叶面喷施甲醇和乙醇对万年青生理特性的影响

为了比较甲醇和乙醇对植物生理特性的影响,分别用5％(V/V)的甲醇和乙醇均匀喷洒于温室条件下盆栽培养的万年青叶片上,以喷施蒸馏水的植物为对照,每周喷施2次,喷施3周后分析这两种醇对万年青叶片生长、生理特性及相关光合基因表达的影响.结果表明,喷施甲醇和乙醇均增加了万年青叶片鲜重、可溶性总蛋白质含量、气孔导度、蒸腾速率、胞间CO2浓度、净光合速率、叶绿素a、b及总叶绿素的含量,降低了过氧化氢的含量,提高了质膜H+ ATPase及H+泵活性,并且乙醇的作用效果比甲醇更加明显.RT-PCR分析结果表明,光合作用相关基因叶绿素a/b结合蛋白基因(Cab)和铁氧还蛋白-2编码的基因(2-FdO)在处理12h时被甲醇抑制但却被乙醇诱导,72 h时被甲醇诱导但却被乙醇抑制;两种醇均诱导光系统Ⅱ反应中心PsbP家族蛋白编码基因(PsbP)的表达.由此推测甲醇和乙醇可能在植物中作为信号分子起作用,且乙醇的刺激效果比甲醇更加显著.     

苦荞黄酮转运相关蛋白基因FtAH4L的克隆及表达分析

为研究苦荞黄酮转运相关基因,以苦荞(Fagopyrum tataricum)品种"西荞二号"为材料,克隆到1条质膜H+-ATPase基因(autoinhibited H+-ATPase isoform 4 like,AH4L),将其命名为FtAH4L。通过开放阅读框(ORF)分析,FtAH4L基因cDNA全长3 398bp,开放阅读框2 898bp,编码966个氨基酸残基,理论分子量为109kD,等电点6.48。氨基酸保守基序比对分析表明,AH4L在植物种间较为保守。在茉莉素诱导处理和5种光(白色荧光、LED白光、LED蓝光、LED红光和UV-B)处理芽期苦荞后,采用半定量RT-PCR和AlCl3比色法分析结果表明,茉莉素处理后的苦荞胚轴和子叶中FtAH4L基因表达量与黄酮含量均显著上升,且二者呈正相关关系;5种光对子叶中FtAH4L表达量无显著影响,但均显著增加其黄酮含量;胚轴中,除LED红光外,各种光均显著提高FtAH4L表达量和总黄酮含量,且LED蓝光与UV-B的影响极显著。该研究结果为深入研究FtAH4L基因参与苦荞黄酮转运奠定了基础。     

Salt modulation of vacuolar H-ATPase and H-Pyrophosphatase activities in Vigna unguiculata

Salt modulation of the tonoplast H⁺-pumping V-ATPase and H⁺-PPase was evaluated in hypocotyls ofVigna unguiculata seedlings after 3 and 7 days of treatment. In 3-day-old seedlings, treatment with 100 mmol/L NaCl decreased the proton transport and hydrolytic activities of both the V-ATPase and the H⁺-PPase. After 7 days, the proton transport and hydrolysis activities of the V-ATPase were higher, while the H⁺-PPase activities were lower in seedlings. Western blot analysis of A- and B-subunits of V-ATPase revealed that the protein content of the two subunits varied in parallel with their activities, i.e. to a higher activity corresponded a higher protein content of the subunits and vice versa. Contrarily, Western blot analysis of H⁺-PPase levels failed to show any correlation with PPase activity, suggesting a partial enzyme inactivation. The results indicate that salt stress induces V-ATPase expression inV. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress. Under the same conditions PPase is inhibited.     

Molecular analysis of two genes between let-653 and let-56 in the unc 22(IV) region of Caenorhabditis elegans

Summary A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na⁺/H⁺ antiporter. Pairs of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.     

The changing sensitivity to auxin of the plasma-membrane H+-ATPase: Relationship between plant development and ATPase content of membranes

The auxin sensitivity of the plasma-membrane H⁺-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H⁺-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H⁺-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H⁺-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA enzyme-linked immunosorbent assay - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T.     

