H+-ATPase

摘要： 腐殖质(HS)是生物体外具备生物学活性的最稳定有机物形式之一.本文综述了HS的化学组成、结构特征和生理活性,依据HS化学组成、结构特征和生理活性的相互关系探讨了HS具有生理活性的内在机制.HS化学组成具有不确定性,超分子结构假设目前被普遍接受.HS通过促进根毛形成和侧根发育、激活质膜H+-ATP酶产生有利的电化学梯度以及自身酸性官能团的螯合作用等促进植物对矿质元素的吸收利用;HS具有类激素活性,植物根际促生细菌(PGPR)分泌物或HS自体组分吲哚-3-乙酸(IAA)能够激活细胞质膜H+-ATPase,HS还能够增加内源植物激素水平,促进植物生长发育;HS通过介导信使NO激活细胞质膜H+-ATPase,清除HS与细胞作用产生的活性氧(ROS),HS通过NO和抗氧化酶调节细胞内ROS稳态,介导根系发育和形态结构.本文还着重探讨了HS生理活性与化学组成的相关性,其中官能团—COOH和—OH活性位点的作用、醌基的电子传递,以及HS亲水疏水平衡与HS生理活性密切相关.未来HS研究工作的重点在于深入研究HS分子结构、明确具备生理活性的"共性结构原件"、阐明HS诱导胞内信号传导通路之间的交叉反馈机制、揭示HS对细胞内物质和能量代谢相关基因表达的调节机制.

摘要： 腐殖质(HS)是生物体外具备生物学活性的最稳定有机物形式之一。本文综述了HS的化学组成、结构特征和生理活性,依据HS化学组成、结构特征和生理活性的相互关系探讨了HS具有生理活性的内在机制。HS化学组成具有不确定性,超分子结构假设目前被普遍接受。HS通过促进根毛形成和侧根发育、激活质膜H+-ATP酶产生有利的电化学梯度以及自身酸性官能团的螯合作用等促进植物对矿质元素的吸收利用;HS具有类激素活性,植物根际促生细菌(PGPR)分泌物或HS自体组分吲哚-3-乙酸(IAA)能够激活细胞质膜H+-ATPase,HS还能够增加内源植物激素水平,促进植物生长发育;HS通过介导信使NO激活细胞质膜H+-ATPase,清除HS与细胞作用产生的活性氧(ROS),HS通过NO和抗氧化酶调节细胞内ROS稳态,介导根系发育和形态结构。本文还着重探讨了HS生理活性与化学组成的相关性,其中官能团—COOH和—OH活性位点的作用、醌基的电子传递,以及HS亲水疏水平衡与HS生理活性密切相关。未来HS研究工作的重点在于深入研究HS分子结构、明确具备生理活性的“共性结构原件”、阐明HS诱导胞内信号传导通路之间的交叉反馈机制、揭示HS对细胞内物质和能量代谢相关基因表达的调节机制。

摘要： 为从分子水平揭示宁夏枸杞钠的吸收积累机理,本试验采用原子吸收分光光度法和实时荧光定量PCR(RT-qPCR)方法,对盐胁迫下宁夏枸杞根中Na^+、K^+含量以及质膜和液泡膜Na^+/H^+转运蛋白与H^+-ATPase基因表达水平进行测定分析。结果表明,相同胁迫时间下,随着NaCl处理浓度的增加,枸杞根系中Na^+浓度总体呈缓慢增加趋势,K^+含量呈先增加后减少趋势,Na^+/K^+比值呈先减少后增加趋势;编码质膜和液泡膜的Na^+/H^+转运蛋白基因LbSOS1、LbNHX1以及液泡膜H^+-ATPase基因LbVHA-C1表达量均呈升高趋势,质膜H^+-ATPase基因LbHA1表达量呈先升高后降低趋势。相同NaCl处理浓度下,随着胁迫时间的延长,Na^+含量总体呈增加趋势,K^+含量呈先增加后减少趋势,Na^+/K^+比值呈增加趋势。LbSOS1、LbNHX1表达量总体呈先升高后降低趋势,LbVHA-C1、LbHA1表达量总体呈降低的趋势。相关性分析显示,不同胁迫时间下,枸杞根中LbSOS1、LbNHX1、LbVHA-C1和LbHA1表达量与Na^+含量存在一定的正相关或负相关。上述结果表明,在低浓度NaCl胁迫时,维持枸杞体内较高的K^+/Na^+比值是宁夏枸杞耐盐的主要方式之一,同时也说明在胁迫初期,质膜和液泡膜的Na^+/H^+转运蛋白与H^+-ATPase参与了枸杞细胞中Na^+及时排出胞外和区隔于液泡,从而保持了根细胞内Na^+的稳定性。此外,随着胁迫时间的延长和NaCl处理浓度的增加,LbSOS1、LbNHX1、LbVHA-C1和LbHA1的表达水平均降低,而Na^+积累量大幅增加,致使枸杞抗盐性降低。本研究揭示了宁夏枸杞的耐盐机理,为利用宁夏枸杞改良宁夏大面积盐碱地提供了理论依据。

摘要： [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.

