番茄calnexin基因的克隆及胁迫表达分析

Calnexin是内质网中重要的类凝集素分子伴侣,其主要作用是辅助糖基化蛋白的折叠和装配,调节内质网中的Ca²⁺稳态平衡和Ca²⁺信号传导过程。从番茄(Lycopersicon esculentum)的cDNA文库中克隆到calnexincDNA全序列,将其命名为Lecnx61.0,并以其3’端DNA片段为探针对番茄基因组进行Southern分析,结果表明Lecnx61.0在该基因组中仅有一个拷贝;Northern和W estern分析表明,Lecnx61.0的表达还受热激、冷害、盐害和内质网应激诱导剂衣霉素的诱导,但对干旱胁迫没有明显的反应。LeCNX 61.0蛋白对Ca²⁺亏缺胁迫的响应呈现组织特异性,但高浓度Ca²⁺并不影响各组织中LeCNX 61.0蛋白的含量,实验结果表明LeCNX 61.0蛋白可能在植物抵抗特定的环境胁迫中发挥作用。     

番茄AT-hook基因家族的鉴定及胁迫条件下的表达分析

AT-hook蛋白家族在植物生长发育、器官构建及逆境胁迫和激素信号应答中发挥重要作用。本研究在番茄基因组范围内,利用生物信息学方法对番茄AT-hook基因家族的成员、分布、结构和功能进行分析。结果表明,番茄AT-hook家族包含32个成员,分为3种类型,其中类型Ⅰ含有13个成员;遗传进化分析表明番茄AT-hook基因成员与拟南芥家族基因具有相似分类。利用实时荧光定量PCR对番茄32个基因开展组织表达分析,结果表明AT-hook基因具有表达差异,主要在根和花中表达较高。氧化胁迫分析结果表明,32个基因受ABA、SA、盐、高温和低温诱导表达,其中部分基因显著上调或下调表达,很可能参与了番茄逆境胁迫条件下的防御应答反应。本研究结果将为番茄AT-hook家族基因的深入研究提供依据,为进一步解析番茄AT-hook基因的功能奠定基础。     

外源Ca(NO_3)_2对NaCl胁迫下番茄幼苗生理特征的影响

以番茄品种'农城906'和"毛粉802'幼苗为材料,利用Hoagland基础培养液,研究在150 mmol/L NaCl胁迫下外源10、20、30和40 mmol/L Ca(NO3)2对番茄植株盐胁迫的缓解作用.结果显示,(1) NaCl胁迫显著降低了2个品种幼苗地上和地下部分干重、叶片叶绿素含量和根系活力,增加了幼苗的可溶性糖含量、细胞膜透性和MDA含量,同时使其CAT、POD、SOD活性显著降低;(2)添加20 mmol/L Ca(NO3)2能够有效提高盐胁迫条件下幼苗的地上和地下部干重,缓解叶绿素的降解和根系活力的下降,增加幼苗体内可溶性糖含量,降低叶片质膜透性和MDA含量,增强幼苗体内POD、CAT等细胞保护酶活性,但随着Ca(NO3)2浓度的继续增加,这种缓解作用逐渐减小.研究表明,一定浓度的外源Ca(NO3)2能有效缓解NaCl对番茄幼苗的生长和生理伤害,在一定程度上增强番茄幼苗对盐胁迫的耐受力,并以20 mmol/L Ca(NO3)2对NaCl胁迫的缓解效果最佳.     

