两相分配法制备玉米根质膜及其纯度鉴定

用Ｄｅｘｔｒａｎ Ｔ５００,ＰＥＧ３３５０两相体系制备玉米根质膜,首先在高盐浓度下选用五种不同的聚合物浓度,研究了玉米根系膜在两相体系中的分配情况,在此基础上研究了ＮａＣｌ浓度对玉米根质膜的纯度及得率的影响。结果表明,制备了玉米根质膜选用６．２％聚合物浓度,７．５ｍｍｏｌ／Ｌ ＮａＣｌ的两相体系比较合适。     

玉米根ABA结合蛋白的亚细胞定位及动力学性质

以玉米（Ｚｅａ ｍａｙｓＬ．）根或胚芽鞘为材料,经匀浆、分级离心得到胞质部分和膜部分（微粒体）,进一步用６．２％ （Ｗ／Ｗ ） Ｄｅｘｔｒａｎ Ｔ５００ 和ＰＥＧ ３３５０ 两相系统制备质膜,用１％ 和８％ （Ｗ ／Ｗ） Ｄｅｘｔｒａｎ Ｔ７０ 梯度离心制备液泡膜． 电镜鉴定及多种标志酶检测表明,制备获得了高纯度正向型质膜和富含液泡膜的组分,其它内膜的污染很少． 用微量放射配体结合（ＭＲＬＢ）实验证明,玉米根微粒体的ＡＢＡ专一性结合位点主要分布在液泡膜和质膜上,这两种膜组分与ＡＢＡ 的特异结合活性分别为２４８５．４ ｆｍ ｏｌ／ｍ ｇ ｐｒｏｔｅｉｎ 和１２５７．３ ｆｍ ｏｌ／ｍ ｇ ｐｒｏ－ｔｅｉｎ,玉米根段胞质部分结合活性最低（差一个数量级）．质膜上ＡＢＡ－ＢＰ与ＡＢＡ 的结合平衡解离常数（ＫＤ）为１．５７ ｎｍ ｏｌ／Ｌ．     

两相分配法制备玉米根质膜及其纯度鉴定

用ＤｅｘｔｒａｎＴ５００,ＰＥＧ３３５０两相体系制备玉米根质膜．首先在高盐浓度（２２ｍｍｏｌ／ＬＮａＣｌ）下选用五种不同的聚合物浓度（５．８％、６．０％、６．２％、６．３％、６．４％,Ｗ／Ｗ）,研究了玉米根质膜在两相体系中的分配情况,在此基础上进一步研究了Ｎａ－Ｃｌ浓度（２、４、５、１１、２２ｍｍｏｌ／Ｌ）对玉米根质膜的纯度及得率的影响．结果表明,制备玉米根质膜选用６．２％（Ｗ／Ｗ）聚合物浓度,７．５ｍｍｏｌ／ＬＮａＣｌ的两相体系比较合适．标志酶鉴定及低ｐＨ值磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡,质膜标志酶ＶＯ３－４－ＡＴＰａｓｅ的活性潜势达８８．９％．     

褪黑素对大鼠海马神经元谷氨酸所致毒性的拮抗作用

在大鼠海马脑片上电刺激Ｓｃｈａｆｆｅｒ 侧支纤维, 胞外记录ＣＡ１ 区锥体细胞层诱发群体锋电位（ｐｏｐｕｌａｔｉｏｎ ｓｐｉｋｅ,ＰＳ） , 观察灌流谷氨酸（Ｇｌｕ） 和褪黑素（ＭＥＬ） 对ＰＳ的影响。结果显示：５－０ ｍｍｏｌ／Ｌ浓度的Ｇｌｕ 可使ＰＳ值下降至对照值的４－１ ％ ; ＭＥＬ（０－４ 、０－５ 和０－６ μｍｏｌ／Ｌ） 与５－０ ｍｍｏｌ／ＬＧｌｕ 混合给药,ＰＳ值分别变化为对照值的１４－７ ％ 、１０５－２％ 、２４－３ ％ ; ＭＥＬ（０－５ μｍｏｌ／Ｌ） 、Ｇｌｕ （５－０ ｍｍｏｌ／Ｌ） , 与赛庚啶（ＣＤＰ,０－５ μｍｏｌ／Ｌ） 混合给药,ＰＳ值下降至０ 。上述结果提示,５－０ ｍｍｏｌ／Ｌ浓度的Ｇｌｕ 有神经毒性作用, 但可为ＭＥＬ拮抗, 这可能由５ＨＴ受体所介导。     