摘要： 研究添加外源抑制剂双环己基碳二亚胺(DCCD)对植物乳杆菌H+-ATPase基因表达水平及酶活力的影响,探讨植物乳杆菌H+-ATPase的调控机制.分别测定加入DCCD前后的亲本菌和低H+-ATPase活力突变菌的生长能力、葡萄糖代谢速率、乳酸产量和H+-ATPase活性,并通过荧光定量PCR对编码H+-ATPase的相关基因进行相对定量分析.结果表明,亲本菌ZUST、突变菌ZUST-1和ZUST-2在添加DCCD后菌体生长能力均减弱,葡萄糖代谢速率减慢,乳酸产量降低,H+-ATPase酶活性也降低.由于受到发酵液中酸性环境以及外源抑制剂DCCD的胁迫,亲本菌ZUST和突变菌ZUST-2编码H+-ATPase的所有基因在稳定期表达水平都高于对数期.与未添加DCCD相比,添加DCCD的亲本菌和突变菌ZUST-2在对数期H+-ATPase各基因的表达水平均下调或基本不变,抑制了H+-ATPase活力从而导致菌株的生长代谢速率减弱.此研究结果为进一步揭示植物乳杆菌中H+-ATPase在酸胁迫下的调控机制奠定了基础.%In this paper,the effects of H+-ATPase activity and genes expression by adding N,N'-dicyclohexyl carbodiimide (DCCD) were explored,and the mechanism of H +-ATPase regulation in Lactobacillus plantarum was studied.The growth ability,glucose consumption rate,lactic acid yield and H +-ATPase activity of wild-type ZUST and mutants ZUST-1,ZUST-2 were measured,respectively.Then,the real-time RT PCR was used to evaluate the relative quantification of the H +-ATPase genes expression.Results revealed that after adding DCCD,the growth ability,glucose consumption rate,lactic acid yield and H +-ATPase activity were lower than those without adding DCCD.All the H +-ATPase coding genes of ZUST and mutant ZUST-2 were higher than those in the exponential phase,which indicated that the expression of all genes was up-regulated due to the dual effects of fermentation acid stress and exogenous inhibitor DCCD.Compared with that without adding DCCD,the H +-ATPase expression levels of Lb.plantarum ZUST and mutant ZUST-2 were reduced or unchanged in the logarithmic phase,the activity of H +-ATPase was inhibited and the growth rate of the strains decreased.These results would pave the way for gaining further insights into the regulation mechanism of the H +-ATPase in Lb.plantarum under acid stress.

摘要： We investigated the influences of exogenous chlorogenic acid (CGA) on the physiological parameters and Al stress-related gene expression of SB (Al-sensitive black soybean) root under Al stress.A major goal of this report was to explore the alleviating molecular mechanism of exogenous CGA on Al toxicity in SB.SB seedings were cultured in water solution and treated with 50 μmol·L-1 Al or CGA plus 50 μmol·L-1 Al.The optimal concentration of CGA alleviating Al toxicity was screened.And we study the effects of optimal exogenous CGA on root tip Al content,the activities of antioxidant enzyme,the expressions of 14-3-3 protein and H+-ATPase,and the activity of H+ pump under 50 μmol·L-1 Al stress.Low concentrations of CGA alleviated inhibition of root elongation caused by Al toxicity and promoted the increase of lateral root numbers under Al stress.However,the CGA-alleviated effect was weaken with high concentration of CGA treatment.It was founded that the alleviation effect of 0.01 g·L-1 CGA was the most significant.Exogenous 0.01 g·L-1 CGA decreased the Al in root tips and MDA contents.And the citric acid content in root exudates of SB under Al stress was significantly increased due to the addition of exogenous 0.01 g·L-1 CGA.Expression analysis showed that exogenous 0.01 g·L-1 CGA enhanced the expression of three 14-3-3 isoforms (14-3-3b,14-3-3m,and 14-3-3k) and increased the expression of GHA2 (plasma membrane H+-ATPase) gene,but inhibited the gene expression of MATE in SB under Al stress.The results of immunoprecipitation(IP) showed that the phosphorylation of the PM H+-ATPase was up regulated by the exogenous 0.01 g·L-1 CGA,which could bind with 14-3-3 protein under Al stress.Meanwhile,plasma membrane H+-ATPase and H+ pump activities were both enhanced by exogenous 0.01 g·L-1 CGA under Al stress.It was suggested that exogenous CGA may enhance the SB tolerance to Al stress by increasing the lateral roots number,compensating inhibition of MATE expression and interaction between them to increase citric acid exudation.%该文研究了外源绿原酸(CGA)对Al胁迫下铝敏感型黑大豆SB根生理生化指标以及根中胁迫相关基因表达的变化,探讨外源CGA缓解SB根铝毒害的效果及分子机理.以不同浓度Al和CGA处理SB,筛选出CGA缓解Al毒害的最佳浓度,测定Al含量、抗氧化系统酶活性、14-3-3蛋白与H+-ATP酶的表达、H+泵活性.结果表明:低浓度CGA能缓解Al胁迫下黑大豆SB根伸长抑制,并促进侧根数目增加,而高浓度CGA的缓解效果下降;0.01 g·L-1 CGA使Al胁迫下SB根尖Al含量与MDA含量下降,促进根系柠檬酸的分泌.RT-PCR和Western Bloting分析表明0.01 g·L-1 CGA促进Al胁迫下SB根中14-3-3b、14-3-3m、14-3-3k和GHA2基因(质膜H+-ATP酶)的表达,抑制MATE基因的表达.同时,0.01 g·L-1 CGA能促进Al胁迫下质膜H+-ATP酶蛋白磷酸化水平以及其与14-3-3蛋白结合,且能提高质膜H+-ATP酶和H+泵活性.因此推测外源CGA可能通过增加侧根数,增强14-3-3蛋白和质膜H+-ATP酶基因蛋白表达水平和互作,弥补Al胁迫下MATE表达的抑制,增加柠檬酸的分泌,增强SB对铝毒害的耐受性.

