青杨(Populus cathayana Rehd.)对逆境胁迫的反应及lea基因的表达分析

杨树具有分布广、适应性强，在生态环境治理和解决木材短缺方面均占有重要位置。青杨(Populus cathayana Rehd.)是青杨派树种的重要成员之一，也是我国的特有种。本研究通过对不同水分梯度的干旱胁迫下青杨形态和生理生化的反应，不同pH值盐碱胁迫下不同海拔和不同气候地区的四个青杨种群在生理生态上的反应差异，以及在干旱和低温胁迫下青杨lea2, lea3组基因表达差异的研究，从形态、生理、生化和分子生物学水平系统地研究了青杨在不同逆境胁迫下的反应和青杨不同种群在盐碱胁迫下的反应差异。主要研究结果如下： 1. 青杨在干旱胁迫下的反应机制：中度和重度干旱胁迫下植株的生长受到明显抑制。表现在光合系统上青杨的净光合同化速率(A)下降，主要原因是气孔导度(gs)，胞间二氧化碳浓度(Ci)下降。另外最大量子产量(Fv/Fm)、光化学猝灭效率(qP)降低反应了干旱胁迫下光合系统II(PSII)受到严重损伤, 而且非光化学猝灭效率(qN)上升，导致可利用化学能产量下降，叶绿体产生淀粉的量减少。qP降低qN上升导致产生的过量电子对光合系统的伤害造成活性氧以及丙二醛(MDA)的含量增加。超微解剖结构显示，干旱胁迫增强时，叶绿体内淀粉粒的数目减少，而且叶绿体、线粒体等细胞器中嗜锇颗粒的数目增加。为清除细胞内的活性氧，植物一般的反应是抗氧化系统酶活性增加，对青杨来讲超氧化物歧化酶(SOD), 抗坏血酸过氧化物酶(APx)活性的增加远大于过氧化物酶(POD)，这显示了在青杨中SOD、APx酶在清除活性氧的作用上大于POD。另外同工酶研究结果显示这些酶活性的升高主要是由于各条同工酶带表达量的增加，而不是诱导新酶带的产生。另外，75% FC水分处理下有些指标非但没有下降，像A和有效光量子产量(Y)的值都略有增加，而且gs同时增加。另外，100% FC比75% FC细胞内淀粉粒的数目少一些，但有少量的嗜锇颗粒。这证明100% FC土壤水分也许并非最适合青杨生长。 2. 盐碱胁迫对不同海拔地区青杨种群的反应差异：青杨高海拔和低海拔种群的各种生理特性随着pH值上升都受到了很大的影响。两种群叶和根中Na+、K+ 含量, Na+/K+比率随着pH值的上升影响显著。在pH值高于10.4时高海拔种群叶和根中Na+/K+比率急剧下降但是低海拔种群中却一直维持在较高水平。两种群中MDA、脯氨酸(Proline)的含量，抗氧化系统酶的活性都受到了严重的影响，证明两个种群都属于盐碱胁迫敏感类型但是高海拔的种群对盐碱胁迫的耐性要高于低海拔。这主要是由于高海拔种群一般具有耐干旱、低温胁迫的能力，而植物的抗逆机制一般都有共通之处。 3. 盐碱胁迫对不同气候地区青杨种群的反应差异：盐碱胁迫下两种群的光合作用受到明显的抑制，具体表现在叶绿素的含量和A 显著下降。净光合速率的下降主要是由于叶片gs，Ci 值降低引起的。与湿润地区的种群相比盐碱胁迫增强时，干旱地区的种群叶绿素含量和光合能力的升高与K+离子含量增加有关。植物维持细胞质高K+/Na+值对植物的抗盐性有很重要的作用。为清除盐碱胁迫产生的活性氧，抗氧化系统酶活性增加。盐碱胁迫下干旱地区的种群在SOD、CAT 和谷胱甘肽还原酶(GR)等酶的活性均显著上升，而湿润地区种群只有谷胱甘肽氧化酶(GST)的活性明显增加，说明干旱种群的抗氧化酶系统在较高盐碱胁迫下的保护作用要强于湿润种群。这主要是由于植物抗盐碱胁迫与抗干旱胁迫在一些方面的机制是一致的，抗旱种群一般也能抵抗一定程度的盐碱胁迫。 4. 青杨lea2、lea3 基因在干旱和低温胁迫下的表达差异：通过荧光定量PCR 分析，lea2、lea3 组基因在干旱和低温胁迫下在mRNA 水平的瞬时表达量明显升高，说明了两基因在青杨耐干旱和低温胁迫上都起显著的作用。而且两基因在干旱胁迫下，表达量的升高和降低的时间近乎同步，表明两基因在干旱胁迫下对植物应急保护机制的启动都发挥着重要的作用。低温胁迫下lea3 基因在mRNA 水平上表达量显著上升的时间要早于lea2，而且lea3 基因的持续作用时间明显长于lea2 组基因，说明了低温胁迫开始时lea3基因在植物应对逆境的作用上要大于lea2 基因。 Poplars play an important role in lumber supply, and are important components of ecosystems due to their wide distribution and well adaptation. Populus cathayana Rehd., which belongs to Populus Sect. Tacamahaca Spach, is one of the most important resources of poplars and is specialist to china. In this study, different altitudes and climates populations of P. cathayana were used as experiment materials to investigate the adaptability to drought and salt-alkali stresses. And the cultures of P. cathayana were used to analyze the lea2 and 3 group genes expression when exposed to drought and low temperature stresses. The results are as follows: 1. A large set of parallel responses to drought stress: Drought stress caused pronounced growth inhibition. A decreased significantly and was mainly the result of gs and Ci down. Besides, Fv/Fm, qP decreased and that reflected the harmful effects to PSII of drought stress. In accordance with qN increasing, decreased useful energy production caused the starch numbers reduction in chloroplast. The qP up and qN down