木薯叶片响应干旱胁迫的磷酸化蛋白质组差异分析

本文以抗旱性较强的‘华南8号’木薯(Manihot esculenta)为材料,分析正常供水、轻度干旱(干旱处理5 d)和重度干旱(干旱处理15 d)胁迫对木薯植株形态及叶片磷酸化蛋白质组的影响。结果表明,随着干旱胁迫程度的增加,木薯叶片从底部开始萎蔫、脱落,但顶端叶片优先保持正常生长。干旱胁迫处理后,共有28个磷酸化蛋白点在叶片中的表达丰度发生了显著变化。质谱(MS)鉴定显示,这些蛋白质主要参与光合作用、能量代谢、碳代谢、胁迫与防御、结合和转录翻译等代谢途径。其中,大部分参与光合作用的蛋白积累量在干旱胁迫后显著降低,而参与能量代谢、碳代谢、胁迫与防御、转录翻译等途径的大部分蛋白质积累量则明显升高。由此推测,木薯应答干旱胁迫可能是通过改变植株形态,抑制叶片中的光合作用相关蛋白,调控叶片碳分配过程,同时,通过有效清除活性氧,防御氧化胁迫损伤,防止蛋白变性和降解等方式。     

干旱胁迫下蒙古沙冬青叶片蛋白质组学研究

通过对蒙古沙冬青[Ammopiptanthus mongolicus(Maxim)Cheng.f]叶组织干旱胁迫下蛋白质组变化的研究,从蛋白表达水平阐释其应答干旱胁迫的分子机制。以20%PEG 6000胁迫处理1 h和72 h,以0 h为对照,提取叶片总蛋白,利用双向电泳和质谱鉴定技术分析差异表达蛋白。共鉴定了40个差异表达蛋白,按功能可分为9类:光合作用,ROS清除,蛋白的合成、加工与降解,物质运输,防御相关,RNA加工,氨基酸代谢,其他相关蛋白和功能未知蛋白质。蒙古沙冬青叶片应答干旱胁迫的核心是叶绿体结构和光合作用的维持。     

脱水胁迫对连翘和冬青卫矛叶片细胞质外体和微环境的影响

用压力室、电导法和原子吸收分光光度分析法综合分析测定了连翘[Forsythiasuspensa (Thunb.) Vahl]和冬青卫矛(Euonymus japonicus Thunb.)在不同程度脱水胁迫时细胞外部微环境的变化.结果表明:(1)在脱水胁迫条件下,叶片细胞随着脱水胁迫强度的加大,细胞内离子外渗的累计量不断加大,但细胞内离子外渗的速度在不同的区间内并无明显的改变,即细胞膜的透性在所测范围内没有明显变化.(2)脱水胁迫同时造成了叶片质外体和共质体溶液中钠、钾离子浓度的增加,但质外体比共质体溶液中钠、钾离子浓度的增加幅度更高,导致细胞内、外离子浓度梯度的改变和离子平衡膜电位的改变,这些改变有可能引起细胞的次生生理变化,并有可能与植物的伤害反应和抗性有关.     

水分胁迫诱导玉米叶片质外体产生H2O2机理

通过组织化学染色、电镜观察、酶活性分析对水分胁迫诱导玉米叶片质外体产生H2O2进行了研究。结果表明:水分胁迫能够诱导玉米叶片内源ABA的积累,ABA参与了水分胁迫诱导的玉米叶片H2O2的产生,质膜NADPH氧化酶、细胞壁过氧化物酶(POD)以及质外体多胺氧化酶(PAO)是水分胁迫诱导玉米细胞在质外体产生H2O2的来源,其中质膜NADPH氧化酶是主要来源;内源ABA的积累参与了水分胁迫激活的质膜NADPH氧化酶、细胞壁POD和质外体PAO活性的提高。研究认为,水分胁迫诱导玉米细胞在质外体产生H2O2可能是由于水分胁迫下内源ABA的积累通过激活质膜NADPH氧化酶、细胞壁POD以及质外体PAO的活性而实现的。     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

