利用氧化剂甲基紫精筛选拟南芥寿限延长突变体

在模式动物中的研究表明,寿限延长与氧化胁迫耐受能力密切有关;在模式植物拟南芥中也发现晚花突变体具有更强的抗氧化胁迫能力,但迄今为止未见有关拟南芥寿限延长突变体的研究报道。本文建立了用氧化剂甲基紫精筛选寿限延长突变体的方法,并对用该方法从经快中子诱变的拟南芥Columbia生态型M_2代群体中筛选获得的突变体SFNA-9-4进行分析,发现该突变体的抗氧化胁迫能力和寿限均显著增加。由此说明,用该方法筛选拟南芥寿限延长突变体是可行的。     

聚乙二醇模拟水分胁迫筛选拟南芥突变体的新方法

在以聚乙二醇（PEG）模拟水分胁迫的条件下,分别在拟南芥种子萌发期和幼苗期以25％和30％为选择浓度,根据一种子是否萌发和幼苗能否存活为指标,筛选拟南芥抗水分胁迫突变体。此法操作简单,方便快捷,特别适合实验室进行大规模筛选。我们用这种方法筛选了T—DNA插入的拟南芥突变体库,得到78株可能是拟南芥抗水分胁迫的突变体。     

拟南芥AMP1负调控植物对高盐胁迫的反应

高盐胁迫严重影响植物的生长发育及农作物产量,因此鉴定盐胁迫响应相关基因至关重要。拟南芥的AMP1编码一个推测的谷氨酸羧肽酶,参与植物的生长发育、光形态建成与种子休眠。研究证明了AMP1的一个新功能,它的缺失提高了缺失突变体amp1的抗高盐胁迫的能力,研究证明amp1突变体的强抗高盐胁迫表型一方面是由于在高盐胁迫下amp1突变体比野生型中积累了更多的甜菜碱和脯氨酸降低了突变体细胞的水势,另一方面高盐胁迫条件下amp1突变体中高盐胁迫响应的下游基因RD29A,以及AHA3的表达量也高于野生型,后者可促进Na+的外排;高盐条件能够对植物造成氧化胁迫,研究发现AMP1的缺失还上调了抗氧化相关基因ZAT10/12的表达量,进而降低了在高盐胁迫条件下amp1突变体内过氧化物的积累水平,减轻对细胞的损伤和生长的抑制,这些都提高了amp1突变体的抗高盐胁迫的能力。以上结果证明在拟南芥中AMP1负调控植物对高盐胁迫的反应过程。     

超表达AhNCED1拟南芥植株在渗透胁迫下抗氧化能力和抗旱相关下游基因表达变化

利用纯系超表达AhNCED1拟南芥植株,在添加300mmol·L-1山梨醇的MS培养基上胁迫培养,分析其表型及抗氧化能力。结果表明,在正常情况及300mmol·L-1山梨醇处理10d条件下,与129B08/nced3突变体和野生型比较,超表达AhNCED1植株生长状况好,主根长度增加,幼苗体内O2ˉ含量水平较低,POD和SOD酶活性较高。说明NCED1基因对于拟南芥体内抗氧化酶系统的维持有重要意义。用sqRT-PCR方法检测结果表明,野生型和超表达AhNCED1拟南芥植物体内KIN1、COR47和RAB18基因表达随脱水延长而明显增强,且在超表达AhNCED1拟南芥中增强幅度大。     