improved the levels of ROS and MDA. Starch numbers in chloroplast reduced and plastoglobuli numbers increased when soil water content decreased. To reduce ROS, the activities of SOD, APX, CAT and PPO were activated. The isozymes results show that the rising activities of the antioxidant enzymes resulted from certain isoform content increased, and not from the new band produced. Interestingly, morphological results show 100%FC maybe wasn’t the favorite water content for P. cathayana growth. 2. Effect of salt-alkali stress on morphological and physiological changes in two different altitudes populations of P. cathayana: We compared the physiological responses of two populations of Populus cathayana Rehder, originating from altitudes 2,840 m and 1,450 m. Our results demonstated that Na+ and K+ contents, and Na+/K+ ratios in leaves and roots are greatly affected by pH values. At pH 10.4, the Na+/K+ ratios in both leaves and roots sharply dropped in the higher altitude population but were always maintained at higher levels in the lower altitude population. The pH values causing maximum malondialdehyde (MDA) level, free proline content and antioxidant enzyme activities were significantly different in two populations. These results indicated that the higher altitude population exhibits greater tolerance to alkalinity stress than does the lower altitude population. 3. Morphological and physiological changes in two different climates populations of P. cathayana when exposed to salt-alkali stress. Salt-alkali stress caused pronounced inhibition of the growth and especially in photosystem. Pigments content and A decreased significantly and at the same time gs and Ci decreased too. Compared with wet climate population, the Chlorophyll content and A increased in drought climate population as pH value rising was related to the K+ content increasing. It is important to resist salt-alkali stress that the K+/Na+ ratio matained at high level in cytoplasm. To reduce ROS content, the SOD, CAT and GR activities rised significantly in drought population but only GST increased in wet population. The drought population showed higher salt-alkali tolerance than the wet population mainly resulted from the fact that drought tolerance was in accordance with salt-alkali tolerance to some extent. 4. The different expressional model of lea2 and lea3 gene when P. cathayana was exposed to drought and cold stress. RT-PCR results show both lea2 and lea3 suddenly expressed significantly in mRNA level under drought and cold stress. The expression level of two genes reached optimal level at the same time. But under cold stress, the earlier significantly rising expressional time and the longer maintained higher level time in lea3 than lea2 elucidated that lea3 may be more important than lea2 in resisting cold stress in short time in P. cathayana.