植物盐胁迫应答蛋白质组学分析

土壤盐渍化是限制植物生长和分布的关键因素之一,揭示植物盐胁迫应答的分子机理是借助分子生物学手段提高植物耐盐性的基础.近年来,人们利用高通量蛋白质组学技术分析了拟南芥、水稻等19种植物的盐胁迫应答蛋白质表达图谱.从植物类群(盐生植物和甜土植物)、组织器官(根、地上部分/茎、胚根和胚轴、叶片、花序和配子体)、细胞(悬浮培养细胞、愈伤组织细胞和单细胞生物)和亚细胞结构(叶绿体、质膜和质外体)几方面整合分析了植物盐胁迫应答蛋白质组表达模式特征,主要特征包括:(1)盐生植物通过全面调节细胞骨架重塑、离子转运和区隔化、渗透平衡、活性氧(ROS)清除、信号转导、光合作用和能量代谢等信号与代谢网络体系,获得相对较高的抗/耐盐能力;(2)植物地上部分(叶片、茎、配子体)或光合组织细胞(悬浮培养细胞、愈伤组织细胞和单细胞盐藻)通过调节参与光合作用、碳和能量代谢、ROS清除过程蛋白质的表达模式应对盐胁迫环境;(3)植物地下部分(根、胚根)通过调控信号转导和离子转运相关蛋白质感知/传递盐胁迫信号并维持离子平衡;(4)花序中参与渗透调节、转录调控、蛋白质加工和ROS清除的蛋白质在盐胁迫条件下变化显著;(5)叶绿体通过调控参与光合作用、蛋白质加工和周转,以及氧化还原系统平衡等过程应对盐胁迫;(6)质外体中参与细胞壁代谢、胁迫防御和信号转导过程的蛋白质受盐胁迫影响明显;(7)细胞膜中参与维持膜结构稳定、物质/离子运输和信号转导过程的蛋白质对植物盐胁迫应答具有重要作用.这些分析为深入研究植物耐盐的分子机制提供了重要信息.     

一种具有热滞活性的女贞叶质外体过氧化物酶

采用浸出离心法从女贞叶中分离的质外体蛋白,约占叶片可溶蛋白的1.63%,其中的质外体过氧化物酶占叶过氧化物酶(POD)总酶活性的3.14%.以凝胶过滤和离子交换层析分离纯化得到一分子量为62 kDa的质外体过氧化物酶,其对H202的表观km值为1.3 mmol-L-1,表观Vmax值为189.33 U·mg-1(蛋白);对愈创木酚的表观Km值为1.78 mmol-L-1,表观Vmax值为215.26U·mg-1(蛋白).在蛋白质浓度为10mg·mL-1时,其热滞活性为0.332℃.此种蛋白的酸性氨基酸含量和亲水性氨基酸含量较高,类似于沙冬青和黑麦的抗冻蛋白.     

沙冬青脯氨酸转运体基因的克隆及表达分析

通过RACE技术从沙冬青中克隆获得了脯氨酸转运体基因Am Pro T(Gen Bank登录号为KJ873133)。序列分析表明,Am Pro T基因开放阅读框为1 329 bp,编码442个氨基酸,预测蛋白质分子量为48.07 k D,等电点为9.32,含有11个跨膜区域,具有典型的脯氨酸转运蛋白的特征。进化分析显示,Am Pro T与大豆Pro T的相似性达到86%。荧光定量PCR分析表明,Am Pro T在地上部的表达量要显著高于地下部,在受到干旱、高盐、脱落酸胁迫后表达量呈上调趋势,推测Am Pro T可能在沙冬青响应干旱、盐等非生物胁迫过程中发挥作用。     

铅污染下芦苇体内铅的分布和铅胁迫相关蛋白

用Pb(NO3 ) 2 10mmol/L处理芦苇幼苗 7d后 ,检测铅在其不同器官、细胞不同区隔和不同化学状态之间的分布以及铅胁迫相关蛋白。结果显示 ,受铅污染后芦苇幼苗根、地下茎、茎和叶片的铅含量增加 ,它们的大小顺序为根 >地下茎 >茎 >叶片。细胞不同部分铅含量的大小顺序为细胞间隙 >细胞壁 >液泡 >细胞质。在根部和叶片中 ,均以活性较低的醋酸可提取态铅和盐酸可提取态铅占优势。在铅胁迫下 ,从芦苇根和叶片中检测出一些铅结合蛋白。另外 ,铅除诱导芦苇合成一种新的不结合铅的蛋白质外 ,还导致一种分子量大约为 72 0 0 0的蛋白质消失。芦苇抗铅胁迫有以下几种机制 :根部比地上部分积累较多的铅 ;铅在体内形成难溶性化合物 ;铅沉淀在质外体内 ;形成铅结合蛋白质和诱导蛋白     

番茄幼苗盐胁迫响应蛋白质组分析

采用营养液栽培,以盐敏感型番茄品种M82为试材,利用双向电泳(2-DE)研究盐胁迫处理下幼苗叶片蛋白质的表达谱,并采用基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF-MS)技术进行差异蛋白质的分离及质谱鉴定。结果表明:(1)盐胁迫处理下,利用2-DE获得差异显著蛋白点20个,其中17个蛋白质点丰度上调表达,3个蛋白质点丰度下调表达。(2)通过质谱分析和蛋白质NCBInr数据库检索,共鉴定出19个差异蛋白,分别为果糖-二磷酸醛缩酶、S-腺苷甲硫氨酸合成酶、甘油醛-3-磷酸脱氢酶等及3个功能未知蛋白;这些鉴定出的差异蛋白质与能量代谢、光合作用、蛋白合成、氧化还原平衡等过程相关,暗示所分离鉴定的蛋白可能参与了番茄的盐胁迫响应,为进一步研究番茄抗逆机制奠定基础。     

Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature

Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expressed in transformed plants. Thermal hysteresis activity (indicating the presence of active AFPs) was present in protein extracts from plants expressing both proteins and was also detected in leaf apoplast fluid from plants expressing AFPs with the signal peptide. Transgenic lines did not demonstrate improved ability to survive freezing when compared to wild-type. However, when cooled under four different regimes, transgenic lines with AFPs in the apoplast fluid froze at significantly lower temperatures than did wild-type, especially in the absence of extrinsic nucleation events.     

Comparative physiological and proteomic response to abrupt low temperature stress between two winter wheat cultivars differing in low temperature tolerance

Abrupt temperature reduction in winter wheat at either autumn seedling stage prior to vernalisation or early spring crown stage can cause severe crop damage and reduce production. Many studies have reported the physiological and molecular mechanisms underlying cold acclimation in winter wheat by comparing it with spring wheat. However, processes associated with abrupt temperature reduction in autumn seedling stage prior to vernalisation in winter wheat are less understood. In this study, physiological and molecular responses of winter wheat seedlings to abrupt low temperature (LT) stress were characterised in the relatively LT‐tolerant winter wheat cultivar Shixin 828 by comparing it with the relatively LT‐sensitive cultivar Shiluan 02‐1 using a combination of physiological, proteomics and biochemical approaches. Shixin 828 was tolerant to abrupt LT stress, while Shiluan 02‐1 exhibited high levels of reactive oxygen species (ROS) and leaf cell death. Significant increases in relative abundance of antioxidant‐related proteins were found in Shixin 828 leaves, which correlate with observed higher antioxidant enzyme activity in Shixin 828 compared to Shiluan 02‐1. Proteomics analysis also indicated that carbohydrate metabolism‐related proteins were more abundant in Shiluan 02‐1, correlating with observed accumulation of soluble sugars in Shiluan 02‐1 leaves. Amino acid analysis revealed a strong response to LT stress in wheat leaves. A negative effect of exogenous sucrose on LT tolerance was also found. This study indicates that high ROS scavenging capacity and high abundance of photosynthesis‐related proteins might play a role in winter wheat response to abrupt LT stress. In contrast, excess accumulation of soluble sugars might be disadvantageous for LT tolerance in the wheat cultivar Shiluan 02‐1.     

Use of intercellular washing fluid to investigate the secreted proteome of the rice–<Emphasis Type="Italic">Magnaporthe</Emphasis> interaction

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice–Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.     

Reproductive Phenology of the Brown Alga Analipus japonicus in Peter the Great Bay (Sea of Japan)

The alga Analipus japonicus (Harv.) Wynne displays a distinct seasonal pattern in its development in Peter the Great Bay (Sea of Japan). In winter and spring, it occurs only in the form of basal crusts, and vertical axes develop in the summer–autumn period. It reproduces mostly asexually from July to November. Algae with unilocular sporangia occur very seldom, only in June and July.     

Accumulation and Loss of Nitrogen in White Clover (Trifolium repens L.) Plant Organs as Affected by Defoliation Regime on Two Sites in Norway

In order to improve the basis for utilising nitrogen (N) fixed by white clover (Trifolium repens L.) in northern agriculture, we studied how defoliation stress affected the N contents of major plant organs in late autumn, N losses during the winter and N accumulation in the following spring. Plants were established from stolon cuttings and transplanted to pots that were dug into the field at Apelsvoll Research Centre (60°42′ N, 10°51′ E) and at Holt Research Centre (69°40′ N, 18°56′ E) in spring 2001 and 2002. During the first growing season, the plants were totally stripped of leaves down to the stolon basis, cut at 4 cm height or left undisturbed. The plants were sampled destructively in late autumn, early spring the second year and after 6 weeks of new spring growth. The plant material was sorted into leaves, stolons and roots. Defoliation regime did not influence the total amount of leaf N harvested during and at the end of the first growing season. However, for intensively defoliated plants, the repeated leaf removal and subsequent regrowth occurred at the expense of stolon and root development and resulted in a 61–85% reduction in the total plant N present in late autumn and a 21–59% reduction in total accumulation of plant N (plant N present in autumn + previously harvested leaf N). During the winter, the net N loss from leaf tissue (N not recovered in living nor dead leaves in the spring) ranged from 57% to 74% of the N present in living leaves in the autumn, while N stored in stolons and roots was much better conserved. However, the winter loss of stolon N from severely defoliated plants (19%) was significantly larger than from leniently defoliated (12%) and non-defoliated plants (6%). Moreover, the fraction of stolon N determined as dead in the spring was 63% for severely defoliated as compared to 14% for non-defoliated plants. Accumulation in absolute terms of new leaf N during the spring was highly correlated to total plant N in early spring (R² = 0.86), but the growth rates relative to plant N present in early spring were not and, consequently, were similar for all treatments. The amount of inorganic N in the soil after snowmelt and the N uptake in plant root simulator probes (PRS^TM) during the spring were small, suggesting that microbial immobilisation, leaching and gas emissions may have been important pathways for N lost from plant tissue.     