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H⁺-ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H⁺-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.     

Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants – a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23–26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185–196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314–320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328–332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents – in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396–402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H⁺, K⁺-ATPase preparations. Intraduodenal injection of AE or BuF (0.5–2.0 g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0 g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5 mg/kg, s.c.)- or bethanecol (1.0 mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05–0.5 g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30 mg/kg, s.c.) or restraint at 4 °C. BuF also decreased the gastric H⁺, K⁺-ATPase activity in vitro proportionately to the concentration (IC₅₀=58.8 μg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309–313], inhibited the in vitro gastric H⁺, K⁺-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.     

酸胁迫下琥珀酸放线杆菌的生理及转录应答

【目的】通过琥珀酸放线杆菌Actinobacillus succinogenes CGMCC1593对酸胁迫的生理应答和转录组学分析,探究琥珀酸放线杆菌酸胁迫的机制。【方法】测定不同pH对细胞生长、H+-ATPase、细胞内pH的影响;测定酸胁迫前后细胞膜和谷氨酸脱氢酶的变化、谷氨酸对琥珀酸放线杆菌生长的影响;通过RNA-seq测序分析酸胁迫条件下的差异表达基因。【结果】随pH值的降低,细胞生长受抑制,H+-ATPase的活性下降。pH 4.7酸胁迫后,细胞膜受到严重损伤,谷氨酸对酸胁迫后的细胞有保护作用,GDH酶活响应酸胁迫后略有增加。酸胁迫后,39个基因差异表达较为显著,其中49%基因属于应激蛋白、转运蛋白,小部分基因与代谢相关。【结论】本文探究了琥珀酸放线杆菌酸胁迫下的生理及转录应答,研究结果可为寻找增强琥珀酸放线杆菌耐酸性策略提供参考。     

黄河三角洲河滩与潮滩芦苇对盐胁迫的生理生态响应

研究黄河三角洲两种生境芦苇对盐胁迫的生理生态响应差异,能为退化滨海湿地生态修复中芦苇植株来源的选择提供重要的理论支持。在盐胁迫(300 mmol/L NaCl)下,比较研究了黄河三角洲河滩芦苇(低盐生境)和潮滩芦苇(高盐生境)叶片中的Na⁺含量、根部分生区Na⁺流速、叶片的光合作用参数、H₂O₂的含量、抗氧化酶的活性、丙二醛和脯氨酸的含量。结果表明:盐胁迫显著提高了河滩芦苇叶片中Na⁺含量,但对潮滩芦苇叶片Na⁺影响不显著。进一步通过非损伤微测技术研究发现,盐胁迫后,潮滩芦苇比河滩芦苇的根部分生区的Na⁺外排流速更高(潮滩芦苇:(1982.05±122.74) pmol cm^-2 s^-1vs.(87.93±12.94) pmol cm^-2 s^-1,P<0.01;河滩芦苇:(1574.16±458.90) pmol cm^-2     

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na⁺-transport in V. alginolyticus subcellular vesicles is discussed.     

Effects of silicon on enzyme activity and sodium,potassium and calcium concentration in barley under salt stress

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown in a hydroponics system containing 120 mol m^-3 NaCl only and 120 mol m^-3 NaCl with 1.0 mol m^-3 Si (as potassium silicate). Compared with the plants treated with salt alone, superoxide dismutase (SOD) activity in plant leaves and H⁺-ATPase activity in plant roots increased, and malondialdehyde (MDA) concentration in plant leaves decreased significantly for both cultivars when treated with salt and Si. The addition of Si was also found to reduce sodium but increase potassium concentrations in shoots and roots of salt-stressed barley. Sodium uptake and transport into shoots from roots was greatly inhibited by added Si under salt stress conditions. However, Si addition exhibited little effect on calcium concentrations in shoots of salt-stressed barley. Thus, Si-enhanced salt tolerance is attributed to selective uptake and transport of potassium and sodium by plants. The results of the present study suggest that Si is involved in the metabolic or physiological changes in plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