过量表达内质网小分子热激蛋白增强番茄的衣霉素抗性

真核细胞内质网腔内未折叠蛋白的过度积累会引起内质网胁迫（ER胁迫）,继而激活未折叠蛋白应答（UPR）信号途径,诱导内质网定位的分子伴侣的大量表达（如BiP和calnexin等）。本工作将CaMV35S启动子驱动的内质网小分子热激蛋白基因（ER-sHSP）导入番茄,发现ER-sHSP的过量表达提高了转基因番茄整株对衣霉素的抗性。衣霉素处理使未转基因番茄中BiP和calnexin基因的表达迅速升高,转基因番茄中这两个基因的表达也有增加,但表达强度明显低于未转基因番茄。说明ER-sHSP能够减轻ER胁迫,并可能参与UPR信号转导途径。     

番茄线粒体和内质网小分子热激蛋白基因的分子克隆

以热激处理的番茄（Lycopersicon esculentum Mill．）花为实验材料,构建了cDNA库,运用ＲＴ－ＰＣＲ方法克隆番茄粒体和内质网小分子热激蛋白cDNA,利用这两个保守区片段为探针,筛选cDNA库,获得线粒体和内质网小分子热激蛋白全序列cDNA。;通过分析线粒体和内质网小分子热激蛋白基因对温度的反应,发现小分子热激蛋白基因在番茄花中的热激应答温度低于它们在叶片中的热激应答温度,并且番茄叶片中的线粒体小分子热激蛋白基因还具有低温应答特性。对线粒体和内质网小分子热激蛋白基因的分子结构特点,小分子热激蛋白基因在番茄花中的特别热激应答温度的调控机理以及线粒体小分子热激蛋白的基因在中片中的低温度应答成因进行了讨论。     

低温胁迫下枇杷幼叶细胞内Ca2+水平及细胞超微结构变化的研究

用焦锑酸钾沉淀的电镜细胞化学方法 ,研究了低温胁迫下枇杷幼叶细胞内 Ca2 水平变化。研究结果表明 ,枇杷幼叶细胞未经低温处理时 ,Ca2 定位分布的沉淀颗粒大量出现在细胞壁、细胞间隙、质膜和液泡 ;叶绿体、细胞质和细胞核中也有一些 Ca2 沉淀颗粒分布。枇杷幼叶经 44h、4℃低温处理后 ,在质膜和液泡膜上 Ca2 的沉淀增多 ,细胞质和细胞核中的Ca2 水平增加。不抗寒品种在低温胁迫条件下可见核孔开口较大 ,有时可观察到核内容物外漏。抗寒品种在低温胁迫条件下核孔未见明显开口 ,叶绿体中类囊体不形成基粒 ,少数片层结合重叠后伸展在整个叶绿体中。而不抗寒品种类囊体则堆积形成明显的颗粒状基粒 ,类囊体片层数量较多。在 2个叶绿体之间还常可见到线粒体紧夹其间 ,但线粒体的内膜模糊不清。不抗寒品种内质网和高尔基体也较多见 ,内膜系统比较发达 ,但低温胁迫条件下膜系统易受破坏 ,膜结构模糊不清。     

铜、镉胁迫下外源NO介导的番茄解毒途径

一氧化氮(NO)作为信号分子,在抵御重金属胁迫中起重要作用,但对不同离子胁迫下的解毒机制尚缺乏研究.本研究采用营养液培养法,研究了铜(Cu)、镉(Cd)单一或复合胁迫下,番茄幼苗对Cu、Cd的吸收转运特性及对外源NO的响应机制.结果表明: 50 μmol·L^-1的Cu²⁺、Cd²⁺均显著抑制番茄植株的生长,其中Cd胁迫对生长的抑制效应远高于Cu胁迫.Cu、Cd单一或复合胁迫均使番茄根系Cu、Cd含量显著升高,但根系对Cu、Cd吸收存在严格选择性.根细胞对必需元素Cu表现出“奢侈吸收”的现象,而对毒性较强的Cd则吸收相对较少,胞内Cd浓度仅为Cu的1/10左右.外源NO处理可不同程度地缓解Cu、Cd胁迫,其中缓解Cd胁迫的效能更强.番茄对被动进入细胞的Cu、Cd具有相似的解毒机制:一方面,Cu、Cd胁迫诱导细胞质中产生谷胱甘肽(GSH)、植物螯合肽(PCs)和金属硫蛋白(MTs),络合过多的Cu、Cd离子,降低其生物毒性;另一方面,过多的Cu、Cd离子或螯合物被转运至液泡区隔化.外源NO通过调控GSH-GSSG(氧化型谷胱甘肽)氧化还原状态及GSH-PCs代谢方向的改变,促进Cu、Cd离子转运至液泡区隔化来缓解胁迫抑制;NO还可诱导植株叶片或根系表达更多的金属硫蛋白、GSH和PCs,而且上述响应普遍存在叠加效应.这可能是NO介导番茄对Cu、Cd胁迫的另一主要解毒途径.     