青杨(Populus cathayana Rehd)雌雄植株对锈病和干旱胁迫的生理响应及差异蛋白质组比较

雌雄异株植物对环境的不同响应一直是一个有趣而新颖的研究领域，由于雌雄个体不同的繁殖成本及不同的生存策略，使得雌雄植株在生长、存活、生殖格局、空间分布、资源配置等方面已经表现出明显的不同，在生理和分子水平上也表现出明显的性别间差异。干旱是制约农林业发展的环境因子之一，叶锈病是对杨树危害最严重的病害之一，由于长期进化的结果，不同性别的植物必然对生物和非生物胁迫有着不同的响应。本文以雌雄异株的青杨为模式植物，研究雌雄间在生理、生化、亚细胞结构和蛋白质水平上对生物和非生物胁迫的差异响应。主要研究结果如下： （1） 青杨雌雄植株对锈病胁迫的生理生化差异响应 在正常的对照组中，雄株叶片比雌株叶片有着较高的活性氧自由基产生速率、较高的SOD、POD、PPO 和较低的CAT 活性；在锈病感染的早期阶段， SOD、POD、CAT 活性、活性氧自由基产生速率、H2O2 含量、膜脂过氧化程度和细胞膜的电渗率在雌雄株中都增加，而PPO 仅在雄株中增加明显，APX 仅在雌株中增加明显，并且雌株比雄株有着更严重的锈病感染程度、细胞膜的伤害程度和光合系统II 的破坏程度，雌株有更多的净光合速率、气孔导度和叶绿素a 含量的降低，在同工酶变化上，雌雄间对锈病也显示出不同的表达模式。结果显示，雄株比雌株对锈病有着更好的抗性和更有效的ROS 清除系统。 （2） 青杨雌雄植株对干旱胁迫的生理生化及亚细胞结构的差异响应 与较好水分条件相比，干旱下雄株比雌株有着更高的A-Ci 响应参数，如Rubisco 最大羧化速率、光呼吸速率、暗呼吸速率和最大电子传递速率等。干旱显著地增加了膜脂过氧化程度和游离脯氨酸含量，并且雄株比雌株表现出较低的膜脂过氧化程度，较高的总蛋白和游离脯氨酸含量。无论是中度干旱还是极度干旱，除了CAT 外，雄株比雌株表现为较强的抗氧化酶活性，在同工酶谱带上，雌雄间表现出不同的变化模式，并且有些条带是干旱影响应的，而有些条带是性别特异性的，这些性别特异性条带能够作为鉴定性别快速而准确的标记。干旱显著地影响了线粒体、叶绿体和细胞壁的结构，尤其在中度干旱胁迫下，雄株线粒体和叶绿体比雌株呈现出较好的完整性，并且雄株细胞壁要比雌株更厚。因此， 雄株比雌株表现出更强的干旱忍耐性和更高效的抗氧化酶系统。 （3） 青杨雌雄植株对干旱胁迫的蛋白质组差异响应 用双相电泳检测到雌雄间近1000 个蛋白点，通过对比发现对照组雌雄间有54 个差异蛋白点，干旱下雌雄间有108 个差异点，其中102 个被质谱成功鉴定。对照组雌雄间的差异蛋白主要集中在与光合作用相关蛋白、抗氧化酶、胁迫防御蛋白和一些调节基因表达的蛋白；干旱胁迫下雌雄间差异蛋白明显增多，主要有参与信号转导、调节基因表达、蛋白质加工、转录产物的转录翻译后修饰的调节性蛋白蛋白和参与氧化还原平衡、抗胁迫、细胞壁合成、光合作用、能量代谢、氨基酸代谢和脂肪酸代谢等的功能性蛋白。干旱下这些蛋白的表达量在雌雄中有的表现出相同的表达模式，如干旱下雌雄株中Rubisco 激活酶、小热激蛋白等表达都增加，而有的表现出相反的表达模式，如Rubisco 大亚基的降解片段、羰酸酯酶等在雄株中表达量上调而在雌株中却是下调。因此，雌雄间在蛋白质水平上对干旱胁迫响应的差异是显著的，也是复杂的。 It is an interesting and novel topic that dioecious plants possess different responses to environmental stress. As for the different productive cost and different survive strategy, different sexual plants have shown obviously morphological, physiological and molecular differences. Drought is one of the most worldwidely important environmental stress factors that limit plant growth and ecosystem productivity. Rust disease is one of the economically important diseases in many trees. As a result of the long evolutionary process, male and female plants should show different responses to abiotic and biotic stress. In this paper, using a dioeious tree of Populus cathayana Rehd as a model, we study the sexual differences to drought and rust disease stress in physiological, biochemical, sub-cellular and proteomics levels. The main results are follows: (1) The sexual differences in physiology and biochemistry of poplar to rust disease In controls, males showed higher production of superoxide radicals, higher activities of SOD, POD, PPO and lower CAT activity. Under rust disease, the activities of antioxidant, the content of ROS and the degree of cellular member destroyed were increased in both sexes, except for PPO in diseased males and APX in diseased females. However, females showed more seriously disease severity and cellular member and PS II destroyed degrees. Net photosynthesis rate, transpiration rate and chlorophyll a content were decreased more in diseased females than in males. There were also some different changes inantioxidant isozymes under rust disease. The results suggested that male poplar possessed a more effectively antioxidant system and were more resistant to rut disease than females. (2) The sexual differences in physiology and biochemistry of poplar to drought stress Under drought stress, there were higher rates of RuBP-saturated CO2 assimilation, dark respiration, photorespiratory release of oxygen, the max electron transportrate in CO2-saturated and carboxylation efficiency in males than in females. And males showed lower TBARS and higher proline content. Except for CAT, the activities of other antioxidants were higher in males than in females. Meanwhile, there were obviously differences in isozyme changes between teo sexes. Drought stress obviously destroyed the integralities of chloroplasts and mitochondria and the sexual differences in sub-cellular level were obviously under the moderate water stress. Male cell walls were more sensitive to drought stress than did female. The results suggested males were more resistant to drought stress. (3) The sexual differences in proteomics of poplar to drought stress By 2-D and MS analysis, we identified 102 different protein spots between males and females. Under control conditions, the different proteins were mainly in photosynthesis related proteins, antioxidants, stress response proteins and some gene expression related proteins. Under drought stress, the different proteins were focused on (i) regulated proteins such as signaling conduction, kinase, HSP, gene expressional regulation and protein modification, (ii) functional proteins such as photosynthesis, energy metabolism, antioxidant, redox, stress response, lipid metabolism and amino acid metabolism. Some protein showed the same expressional pattern, while some showed contrary expressional pattern. Thus, the results suggested that sexual differences in proteomics were significant and complex.