摘要： [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atpA基因均有22个位点发生突变,而ZUST-2的atpC基因有6个位点发生突变.突变菌ZUST-1和ZUST-2的atpA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atpC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atpC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atpC不表达外),而且atpA和atpC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.

摘要： 从肉鸭消化道内分离出的7株酿酒酵母益生菌中,筛选出高耐酸性的菌株,并探究酵母细胞内Na+、K+浓度及H+-ATPase活性与环境pH的相关性.结果显示:不同酵母菌株酸胁迫耐受性存在差异.酵母菌株通过泵出H+吸收K+来维持胞内pH稳定,表明膜H+-ATPase和K+在酵母菌株耐酸中发挥作用.7株酿酒酵母细胞膜H+-ATPase活性与其生长环境的pH呈负相关.Y-2和Y-7两株酵母菌株相对于其他菌株耐酸性更佳,可作为后续肉鸭专用饲用酵母益生菌的备选菌株.

摘要： In order to study the relation between the H+-ATPase and H+-PPase activities of plasmalemma and tono-plast and the sugar accumulation in fruit of Ziziphus jujuba cv.Lingwuchangzao,the H+-ATPase and H+-PPase activities of plasmalemma and tonoplast and the sugar content in fruit were determined with different developmental periods fruit as materials.The results were as follows:(1)The fruit mainly accumulated glucose and fructose before the second rapid growth period,sucrose accumulated rapidly after the second rapid growth period,at the same time, the content of glucose and fructose decreased gradually,the mature period fruit mainly accumulated sucrose.(2)The plasmalemma H+-ATPase activity was the lowest in the slower growth period S1 and the highest in the first rapid growth period,but it droped in the slower growth period S2 and rose to higher in the second rapid growth period and droped substantially in the mature period.(3)The variation trend of tonoplast H+-ATPase activity was similar to tonoplast H+-PPase activity during fruit development.Tonoplast H+-ATPase and H+-PPase activities were lower in the slower growth period S1 ,they droped slowly to the lowest from the slower growth period S1 to the first rapid growth period and increased gradually from the first rapid growth period,tonoplast H+-PPase activity was higher throughout than tonoplast H+-ATPase activity except the second rapid growth period.Hence,the plasmalemma H+-ATPase and the tonoplast H+-ATPase and H+-PPase played an important regulative role in secondary active transport sugar across membrane in fruit of Z.jujuba cv.Lingwuchangzao.%以不同发育时期灵武长枣(Ziziphus jujuba cv．Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜 H＋-ATPase和 H＋-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜 H＋-ATPase和 H＋-PPase活性与糖积累特性的关系.结果表明：(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖.(2)在果实发育的缓慢生长期S1,质膜H＋-ATPase活性最低；第一次快速生长期,质膜H＋-ATPase活性最高；缓慢生长期S2,其活性降低；第二次快速生长期,质膜 H＋-ATPase活性升至次高；完熟期,质膜 H＋-ATPase 活性下降幅度较大.(3)在果实发育过程中,液泡膜 H＋-ATPase和 H＋-PPase活性的变化趋势相似.缓慢生长期S1,液泡膜 H＋-ATPase和 H＋-PPase活性较低；从缓慢生长期S1至第一次快速生长期缓慢下降至最低；从第一次快速生长期开始,液泡膜 H＋-ATPase和 H＋-PPase活性呈现为逐渐增高的变化趋势；除第二次快速生长期以外,液泡膜 H＋-PPase 活性始终高于 H＋-ATPase.由此推测,质膜 H＋-ATPase 和液泡膜 H＋-ATPase、H＋-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用.