热激转录因子HSFA1d参与植物内质网胁迫应答

环境胁迫是全球范围内影响植物正常生长发育的重要因素之一.研究植物的抗逆机制,运用生物技术提高作物的抗逆性,不仅具有重要的科学意义,也对农业生产具有重要的促进作用.内质网是蛋白合成和加工的重要场所,当环境胁迫引起内质网中错误折叠蛋白积累时,就会引起一个保守的未折叠蛋白应答(UPR)机制,帮助细胞达到一个新的平衡状态.热激转录因子HSFA1s在植物细胞质热激反应过程中起着重要的作用.本研究证明,拟南芥HSFA1d的表达受内质网胁迫的诱导,过表达HSFA1d的转基因植株对内质网胁迫诱导剂衣霉素(TM)具有一定程度的抗性.统计显示,在TM板上生长的转基因植株出真叶率明显提高.亚细胞定位显示,HSFA1d在正常条件下定位于细胞质中,当受到内质网胁迫时,HSFA1d从细胞质转移到细胞核中.HSFA1d可以调控UPR下游基因的表达.这些结果表明,经典的热激反应相关热激转录因子HSFA1d也参与了植物的内质网未折叠蛋白应答过程.     

H_2S与Ca~(2+)协同增强谷子对Cr~(6+)胁迫的耐受

继一氧化氮(NO)和一氧化碳(CO)之后,第三种气体信号分子硫化氢(H2S)对植物体生长发育和环境胁迫应答的调控正在受到越来越多的关注。钙离子(Ca2+)是重要的第二信使,参与植物对多种胁迫的响应。该实验以谷子这种抗逆性较强的作物为材料,对其响应六价铬(Cr6+)胁迫过程中H2S和Ca2+信号的互作进行了研究。结果表明,Cr6+胁迫显著激活谷子幼苗的H2S产生系统,外源H2S预处理能明显降低Cr6+胁迫对谷子根尖细胞的损伤,而H2S的合成抑制剂羟胺(HA)预处理,使得Cr6+对谷子的毒害增强;进一步实验发现,H2S能激活Ca2+信号下游相关基因的表达,同时Ca2+能增强H2S的产生,表明在植物体内H2S和Ca2+信号存在复杂的联系。该研究也证明,H2S和Ca2+可以通过调节重金属离子转运蛋白增强谷子对Cr6+的耐受。     

NaCl胁迫对番茄嫁接苗生物量及离子含量的影响

以影武者为砧木,宝大903为接穗,研究了NaCl胁迫下番茄嫁接苗与接穗自根苗在生物量及Na 、K 、Ca2 、Mg2 、Cl-等含量方面的差异.结果显示,NaCl胁迫下,嫁接苗生长受抑制程度较轻;嫁接苗和自根苗Na 、Cl-含量增长幅度最大的部位均是叶片,嫁接苗增长幅度小于自根苗;K 、Ca2 、Mg2 含量下降幅度最大的部位均是根系,嫁接苗下降幅度小于自根苗;嫁接苗的K /Na 值、Ca2 /Na 值显著高于自根苗,说明嫁接苗选择性吸收K 、Ca2 能力显著强于自根苗.结果表明,NaCl胁迫下嫁接苗体内离子分布的区域化优于自根苗,耐盐性强于自根苗.     