拟南芥ANAC055基因突变体对高盐和渗透胁迫的响应

高盐和渗透等非生物胁迫是影响农作物产量和品质的重要因素,非生物胁迫发生时,植物通过体内各类转录因子启动胁迫应答反应,进而降低非生物胁迫对植物的损伤。本研究筛选出植物特异性转录因子ANAC055编码基因的纯合T-DNA插入突变体SALK_152738,测序分析发现T-DNA插在ANAC055基因的3'UTR区域。实时荧光定量PCR结果表明叶中ANAC055基因表达量最高;与野生型相比,突变体叶、茎和花中ANAC055基因表达量分别下降了40%、50%和70%。高盐胁迫后,野生型和突变体叶中ANAC055基因表达量分别比对照上升了320%和55.4%;而渗透胁迫时,该基因叶中的表达量分别比对照下降了47.7%和56.3%;电子表达谱分析发现该基因根中的表达可受高盐和渗透等多种非生物胁迫的诱导表达。高盐和渗透胁迫时野生型和突变体幼根的生长均受到明显抑制,但高盐胁迫对突变体根生长的抑制作用比对野生型根生长的抑制作用更大。上述分析表明拟南芥ANAC055基因可受高盐和渗透等非生物胁迫的诱导表达,并且其在拟南芥幼根的生长发育过程中具有一定的作用,本研究有助于进一步明确其在非生物胁迫过程中的作用。     

拟南芥神经酰胺酶基因对氧化胁迫的响应

以拟南芥哥伦比亚生态型（Col）和神经酰胺酶突变体为实验材料,通过对突变体的一系列生理生化指标的测定,来研究拟南芥神经酰胺酶基因（AtCER）对H2O2的响应。利用PCR和Northern blot获得了9个AtCER纯合单突变体。不同浓度H2O2处理野生型和突变体后,发现突变体对H2O2的反应比野生型更加敏感。H2O2处理后突变体叶片出现比野生型更严重的黄化现象和坏死斑点,总叶绿素含量也比野生型下降的更快,电导率测定也发现突变体比野生型的电导率增加得更多。抗氧化酶活性的分析结果发现H2O2处理后,突变体的抗氧化酶活性比野生型提高了1．5～3倍。上述研究结果说明AtCER参与了H2O2诱导的氧化胁迫反应。     

拟南芥H_2O_2突变体的筛选及特性分析

通过化学诱变剂甲基磺酸乙酯(EMS)诱变模式植物拟南芥(Arabidopsis thaliana)获得突变体筛选群体.在5 mmol/L H2O2胁迫下,以叶片温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对H2O2不敏感突变体hpi1(hydrogen peroxide-insensitive1)和敏感突变体hps1(hydrogen peroxide-sensitive1).进一步研究发现,两种突变均为单基因隐性突变,气孔密度同野生型一样,而叶片温度、气孔开度和叶片失水率则有明显的差异.种子萌发实验表明,hpi1对甘露醇(Man)和NaCl不敏感而对ABA敏感,hps1则对3种胁迫都表现出敏感特性.     

拟南芥活性氧不敏感型突变体筛选与特性分析

采用EMS化学诱变方法与H2O2氧化胁迫选择,以根在重力作用下的弯曲生长为指标,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明,突变株对活性氧不敏感性状为隐性单基因突变所致;生理生化分析表明突变体对H2O2有很强的抗性,表现为气孔开度对H2O2不敏感和H2O2胁迫时较低的膜脂过氧化水平。运用LSCM技术并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞内产生H2O2的情况,结果显示突变体体内荧光强度比对照低,暗示了突变体体内消除H2O2的能力可能有所提高,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料,而且还将大大加深人们对信号转导途径的再认识。     

拟南芥活性氧不敏感型突变体的筛选与特性分析

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 ,而且还将大大加深人们对信号转导途径的再认识     

ATHKl基因调节拟南芥渗透胁迫信号转导过程

以拟南芥ATHKl基因T-DNA插入所产生的缺失突变体和野生型ws(wassilewskija)生态型为材料,分析了它们在生理和基因表达方面的差异.结果表明突变体的离体叶片失水率明显大于野生型;在30%PEG-6000胁迫后,野生型和ATHKJ突变体的细胞膜离子外渗率比胁迫前分别增加了50%和80%.PEG胁迫48 h时突变体的萎蔫程度明显大于野生型ws.以上结果说明ATHKl突变体的抗渗透胁迫能力低于野生型,即ATHKl基因参与了拟南芥适应逆境的调节反应.利用DDRT-PCR技术研究二者在PEG胁迫36h后的基因表达差异,分离到9个在野生型中被PEG诱导表达而在突变体中未被诱导的参与逆境应答的基因片段,其中包括MAPKKKl8和丝氨酸/苏氨酸蛋白激酶基因,即ATHKJ基因失活引起下游基因响应渗透胁迫的能力减弱,进一步说明ATHKJ基因参与拟南芥适应逆境的调节反应,并且ATHKl可能在逆境信号转导组分MAPK的上游起作用,很可能是植物体中的渗透感受器.     