X射线致HeLa细胞损伤过程中NADPH氧化酶的作用

以HeLa细胞为实验材料,探讨了NADPH氧化酶在X射线诱导细胞损伤过程中的作用。结果显示,12Gy X射线辐照后细胞内活性氧(ROS)明显增加,在用NADPH氧化酶抑制剂处理后再辐照,则细胞内ROS降低到未辐照水平;同时辐照后NADPH氧化酶细胞质亚基p47phox在细胞质积聚并和细胞膜亚基gp91phox结合;Western blotting检测结果显示,NADPH氧化酶的关键亚基gp91phox的表达量明显增加。以上结果说明,NADPH氧化酶可以被X射线激活,由其介导产生的ROS在X射线诱导HeLa细胞损伤过程中扮演重要角色。

培基介导的辐射旁效应及机理初探

采用ICR小鼠,转移受照射细胞的条件培养液来培养未受照射细胞,检测YAC?I(小鼠T淋巴瘤)细胞对小鼠自然杀伤(NK)细胞的敏感性变化,测定小鼠脾细胞增殖能力,观察自由基清除剂二甲基亚砜(Dimethylsulfoxide,DMSO)照射前处理细胞的影响。初步研究了电离辐射旁效应(Bystandereffect,BE)及其可能的机理。结果显示,条件培养液培养的Yac?I细胞对NK细胞的敏感性明显升高。用条件培养液培养的小鼠脾细胞的增殖能力也明显受到抑制,提示辐射旁效应的存在,观察到DMSO对旁效应有一定的抑制作用,说明活性氧(Reactiveoxygenspecies,ROS)等自由基对旁效应有一定贡献。然而在淋巴细胞增殖实验ICM+1%DMSO组的数据显示DMSO对旁效应的抑制作用非常有限,表明除自由基外必定存在其他因素介导旁效应的发生。