Sphingosine kinase 1 promotes cerebral ischemia-reperfusion injury through inducing ER stress and activating the NF-κB signaling pathway

Endoplasm reticulum stress and inflammation response have been found to be linked to cerebral ischemia-reperfusion (IR) injury. Sphingosine kinase 1 (SPHK1) has been reported to be a novel endoplasm reticulum regulator. The aim of our study is to figure out the role of SPHK1 in cerebral IR injury and verify whether it has an ability to regulate inflammation and endoplasm reticulum stress. Hydrogen peroxide was used to induce cerebral IR injury. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, western blots, and immunofluorescence were used to measure the alterations of cell viability, inflammation response, and endoplasm reticulum stress. The results demonstrated that after exposure to hydrogen peroxide, cell viability was reduced whereas SPHK1 expression was significantly elevated. Knockdown of SPHK1 attenuated hydrogen peroxide-mediated cell death and reversed cell viability. Our data also demonstrated that SPHK1 deletion reduced endoplasm reticulum stress and alleviated inflammation response in hydrogen peroxide-treated cells. In addition, we also found that SHPK1 modulated endoplasm reticulum stress and inflammation response to through the NF-κB signaling pathway. Inhibition of NF-κB signaling pathway has similar results when compared with the cells with SPHK1 deletion. Altogether, our results demonstrated that SPHK1 upregulation, induced by hydrogen peroxide, is responsible for cerebral IR injury through inducing endoplasm reticulum stress and inflammation response in a manner working through the NF-κB signaling pathway. This finding provides new insight into the molecular mechanism to explain the neuron death induced by cerebral IR injury.     

钙网蛋白的生理及病理生理学作用

钙网蛋白（calreticulin,CRT）是内质网／肌浆网主要的Ca2^＋结合蛋白,通过协助蛋白质正确折叠和维持细胞Ca^2＋稳态而参与调节细胞凋亡、黏附、类固醇敏感性基因表达和自身免疫反应等,并与多种人类疾病的发生、发展和预后相关。本文综述钙网蛋白的生理功能及其在心肌肥大与衰竭、血管新生和应激等病理状态下的变化。     

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT''s association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.     

内质网应激反应分子机理研究进展

内质网应激是导致心脑组织缺血梗塞、神经退行性疾病等发生的重要环节 .目前发现同型半胱氨酸、氧化应激、钙代谢紊乱等都能引起内质网应激级联反应 ,表现为蛋白质合成暂停、内质网应激蛋白表达和细胞凋亡等 .这些表现包括在未折叠蛋白反应 (UPR)、整合应激反应 (ISR)和内质网相关性死亡 (ERAD)三个相互关联的动态过程中 ,每一过程的分子机理现已逐步被揭示 .作为细胞保护性应对机制的内质网应激体系一旦遭到破坏 ,细胞将不能合成应有的蛋白质 ,亦不能发挥正常的生理功能 ,甚至会出现细胞凋亡 .掌握内质网应激过程对进一步理解多种疾病的发生机理有十分重要的理论意义     

植物内质网胁迫研究进展

内质网是蛋白质折叠和蛋白质糖基化修饰的重要场所。在内质网中存在多种调控机制来确保其中的蛋白质被正确地折叠、修饰和组装,以维持内质网稳态,这对于细胞正常的生理活动十分重要。然而,多种物理、化学因素均可使内质网稳态失衡,即在应激条件下,错误折叠和未折叠蛋白质的大量积累将导致内质网胁迫(endoplasmic reticulum stress, ERS),进而会引起未折叠蛋白质响应(unfolded protein response, UPR),极端情况下还会启动细胞程序性死亡(program cell death, PCD)。目前,植物内质网胁迫方面的研究较酵母和动物滞后,因此,从内质网质量控制系统和未折叠蛋白质响应2个方面对植物内质网胁迫现有研究进行了综述,以期为进一步理解内质网胁迫与植物逆境胁迫的关系提供参考。     

SelK is a novel ER stress-regulated protein and protects HepG2 cells from ER stress agent-induced apoptosis

Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.     