2,4—D和乙烯利对玉米幼苗抗旱性效应的研究

水分胁迫下,２０ｍｇ／Ｌ２,４－Ｄ和４００ｍｇ／Ｌ乙烯利（ＣＥＰＡ）分别不同程度地降低和提高玉米幼叶生长部位的相对含水量（ＲＷＣ）、水势（ψｗ）和渗透调节能力（ＯＡ）。胁迫后期,玉米幼叶中脯氨酸含量（Ｐｒｏ）为：ＣＥＰＡ〉对照（ＣＫｓ）〉２,４－Ｄ。随胁迫宾进行,２,４－Ｄ处理幼叶的膜相对透性（ＲＰ）始终处于较高水平且增长幅度较大,叶片延伸生长速率（ＬＥＲ）对ＲＰ的变化较为敏感;而ＣＥＰＡ处理则与     

百草枯和苯甲酸钠对渗透胁迫下抗旱性不同玉米品种愈伤组织的影响

两个品种玉米愈伤组织经０．５和５ｍｍｏｌ／Ｌ的百草枯处理３ｈ后,在渗透胁迫下处理２４ｈ电解质泄漏率增加;Ｈ２Ｏ２和ＭＤＡ积累;ＡｓＡ和ＣＡＲ含量的减少加剧。０．５和５ｍｍｏｌ／Ｌ的苯甲酸钠减轻渗透胁迫诱导的氧化伤害,促进ＣＡＴ活性的增加;使ＳＯＤ、ＧＲ、ＡＰ和ＰＯＤ能维持较高的活性;ＡｓＡ和ＣＡＲ含量降低的幅度减小。     

鸡蛋果幼叶细胞质丙酮酸激酶的调节特性

鸡蛋果幼叶ＰＫｃ的Ｋｍ（ＰＥＰ）为０．０３７ｍｍｏｌ／Ｌ,Ｋｍ（ＡＤＰ）为０．０５ｍｍｏｌ／Ｌ,与成长叶相同。幼叶ＰＫｃ的效应物与成长叶的有较大差异,草酸和Ｃａ２＋对幼叶ＰＫＣｃ抑制机制与成长叶相同,但亲和力不同,这可能反映了酶的非活性部位具有不同的构象。     

鸡蛋果幼叶细胞质丙酮酸激酶的调节特性

鸡蛋果幼叶ＰＫｃ的Ｋｍ（ＰＥＰ）为０．０３７ｍｍｏｌ／Ｌ,Ｋｍ（ＡＤＰ）为０．０５ｍｍｏｌ／Ｌ,与成生叶相同。幼叶ＰＫｃ的效应物与成长叶的有较大差异,草酸和Ｃａ＾２＋对幼叶ＰＫｃ的抑制机制与成长叶相同,但亲和力不同,这可能反映了酶的非活性部位具有不同的构象。     

褐黑素对大鼠海马神经元谷氨酸所致毒性的拮抗作用

在大鼠海马脑片上电刺激Ｓｃｈａｆｆｅｒ侧支纤维,胞外记录ＣＡ１区锥体细胞层诱发群体锋电位,观察灌流谷氨酸和褪黑素对ＰＳ的影响。结果显示：５．０ｍｍｏｌ／Ｌ浓度的Ｇｌｕ可使ＰＳ值下降至对照值的４．１％;ＭＥ（０．４、０．５和０．６μｍｏｌ／Ｌ）一５．０ｍｍｏｌ／Ｌ浓度的Ｇｌｕ可使ＰＳ值下降至对照值的１４．７％、１０５．２％、２４．３％;ＭＥＬ、Ｇｌｕ,与赛庚啶混合给药,ＰＳ值下降至０。上述结果提示。     

盐生杜氏藻甘油-3-磷酸脱氢酶的分离纯化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,ＢｌｕｅＳｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化盐生杜氏藻（Ｄｕｎａｌｉｅｌａｓａｌｉｎａ（Ｄｕｎａｌ）Ｔｅｏｄ．）甘油三磷酸（Ｇ３Ｐ）脱氢酶（ＥＣ１．１．１．８）,得到比活为１２．６Ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４％～２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶催化磷酸二羟丙酮（ＤＨＡＰ）还原的最适ｐＨ值为７．５,催化Ｇ３Ｐ脱氢的最适ｐＨ值为１０。该酶对４个底物还原型辅酶Ⅰ（ＮＡＤＨ）,二磷酸吡啶核苷酸（ＤＨＡＰ）,辅酶Ⅰ（ＮＡＤ）,Ｇ３Ｐ的表观Ｋｍ值分别为６３μｍｏｌ／Ｌ,２７２μｍｏｌ／Ｌ,１．５３ｍｍｏｌ／Ｌ,６．５２ｍｍｏｌ／Ｌ。该酶在保存过程中易失活。ＮＡＤＨ能降低酶失活的速度,而ＮＡＤ则不然。低浓度ＮａＣｌ对酶略有保护作用,但高浓度ＮａＣｌ加快酶的失活,且浓度越高效应越明显。     