用培养基介导法研究辐射旁效应及其机理

目的通过受照射的细胞培养液培养靶细胞,并用二甲基亚砜(DMSO)对靶细胞预处理,检测其对自然杀伤(NK)细胞活性的影响,研究旁效应是否存在及其产生机理。方法以健康雄性ICR小鼠脾NK细胞活性检测经过不同剂量照射的细胞培养液对小鼠淋巴瘤YacⅠ细胞的损伤程度,同时观察DMSO对该细胞的保护作用,即对旁效应的抑制作用。结果应用不同剂量60Coγ射线照射的细胞培养液来培养YacⅠ细胞,引发该细胞不同程度的损伤,表现在YacⅠ细胞对小鼠脾NK细胞的敏感性增强(P<0.01);DMSO预处理细胞对旁效应有一定的抑制作用,特别在0.25和0.5Gy组,对细胞的保护作用明显。结论60Coγ射线照射能诱发YacⅠ细胞的培养液介导的旁效应。活性氧(ROS)是诱发辐射旁效应的因素之一,清除ROS可部分抑制辐射旁效应。

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane Subunit gp91(phox) was dose-dependent. Meanwhile, the cytoplasmic subunit p47(phox) was translocated to the cell membrane and localized with p22(phox) and gp91(phox) to form reactive NADPH oxidase. Our data Suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O-2(-) (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 mu M) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm(2) strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm(2)). Also, H2O2 (200 similar to 500 mu M) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O-2(-) and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O-2(-) mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.

辐照诱导的人正常肝细胞系HL-7702细胞远后效应

近年来，放射治疗在肿瘤治疗中的作用受到了越来越多的重视，放疗技术和手段的迅速发展为人们选择放疗创造了更多的机会[1]。放疗是利用电离辐射对细胞，特别是细胞中的遗传物质DNA的损伤作用，诱发病灶部位不正常细胞的凋亡和坏死来治疗肿瘤的。即使制定了周密的放疗方案和计划，放疗过程中难免也会对正常组织和细胞造成一定程度的损伤，而且辐射的远后效应和旁效应的发现，也使得放射治疗肿瘤的安全性受到了很大的关注。要想充分发挥放疗的优势，而又使其对人体正常组织的毒副作用尽可能降低，就必须搞清楚电离辐射对细胞造成的各种损伤的类型及其分子机理。自从电离辐射的远后效应和旁效应被发现以来就一直是辐射生物医学领域的研究热点，国内外的科研人员进行了大量的实验试图解释它们的本质，可是几十年来相关领域的研究一直局限于细胞学的水平，在向分子水平前进的道路上遇到了巨大的困难[2,3]。目前对于电离辐射远后效应和旁效应的研究是相对独立进行的，很少有探讨二者相互关系的论文发表。本文试图在总结前人科研成果并结合对自己所取得的实验数据进行分析的基础上对电离辐射的远后效应、旁效应、基因不稳定性与电离辐射所产生的活性氧自由基的关系进行初步的探讨。实验方法与结果： 1.用X射线辐照人正常肝细胞系HL-7702细胞，运用胞质分离阻滞微核实验检测细胞微核率，AnnexinV-FITC细胞凋亡检测试剂盒检测细胞凋亡率，细胞微核率和凋亡率随着辐照剂量的增加而显著增加。X射线照射后细胞继续传代培养，第七代时不同剂量辐照后子代细胞微核率和凋亡率同未辐照细胞的微核率和凋亡率相比已经没有明显区别。对不同剂量辐照后传代七代的细胞再次照射2.5Gy相同的剂量，发现受初次不同剂量辐照的细胞其微核率和凋亡率再次出现明显差异，初次辐照剂量高的细胞再次相同剂量辐照后的微核率和凋亡率也高。 2.对不同剂量X射线辐照后的细胞继续传代到第十五代时用H2O2浓度为1ul/ml的培养基处理15min，发现受初次不同剂量辐照的细胞其微核率再次出现比较明显差异，初次辐照剂量高的细胞H2O2处理后的微核率也高。 3.对受到不同剂量X射线辐照的人正常肝细胞存活后代进行二次辐照，分两组分别给予1.5GyX射线和1Gy碳离子束辐照，并检测二次辐照后细胞的微核率，发现重离子束二次辐照后并不像X射线一样可以诱发初次X射线辐照造成的损伤信息的表达，而只是表现出二次重离子辐照所造成的损伤。结论： 1.X射线辐照导致了HL-7702细胞基因组不稳定性这一辐射远后效应，X射线二次辐照辐照存活细胞的子代细胞可以诱发辐射的远后效应（如基因组不稳定性）明显的表现； 2.X射线辐照导致的HL-7702细胞后代基因组不稳定性，可以通过H2O2处理而得以诱发，揭示电离辐射过程中产生的氧自由基可能与辐射的远后效应存在密切的联系； 3.电离辐射产生的ROS在诱发细胞产生微核中具有重要的作用，但是并非唯一的影响因素。 4.X射线和12C6+重离子束辐照后存活细胞的后代中的细胞损伤类型可能存在着本质上的区别