小鼠脑组织及培养神经元Neuro-2a 在内质网应激反应时的基因表达谱分析

内质网是真核细胞的重要细胞器。某些细胞内外因素如病原体感染等能引起从内质网到胞浆和胞核的信号传导途径活化,即内质网应激反应。但是,目前国内外尚无针对内质网应激反应的基因表达谱分析报道。本研究中,用3种已报道的内质网应激反应诱导剂,包括蛋白质糖基化抑制剂衣霉素(tunicamycin)、内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素（thapsigargin）和乙脑病毒(Japanese encephalitis virus, JEV),分别处理小鼠颅腔和小鼠脑神经瘤细胞（Neuro-2a）,试剂处理组与未处理组的第二代RNA测序分析发现,衣霉素、毒胡萝卜素和乙脑病毒在体外和体内均引起分子伴侣基因Hsp70表达上调,诱导内质网应激反应。衣霉素、毒胡萝卜素和乙脑病毒体外处理诱导的内质网应激反应信号通路中,基因差异表达相似性高于体内处理组。乙脑病毒和糖基化抑制剂衣霉素体内外处理,主要诱导内质网应激反应的非折叠蛋白质反应信号通路,引起相关基因Atf4、Bip、Edem和Perk等表达上调。内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素主要诱导内质网超负荷反应,激活NF-κB信号通路。乙脑病毒诱导的内质网应激反应相关差异表达基因数量最多,体外与体内合计有40种。乙脑病毒体内外处理上调的基因包括Bax、Casp12、Atf4、Bip、Edem和Perk等,下调的基因包括Sec23/24、Nef、Svip和Jnk等。糖基化抑制剂衣霉素体内外处理上调基因包括Gadd34、Atf4、Ermani和Bip等,下调基因包括Grp94、Atf6、Sec23/24和Nef等。内质网Ca ^-2+-ATPases抑制剂毒胡萝卜素体内外处理上调的基因包括Sec61、Trap和Ask1等。衣霉素、毒胡萝卜素和乙脑病毒体内外处理也通过内质网应激反应,调控与炎症或凋亡相关的MAPK信号通路和P53信号通路。本研究首次通过使用3种内质网应激反应诱导剂分别处理小鼠和细胞,揭示了体内外内质网应激反应引起的基因表达谱变化,为内质网应激反应相关疾病的治疗提供了新思路。     

Apoptosis- and endoplasmic reticulum stress-related genes were regulated by estrogen and progesterone in the uteri of calbindin-D(9k) and -D(28k) knockout mice

Calcium (Ca(2+)) is an important regulator of apoptotic signaling. Calbindin-D(9k) (CaBP-9k) and -D(28k) (CaBP-28k) have a high affinity for Ca(2+) ions. Uterine calbindins appear to be involved in the regulation of myometrial activity by intracellular Ca(2+). In addition, uterine calbindins are expressed in the mouse endometrium and are regulated by steroid hormones during implantation and development. The aim of the present study was to evaluate the regulation of apoptosis in the uteri of CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice. Our findings indicated that Bax protein was enhanced in the uteri of CaBP-28k and CaBP-9k/28k KO mice compared to wild-type (WT) and CaBP-9k KO mice, but no difference was observed in Bcl-2 protein expression. The expressions of caspase 3, 6, and 7 proteins were higher in both CaBP-28k and CaBP-9k/28k KO mice than in WT and CaBP-9k KO mice. These results suggest that the absence of CaBP-28k increases apoptotic signaling. We also investigated the expression of endoplasmic reticulum (ER) stress genes by Western blot analysis in calbindin KO mice. C/EBP homologous protein and immunoglobulin heavy chain-binding protein protein levels were elevated in CaBP-28k KO mice compared to WT mice. When immature mice were treated with 17β-estradiol (E2) or progesterone (P4) for 3 days, we found that the expressions of Bax and caspase 3 protein were increased by E2 treatment in WT and CaBP-9k KO mice, and by P4 treatment in CaBP-28k KO mice. These results indicate that CaBP-28k blocks the up-regulation of apoptosis-related genes and ER stress genes, implying that CaBP-28k may decrease the expression of genes involved in apoptosis and ER stress in murine uterine tissue.