水分胁迫对玉米幼叶生长区细胞质膜H~+-ATPase活性的影响

PEG/Hoagland(-0.1 MPa)溶液根际胁迫处理5叶期玉米幼苗24h,明显刺激第5幼叶生长区细胞H?的分泌,比对照高约1.5倍,钒酸钠对此过程有强烈抑制作用。用水溶性多聚物(PEG4000/Dextran T 500)两相分配法分离玉米第5幼叶生长区的质膜,胁迫处理增加了质膜H~+—ATPase对底物的亲和力,K_m降低到对照的?;活力增加约1.5倍。亚胺环己酮对胁迫处理引起的质膜H~+—ATP_(ase)活性增加没有抑制作用,不同程度的胁迫处理会导致膜制剂磷脂/蛋白比率的变化,其比率在0.83～1.05之间时.随比率的增加,质膜 H~+—ATP_(ase)活性迅速上升;比率在1.05～1.37之间时,随比率值增加,活性迅速下降。暗示质膜的磷脂含量变化可能是胁迫导致质膜 H~+—ATP_(ase)活性增加的主要原因。     

Effect of Salt Stress on Ion Channel Selective Permeability of Plasmalemma in Sorghum Roots

100 mmol/L, 200 mmol/L and 300 mmol/L NaC1 was used in proper order to treat three-day old seedlings of Sorghum vulgare Pets. The plasma membrane of roots was isolated and purified by aqueous biphasic partition device. The plasmalemma was incorporated into planar bilayer lipid membrane and ion channels were measured by electrical methods. Ion selective permeabilities (PK: PNa) were assayed in asymmetrical solutions containing 100 mmol/L NaC1 in Cis chamber and 100 mmol/L KC1 in Tran with chamber and were calculated from Goldman-Hodgkin-Kaltz equation. PK: PNa was 3.5 in plasmalemma of control roots and 1.5 in plasmalemma of salt stressed roots. It showed that the changes of ion selective permeability was very significant under salt stress. The importance of the change of ion selective permeability is discussed.     

用两相法纯化玉米精细胞质膜

纯系玉米(Zea mays L.)708的新鲜花粉经蔗糖溶液等渗温育、低渗胀破,用30％Percoll不连续密度梯度离心,制备大量的生活精细胞。精细胞用French压力室破碎后离心(90000×g,4℃),获得微粒体。随后,用16g的(6.2％Dextran T500/6.2％PEG3350)两相系统加以纯化。一级分离后,上相液(U_1)的K~ 刺激的Mg~(2 )-ATP酶活性稍低于下相液(L_1);对U_1进行二级分离后,U_2的K~ 刺激的Mg~(2 )-ATP酶活性远远高于下相液(U_2L),分离率达到61.1％。膜蛋白质的分离趋势与K~ 刺激的Mg~(2 )-ATP酶相似。选用细胞色素C氧化酶作为线粒体的标志酶,虽只经一级分离,但在U_1中已检测不到该酶活性。精细胞质膜的磷钨酸特异染色电镜观察结果显示,由二级分离所得质膜的纯度达到90％以上。     

水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.     

Chlorophyll Biosynthetic Reactions during Senescence of Excised Barley (Hordeum vulgare L. cv IB 65) Leaves

The inhibitor sensitivity of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium pumps of red beet (Beta vulgaris L.) were studied by measuring the ATP-driven accumulation of 45Ca2+ into isolated membrane vesicles. Both transporters were strongly inhibited by 50 [mu]mol m-3 erythrosin B, but only by 50% in the presence of 100 mmol m-3 vanadate. A number of inhibitors considered to be specific for the sarcoplasmic reticulum (SR)/ER-type calcium pump in animal cells were used to further characterize the PM and ER Ca2+-ATPases in red beet and were compared with their effect on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-1,4-benzohydroquinone and 2,5-di(tert-amyl)-1,4-benzohydroquinone produced around 20 and 40% inhibition of activity, respectively, of the PM and ER calcium pumps and the PM H+-ATPase when present at concentrations of 30 mmol m-3. In contrast, the vacuolar proton pump displayed a much higher sensitivity to these two compounds. Nonylphenol appeared to have a general inhibitory effect on all four membrane transport proteins and gave almost complete inhibition when present at a concentration of 100 mmol m-3. Thapsigargin and the structurally related compound trilobolide produced 50% inhibition of both the ER and PM calcium pumps at concentrations of 12.5 and 24 mmol m-3, respectively. The PM and tonoplast proton pumps were also sensitive to these compounds. The ER and PM calcium pumps were almost completely insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 mmol m-3. When present at 100 mmol m-3 CPA caused 30% inhibition of the transport properties of all four ATPases. The high concentrations of all of the inhibitors of the SR/ER Ca-ATPase required to inhibit the red beet ER calcium pump, together with the similar effects on the PM calcium pump and the PM and tonoplast proton pumps, suggests that these hydrophobic compounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions.     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchR1表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱（acetylcholine,Ach）及拮抗剂阿托品（atropine,Atro）对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型（mAchR1）和c-fos的表达差异。结果表明,1mmol／L Ach对SK-N-SH细胞有明显促增殖作用,而1mmoL／L Atro阻滞细胞从S期向G2／M期移行;1mmol／L Ach与1mmol／L Atro均反馈调节mAchR1的蛋白水平。但mAchR1 mRNA的表达不受影响;1mmol／L Ach显著上调c-fos mRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