辐射诱导的HeLa细胞旁效应信号通路研究

随着肿瘤认识的不断深入，肿瘤外科学、肿瘤放射治疗学、肿瘤化学治疗学构成了现代肿瘤治疗学的三大支柱，而放射治疗学的研究对于肿瘤的治疗有重要的临床意义。本文通过对HeLa进行辐射来观察其产生的旁效应信号通路，从而对临床工作的起到一定的帮助。辐照过的细胞通过释放信号分子引起周围未辐照细胞产生一系列的生物学反应的现象，称之为辐射诱导的旁效应。活性氧（Reactive oxygen species, ROS）和一氧化氮（Nitric oxide, NO）是辐射诱导的旁效应信号通路中的两个重要信号分子。本文研究了这两种重要的信号分子在辐射诱导的HeLa细胞旁效应信号通路中的关系。通过微核实验，我们发现X射线以及12C6+ 辐照过的HeLa细胞及其旁观者细胞的微核形成明显增加，而1%的二甲亚砜（Dimethyl sulfoxide，DMSO，ROS清除剂）预处理X射线辐照的细胞则显著抑制了受辐照细胞及其旁观者细胞的微核形成。1 Gy的X射线辐照能够抑制细胞的增殖速率而0.5%和1%的DMSO预处理则能减少X射线的增殖抑制作用，并且DMSO预处理的效果与浓度有关：1%的DMSO比0.5%的DMSO处理更大程度的恢复了受辐照细胞的增殖速率。另一方面，接受条件培养基（Conditioned medium）的旁观者细胞的增殖速率增加，而DMSO预处理产生条件培养基的受辐照细胞则使旁观者细胞的增殖速率降低，且DMSO预处理的效果同样与其浓度相关：浓度越高，条件培养基的刺激生长作用越小。Western blotting和DAF-FM DA荧光探针检测分别显示了辐照过后细胞的诱导型一氧化氮合酶（Inducible nitric oxide synthase, iNOS）和NO水平均升高，而DMSO预处理则降低其水平。因此，我们推测在X射线辐照的HeLa细胞旁效应信号通路当中ROS是NO的上游信号。另外，我们采用培养基转移后立即加DMSO或BMS－345541（IKK/NF-κB抑制剂）的方法研究了旁观者细胞当中的旁效应信号通路。我们发现DMSO和BMS－345541均显著抑制了旁观者细胞的NO水平。因此，在旁观者细胞当中ROS与NF-κB均为NO的上游信号