Oxidative stress in Caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.     

Matricide in Caenorhabditis elegans as an example of programmed death of whole animal organism: role of mitochondrial oxidative stress

Nematodes Caenorhabditis elegans is a widely used model for studying the genetic and molecular mechanisms that determine the lifespan. The choice between the two vital program strategies of adult hermaphrodite C. elegans--normal aging and matritcide (programmed death), is largely affected by the availability of food, and also depends on a variety of stresses. We decided to test the hypothesis that, in line with the phenoptosis theory, oxidative stress increases probability of the programmed death of the whole organism. It is shown that high concentrations of paraquat (strong mitochondrial stress) significantly increase the propensity to matricide. In this case, mutants with a reduced antioxidant capacity of mitochondria (nnt) are more sensitive to the reagent. On the other hand, the concentrations of paraquat, necessary for the manifestation of this effect, are toxic to the offspring, while at low concentrations matricide of mutant worms and wild-type worms occurs with equal frequency. Therefore it is safe to conclude that oxidative stress is not the key initiating mechanism of matricide under normal conditions.     

Genetic analysis of extended lifespan in Drosophila melanogaster III. On the relationship between artificially selected and wild stocks

Adult lifespans, age‐specific survival, age‐specific mortality, survival times on paraquat, and survival times on DDT were assayed in seven lines of Drosophila melanogaster, including two genetically heterogeneous wild lines recently collected from nature, and three inbred and recombinant inbred lines derived from an artificial selection experiment for increased lifespan. Survival on paraquat is positively correlated with adult lifespan. DDT resistance is uncorrelated with either paraquat resistance or lifespan. The wild lines are unexceptional with respect to average lifespan, paraquat resistance, age‐specific survivorship, and leveling off of mortality rates at advanced ages, but have high levels of DDT resistance. Cluster analysis groups the wild lines with three unselected laboratory stocks in one cluster, while two long‐lived elite recombinant inbred lines form a second cluster. Long‐lived laboratory‐adapted lines are quantitatively differentiated from the wild stocks, both with respect to average adult lifespans and resistance to an oxidizing agent. We reject the ‘recovery’ hypothesis, which proposes that Drosophila artificially selected for long life have phenotypes that merely recover the wild state. This revised version was published online in July 2006 with corrections to the Cover Date.     

Contributions of individual σB-dependent general stress genes to oxidative stress resistance of Bacillus subtilis

The general stress regulon of Bacillus subtilis comprises approximately 200 genes and is under the control of the alternative sigma factor σ(B). The activation of σ(B) occurs in response to multiple physical stress stimuli as well as energy starvation conditions. The expression of the general stress proteins provides growing and stationary nonsporulating vegetative cells with nonspecific and broad stress resistance. A previous comprehensive phenotype screening analysis of 94 general stress gene mutants in response to severe growth-inhibiting stress stimuli, including ethanol, NaCl, heat, and cold, indicated that secondary oxidative stress may be a common component of severe physical stress. Here we tested the individual contributions of the same set of 94 mutants to the development of resistance against exposure to the superoxide-generating agent paraquat and hydrogen peroxide (H(2)O(2)). In fact, 62 mutants displayed significantly decreased survival rates in response to paraquat and/or H(2)O(2) stress compared to the wild type at a confidence level of an α value of ≤ 0.01. Thus, we were able to assign 47 general stress genes to survival against superoxide, 6 genes to protection from H(2)O(2) stress, and 9 genes to the survival against both. Furthermore, we show that a considerable overlap exists between the phenotype clusters previously assumed to be involved in oxidative stress management and the actual group of oxidative-stress-sensitive mutants. Our data provide information that many general stress proteins with still unknown functions are implicated in oxidative stress resistance and further support the notion that different severe physical stress stimuli elicit a common secondary oxidative stress.     