接种土壤微生物对铜胁迫下海州香薷生长及光合生理的影响

采用框栽试验方法,模拟Cu胁迫条件下,探讨接种土壤微生物对海州香薷(Elsholtzia splendens)生长和光合生理的影响。结果表明:(1)在Cu胁迫下,海州香薷株数、株高、基径、生物量、茎重比均显著低于对照;与Cu胁迫相比,接种土壤微生物能显著缓解Cu胁迫对海州香薷生长的抑制作用,使植株的株数、株高、生物量、茎重比显著提高。Cu胁迫下,接种土壤微生物均降低了植株体内不同器官Cu含量,茎和叶Cu的累积量显著减少,但对其它器官的Cu含量影响不显著。(2)秋季,各处理的海州香薷的净光合速率(Pn)日变化均呈"单峰"曲线,接种土壤微生物显著提高了Cu胁迫下海州香薷的日均Pn、日均蒸腾速率(Tr),而日均气孔导度(Gs)、日均胞间CO2浓度(Ci)显著降低。(3)Cu胁迫下,接种土壤微生物显著提高了植株的最大净光合速率(Pnmax)、光饱和点(LSP)、表观量子效率(AQY)、最大羧化速率(Vcmax)、最大电子传递速率(Jmax)、磷酸丙糖利用率(TPU),且使光补偿点(LCP)显著降低。表明接种土壤微生物通过提高光能利用率、利用弱光和碳同化能力来增强光合作用能力及有机物的积累,缓解Cu胁迫对海州香薷的毒害。因此,接种土壤微生物可促进Cu胁迫下海州香薷的生长,在重金属污染土壤的植物修复中具有较好的应用潜力。     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Diet composition of the juvenile chub mackerel (Scomber japonicus) in the Aegean Sea (Izmir Bay, Turkey)

Stomach contents of 296 juvenile chub mackerel (Scomber japonicus Houttuyn, 1782) specimens were examined based on samplings carried out in Izmir Bay (Aegean Sea, Turkey) during 2001. In terms of percentage weight (W%), fishes were the main food during summer and autumn. Thaliaceans (Salpa sp.) constituted the most important food source in winter, whereas planktonic crustaceans (Amphipoda, Copepoda) were the main prey during spring. According to the Bray–Curtis similarity index, diet of the chub mackerel was 64.1% similar during the summer, winter and autumn seasons.     

不同水深条件下刺参养殖池塘大型底栖动物群落结构季节性变化特征

于2012年7月至2013年4月调查了荣成靖海湾3个不同水深的刺参(Apostichopus japonicus)养殖池塘内大型底栖动物的构成,以了解不同水深对刺参养殖池塘环境条件的影响以及由此引起的大型底栖生物群落结构的改变。结果表明:3个不同水深梯度池塘(1#浅水位、2#正常水位和3#高水位)底部光照强度、叶绿素a(Chla)和总有机物(TOM)含量存在显著差异,各池塘水温差异不显著。光强、Chla和TOM含量在夏季、冬季和春季均表现为1#池塘显著高于3#池塘;秋季各池塘间光强和TOM含量差异不显著,Chla含量则表现为3#池塘显著高于1#池塘。各季节3个池塘间大型底栖动物在种类组成、丰度、生物量和多样性指数上均存在显著性差异。大型底栖动物丰度和生物量夏季均表现为1#池塘显著高于3#池塘,秋季和冬季则相反;春季1#池塘丰度显著高于3#池塘,生物量则差异不显著。这些差异主要与其各自优势种及其优势度指数大小有关。大型底栖动物多样性指数夏季和秋季均表现为1#池塘高于3#池塘,春季则相反,冬季各池塘间多样性指数差异不显著。单因子相似性分析(ANOSIM)表明,各季节3个池塘间大型底栖动物群落结构均存在显著差异,表明水深梯度对刺参养殖池塘大型底栖动物群落结构造成显著性影响。相似性百分比分析(SIMPER)显示,各季节对3个池塘间大型底栖动物群落差异起主要作用的物种为各个池塘的优势种。典范对应分析(CCA)表明,水深、Chla和TOM含量为影响大型底栖动物群落的主要环境因子。