CO_2和O_3浓度升高对春小麦活性氧代谢及抗氧化酶活性的影响

【目的】揭示CO2和O3浓度升高及其复合作用对植物活性氧(ROS)代谢及抗氧化酶活性的影响机理。【方法】以春小麦(Triticum aestivum L.)为试材,利用开顶式气室(OTCs)研究CO2和O3浓度升高及其复合作用下,春小麦叶片膜脂过氧化程度,活性氧产生速率、含量及抗氧化酶活性的变化。【结果】在整个生育期内,与对照相比,高浓度CO2[(550±20)μmol·mol-1]处理下,春小麦叶片相对电导率、MDA含量减小,产生速率、H2O2含量下降,SOD、CAT、POD和APX活性增强;而在O3浓度为(80±10)nmol·mol-1的条件下,春小麦叶片相对电导率、MDA含量增大,产生速率、H2O2含量升高,SOD、CAT、POD和APX活性总体上有所减弱;CO2和O3浓度升高复合[(550±20)μmol·mol-1+(80±10)nmol·mol-1]处理下,春小麦叶片MDA含量、产生速率和SOD活性总体上低于对照,而相对电导率、H2O2含量以及CAT、POD和APX活性总体上增加。【结论】CO2浓度升高抑制了春小麦叶片活性氧的代谢速率,提高了抗氧化酶的活性,对春小麦表现为保护效应,而O3浓度升高促进了春小麦叶片活性氧的代谢速率,降低了抗氧化酶的活性,对春小麦表现为伤害效应。在CO2和O3浓度升高复合处理下,CO2浓度升高在一定程度上缓解了O3浓度升高对春小麦的伤害效应,而O3浓度升高亦在一定程度上削弱了CO2浓度升高对春小麦的保护效应。

CO_2和O_3浓度升高及其复合作用对玉米(ZeamaysL.)活性氧代谢及抗氧化酶活性的影响

为了揭示CO2和O3浓度升高及其复合作用对植物活性氧(ROS)代谢及抗氧化酶活性的影响机理,以玉米(Zea maysL.)为研究材料,利用开顶式气室(OTCs)研究了CO2和O3浓度升高及其复合作用下,玉米叶片活性氧产生速率、含量,膜脂过氧化程度,抗氧化酶活性,净光合速率及玉米籽粒产量的变化。结果表明,在整个生育期内,与对照相比,高浓度CO2((550±20)μmo.lmol-1)处理下,玉米叶片净光合速率升高,O2-.产生速率、H2O2含量下降,MDA含量、相对电导率减小,SOD、CAT、POD活性增强,玉米百粒重和穗粒数增加;而在O3浓度为(80±10)nmo.lmol-1的条件下,玉米叶片净光合速率下降,O-2.产生速率、H2O2含量升高,MDA含量、相对电导率增大,SOD、CAT、POD活性减弱,玉米百粒重和穗粒数降低;CO2和O3浓度升高复合((550±20)μmo.lmol-1+(80±10)nmo.lmol-1)处理下,玉米叶片的净光合速率、H2O2含量、SOD活性先升高后降低,MDA含量、相对电导率、CAT活性增加,POD活性减弱,而O-2.产生速率几乎不变化,且玉米的百粒重和穗粒数略低于对照。以上结果说明,CO2浓度升高抑制了玉米叶片活性氧的代谢速率,提高了抗氧化酶的活性,从而增强了光合作用,使玉米籽粒产量增加,对玉米表现为保护效应,而O3浓度升高促进了玉米叶片活性氧的代谢速率,降低了抗氧化酶的活性,抑制了光合作用,使玉米籽粒产量下降,对玉米表现为伤害效应。在CO2和O3浓度升高复合处理下,CO2浓度升高在一定程度上缓解了O3浓度升高对玉米的伤害效应,而O3浓度升高亦在一定程度上削弱了CO2浓度升高对玉米的保护效应。