Matricide in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as an example of programmed death of an animal organism: The role of mitochondrial oxidative stress

Nematode Caenorhabditis elegans is a widely used model for studying genetic and molecular mechanisms of lifespan regulation. The choice between two life strategies—normal aging and matricide (programmed death)-made by an adult hermaphroditic C. elegans organism is based on food availability and is also affected by different kinds of stress. We have tested a hypothesis concerning an increase in matricide probability as a result of oxidative stress; this hypothesis is based on the phenoptosis theory. High concentrations of paraquat, a strong mitochondrial stressor, were shown to increase matricide propensity. Mutants with a decreased antioxidant potential of mitochondria (nnt) were more sensitive to the reagent. On the other hand, paraquat concentrations required for this effect to be observed are toxic for the progeny, whereas at low paraquat concentration matricide frequency is the same in wild-type worms and nnt mutants. Therefore, one can assume that the molecular mechanisms of matricide initiation under normal conditions are not necessarily connected to mitochondrial oxidative stress.     

G protein–coupled receptor kinase 2 modifies the ability of Caenorhabditis elegans to survive oxidative stress

Survival and adaptation to oxidative stress is important for many organisms, and these occur through the activation of many different signaling pathways. In this report, we showed that Caenorhabditis (C.) elegans G protein–coupled receptor kinases modified the ability of the organism to resist oxidative stress. In acute oxidative stress studies using juglone, loss-of-function grk-2 mutants were more resistant to oxidative stress compared with loss-of-function grk-1 mutants and the wild-type N2 animals. This effect was Ce-AKT-1 dependent, suggesting that Ce-GRK2 adjusted C. elegans oxidative stress resistance through the IGF/insulin-like signaling (IIS) pathway. Treating C. elegans with a GRK2 inhibitor, the selective serotonin reuptake inhibitor paroxetine, resulted in increased acute oxidative stress resistance compared with another selective serotonin reuptake inhibitor, fluoxetine. In chronic oxidative stress studies with paraquat, both grk-1 and grk-2 mutants had longer lifespan compared with the wild-type N2 animals in stress. In summary, this research showed the importance of both GRKs, especially GRK2, in modifying oxidative stress resistance.     

The DAF-2 insulin-like signaling pathway independently regulates aging and immunity in C. elegans

The Caenorhabditis elegans DAF-2 insulin-like signaling pathway, which regulates lifespan and stress resistance, has also been implicated in resistance to bacterial pathogens. Loss-of-function daf-2 and age-1 mutants have increased lifespans and are resistant to a variety of bacterial pathogens. This raises the possibility that the increased longevity and the pathogen resistance of insulin-like signaling pathway mutants are reflections of the same underlying mechanism. Here we report that regulation of lifespan and resistance to the bacterial pathogen Pseudomonas aeruginosa is mediated by both shared and genetically distinguishable mechanisms. We find that loss of germline proliferation enhances pathogen resistance and this effect requires daf-16, similar to the regulation of lifespan. In contrast, the regulation of pathogen resistance and lifespan is decoupled within the DAF-2 pathway. Long-lived mutants of genes downstream of daf-2, such as pdk-1 and sgk-1, show wildtype resistance to pathogens. However, mutants of akt-1 and akt-2, which we find to individually have modest effects on lifespan, show enhanced resistance to pathogens. We also demonstrate that pathogen resistance of daf-2, akt-1, and akt-2 mutants is associated with restricted bacterial colonization, and that daf-2 mutants are better able to clear an infection after challenge with P. aeruginosa. Moreover, we find that pathogen resistance among insulin-like signaling mutants is associated with increased expression of immunity genes during infection. Other processes that affect organismal longevity, including Jun kinase signaling and caloric restriction, do not affect resistance to bacterial pathogens, further establishing that aging and innate immunity are regulated by genetically distinct mechanisms.