乙酰胆碱及其拮抗剂对人SK-N-SH细胞增殖及mAchRl表达的影响

用CCK-8比色法和流式细胞术,检测乙酰胆碱(acetylcholine,Ach)及拮抗剂阿托品(at-ropine,Atro)对SK-N-SH细胞增殖活性和周期分布的调节作用;进一步用荧光定量PCR、免疫印迹和流式细胞间接免疫荧光技术,分析SK-N-SH细胞毒蕈碱受体亚型Ⅰ型(mAchR1)和c-fos的表达差异。结果表明,1mmol/LAch对SK-N-SH细胞有明显促增殖作用,而1mmol/LAtro阻滞细胞从S期向G_2/M期移行;1mmol/LAch与1mmol/LAtro均反馈调节mAchR1的蛋白水平,但mAchR1mRNA的表达不受影响;1mmol/LAch显著上调c-fosmRNA和Fos蛋白的表达,但这种作用可被Atro逆转。提示胆碱类受体参与配基对肿瘤细胞的促增殖作用。     

Effect of Plastic Film Mulching on the Grain Filling and Hormonal Changes of Maize under Different Irrigation Conditions

Plastic film mulching (PM) is widely utilized for maize production in China. However, the effect of PM on the grain yield of crops has not been established, and the biochemical mechanism underlying the increase or decrease in grain yield under PM is not yet understood. Grain filling markedly affects the grain yield. The objective of this study was to investigate the effects of PM on maize grain filling under different irrigation levels and the relationship of such effects with hormonal changes. In the present study, PM was compared with traditional nonmulching management (TN) under 220 mm, 270 mm and 320 mm irrigation amount, and the grain filling characters of the grains located in various parts of the ear and the hormonal changes in the grains were measured. The results indicated that at 220 mm irrigation, PM significantly increased the grain filling rate of the middle and basal grains and decreased the grain filling rate of the upper grains. At 270 mm irrigation, the PM significantly increased the grain filling rate of the all grains. At 320 mm irrigation, the PM only significantly increased the grain filling rate of the upper grains. The IAA, Z+ZR and ABA content in the grains was positively correlated with the grain weight and grain-filling rates; however, the ETH evolution rate of the grains was negatively correlated with the grain weight and grain-filling rates. These results show that the effect of PM on maize grain filling is related to the irrigation amount and that the grain position on the ear and the grain filling of the upper grains was more sensitive to PM and irrigation than were the other grains. In addition, the PM and irrigation regulated the balance of hormones rather than the content of individual hormones to affect the maize grain filling.     

玉米氮素吸收的基因型差异及其与根系形态的相关性

采用溶液培养的方法,选用在田间、土培试验中对氮反应有典型差异的玉米自交系：478、H21、Wu312、Zong31、Baici,在4个供N水平（0.04、0.4、2.4mmol/L）下,研究了玉米苗期氮素吸收、分配的基因差异以及与根系形态之间的相关关系,结果表明：在一定的NO3^-浓度范围内（0.04-2mmol/L）,根系生物量随N水平的提高而增加,而高N不同程度地降低了5个自交系根系干重。低N下（0.04mmol/L）,与其它自交系相比,N高效基因型478具有较大的根系生物量,其根系干重分别为H21、Wu312、Zong31、Baici的1.1、1.74、1.6、1.18倍,并往根系分配了较大比例的N素,根系N累积占总N量的百分率比Wu312、Zong31分别高18.34％、17.08％,而N低效基因型Wu312、Zong31则往地上部分配了较大比例的氮素。随N水平的增加,显著促进了地上部的生长,并在地上部分配了较大比例的N素。当N水平增至4mmol/L时,地上部N素分配的基因型差异减小。低N下,5个自交系根系干重、总根长、根轴总长与总吸N量显著线性相关,而高N下不表现相关关系,说明在N素胁迫的条件下,根系形态对N吸收效率起重要作用。