氯化镁预处理对美人蕉根部质膜H+-ATPase活性与农田废水硝态氮吸收效率的影响

以湿地挺水植物七叶红色大花美人蕉(Cana generalis Bailey)幼苗为实验材料,在温室中用150μmol/L氯化镁溶液预处理12h,然后置于硝态氮(NO3--N)浓度为10mg/L的农田废水中处理,考察氯化镁预处理对美人蕉吸收农田废水中硝态氮效率的影响,以及处理期间根部质膜H+-ATPase活性、H+-泵活性、14-3-3蛋白与质膜H+-ATPase相互作用。结果显示:经过150μmol/L氯化镁溶液预处理12h后,美人蕉吸收农田废水中硝态氮的效率比对照增加11%;与没有预处理的植株相比,氯化镁预处理美人蕉根中质膜H+-ATPase和H+-泵的活性均显著提高,质膜H+-ATPase和14-3-3蛋白的相互作用显著增强。研究表明,氯化镁能够通过增强质膜H+-ATPase与14-3-3蛋白的相互作用来提高质膜H+-ATPase的活性,从而增加美人蕉对硝态氮的吸收效率。     

长叶红砂液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特性

为研究长叶红砂(Reaumuria trigyna)离子转运分子机制,利用RT-PCR和RACE技术,克隆到其液泡膜Na+/H+逆向转运蛋白基因(NHX1)的全长cDNA片段,命名为RtNHX1(NCBI序列号为KR919802)。结果表明:RtNHX1的cDNA片段全长2 622bp,开放阅读框1 662bp,5′非编码区509bp,3′非编码区451bp,编码553个氨基酸,推测分子量为60.91kD。该蛋白含有12个跨膜结构域,为疏水蛋白,与其他植物液泡膜Na+/H+逆向转运蛋白NHX1的亲缘关系较近。实时荧光定量PCR对其在NaCl胁迫下的表达检测显示,不同时间和不同浓度NaCl胁迫下,RtNHX1表达量变化均呈先升高后降低趋势,在100mmol/L NaCl胁迫6h和200mmol/L NaCl胁迫后达到最高,表达量分别超过或约是对照的3倍,一定程度反应出RtNHX1参与长叶红砂的盐胁迫应答,是该植物离子转运体系的重要元件。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

低H~+_-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析

【目的】筛选H~+_-ATPase活性降低的植物乳杆菌突变菌,比较其与亲本菌基因表达水平的差异,进一步探索H~+_-ATPase的调控机制。【方法】利用硫酸新霉素诱变、筛选突变菌,并对亲本菌(ZUST)和突变菌(ZUST-1、ZUST-2)进行生长、产酸能力及H~+_-ATPase活性的测定。分别提取亲本菌和突变菌的基因组DNA,扩增H~+_-ATPase全部编码基因并测序。通过荧光定量PCR对H~+_-ATPase全部编码基因进行相对定量分析。【结果】突变菌的生长和产酸能力均低于亲本菌,突变菌ZUST-1和ZUST-2的H~+_-ATPase活性比亲本菌分别降低了10.1%和28.8%。突变菌ZUST-1和ZUST-2的atp A基因均有22个位点发生突变,而ZUST-2的atp C基因有6个位点发生突变。突变菌ZUST-1和ZUST-2的atp A在对数期基因表达水平分别比亲本菌ZUST下调了41.1%和35.7%,在稳定期分别下调了43.6%和14.2%;ZUST-1的atp C基因在对数期的表达水平比ZUST略高,在稳定期比ZUST上调了30%,而ZUST-2的atp C基因未表达。【结论】突变菌H~+_-ATPase活性减弱会导致其全部编码基因在稳定期表达水平上调(除ZUST-2的atp C不表达外),而且atp A和atp C基因突变导致的基因表达水平的差异是影响H~+_-ATPase活性的主要因素,此研究结果为进一步研究植物乳杆菌中H~+_-ATPase的调控机制奠定了基础。