O_3和CO_2浓度升高对油松针叶抗氧化系统的影响

利用开顶箱模拟大气O3与CO2浓度升高,对油松进行了连续4个月的熏蒸实验,探讨了油松针叶抗氧化系统化系统的响应。结果表明:1)高浓度O3显著增加了油松针叶过氧化氢的积累,到处理后期过量的过氧化氢显著地抑制了抗氧化酶活性,如SOD和APX,并且抗坏血酸被耗竭,加剧了膜质过氧化,最终导致了严重氧化伤害;2)高浓度CO2处理中油松针叶抗氧化酶活性普遍低于对照,ASA含量显著高于对照,可能是高CO2浓度促进ASA合成,或者是ASA的消耗减少,到处理后期使H2O2含量比对照降低了15.5%,从而减轻了膜质过氧化产物丙二醛含量,减轻了氧化伤害;3)与O3单因素相比,在协同处理中油松针叶具有较高的抗氧化酶活性和ASA含量,说明高CO2浓度减轻了高O3对抗氧化酶活性的抑制作用,并且提高了针叶内ASA含量,增强了针叶的抗氧化能力,有效地控制了ROS的产生与清除平衡,缓解了高O3带来的氧化伤害。

臭氧浓度升高对玉米活性氧代谢及抗氧化酶活性的影响

为了揭示臭氧(O3)浓度升高对玉米活性氧(ROS)代谢及抗氧化酶活性的影响机理,以玉米(ZeamaysL.)为研究材料,利用开顶式气室(OTC)研究了臭氧浓度升高处理[(80±10)nmol·mol-1]下,玉米叶片活性氧产生速率、含量及抗氧化酶活性的变化。结果表明,与对照相比,高浓度臭氧处理下,抽雄期和灌浆期玉米叶片中超氧阴离子(O)产生速率显著增加(P<0.05),过氧化氢(H2O2)含量极显著增加(P<0.01)。处理至灌浆期时,丙二醛(MDA)含量增加了22.6%,显著高于对照(P<0.05),而相对电导率在抽雄期和灌浆期均极显著高于对照(P<0.01),与对照相比,分别增加了20.0%和26.3%,表明细胞膜受损严重。在各个生育时期内,臭氧处理玉米叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性均低于对照,表明臭氧浓度升高能够抑制玉米叶片抗氧化酶的活性。本试验中抗氧化酶活性在整个处理期间呈现出逐渐升高的趋势,说明玉米植株对O3胁迫具有保护性机制,在一定程度上缓解臭氧对其造成的伤害。!2

Stress Resistance and Disease Resistance in Seaweeds:The Role of Reactive Oxygen Metabolism

It has become clear that the last 15-20 years that the immediate effect of a wide range of environmental stresses,and of infection,on vascular plants is to increase the information of reactive oxygen species(ROS) and to impose oxidative stress on the cells.Since 1994,sufficient examples similar responses in a broad range of marine macroalgae have been decribed to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond(and become resistant) to stress and infection.Desiccation,freezing,low temperatures,high light,ultraviolet radiation,and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen.The response to other stresses (infection or oligosaccharides which signal that infection is occurring,mechanical stress,hyperosmotic shock) is quite different-a more rapid and intence,but short-lived production of ROS ,discribed as an "oxidative burst"-which is attributed to activation of NADPHoxidases in the plasma membrane.Seaweed species that are able to survive such stresses or resist infection have the capacity to remove the ROS through a high cellular content of antioxidant compounds,or a high activity of antioxidant enzymes.

Protective immunity induced by CpG ODNs against white spot syndrome virus (WSSV) via intermediation of virus replication indirectly in Litopenaeus vannamei

The worldwide shrimp culture is beset with diseases mainly caused by white spot syndrome virus (WSSV) and suffered huge economic losses, which bring out an urgent need to develop the novel strategies to better protect shrimps against WSSV. In the present study, CpG-rich plasmid pUC57-CpG, plasmid pUC57 and PBS were employed to pretreat shrimps comparatively to evaluate the protective effects of CpG ODNs on shrimps against WSSV. The survival rates, WSSV copy numbers, and antiviral associated factors (Dicer, Argonaute, STAT and ROS) were detected in Litopenaeus vannamei. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in pUC57-CpG-pretreatment shrimps than those in pUC57- and PBS-pretreatment shrimps after WSSV infection. The Argonaute mRNA expression in pUC57-CpG-, pUC57- and PBS-pretreatment shrimps after WSSV infection was significantly higher than that of shrimps post PBS stimulation on the first day. The ROS levels in pUC57-CpG-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection on the first day. These results together demonstrated that pUC57-CpG induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei. (C) 2009 Elsevier Ltd. All rights reserved.