvLEA‐18 was localized in the cytoplasm and nucleus of nodule cortex cells, and preferentially in cells of the vascular bundles, showing enhanced accumulation under water deficit. To our knowledge, this is the first time that a LEA protein has been identified in legume nodules.     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

耐盐突变体小麦后代耐盐稳定性分析研究

以卫星搭载小麦种子为原始材料,利用其幼穗、幼胚诱导的愈伤组织进行耐盐突变体的筛选,对耐盐愈伤组织再生植株后代进行耐盐稳定性生理生化特性分析。结果表明：(1)耐盐系后代在土壤高盐浓度条件下,游离脯氨酸含量稳定增加,且高于对照系;(2)耐盐系再生植株后代保持较高的K^ ／Na^ 比;(3)与对照相比,种子醇溶蛋白电泳带谱中的b2,b3,b5,b7带为耐盐系所特有,b8带消失;(4)耐盐系再生植株后代可溶蛋白电泳带为26条,而对照系为23条蛋白带。其中98kD、75kD、52kD、49kD和32kD为耐盐系的特有蛋白带。而38kD和35kD蛋白带为对照系所特有。     

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.     

鸡骨骼肌发育过程生肌素的表达

ＭｙｏＤ家族生肌因子ＭｙｏＤ、生肌素（ｍｙｏｇｅｎｉｎ）、ｍｙｆ－５和ｍｙｆ－６／ｈｅｒｃｕｌｉｎ对脊椎动物肌细胞分化和骨骼肌系统的发育成熟具有重要意义,其中生肌素的作用尤为突出,是肌细胞终末分化的关键因素。采用Ｎｏｒｔｈｅｒｎ和Ｗｅｓｔｅｒｎ印迹技术检测鸡胚骨骼肌发育过程中生肌素在转录水平的表达动力学,发现在胚胎发育第９ｄ已有鸡肢体骨骼肌生肌素ｍＲＮＡ表达,第１３ｄ达到高峰,第１５ｄ开始下降,第１８ｄ至出生后两周几无可检测的ｍＲＮＡ;而生肌素蛋白则在胚胎发育第１３ｄ方可被检测到,第１５～１８ｄ达到高峰,至出生后两周仍维持在一定水平,表明生肌素的表达存在翻译和翻译后水平的调控。利用鸡生肌素基因上游－２２３／＋４０调控片段为探针对发育不同时期的鸡肌肉组织细胞核抽提物进行Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹分析。发现一种约３５ｋＤ的核内蛋白与探针有较强结合,结合活性第９ｄ时出现,至第１８ｄ达到峰值,出生后两周时降至难检测水平;另外有一种约１１ｋＤ的核内蛋白的ＤＮＡ结合活性恰好在转录活性下降的第１５～１８ｄ发生,按此情况两种核因子可能对生肌素的转录激活分别起正负调控作用。