Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by micropro-jectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclo     

渗透胁迫下脱水蛋白DHN1在猕猴桃细胞中表达及亚细胞分布的动态变化

以绿色荧光蛋白(GFP)为报告分子, 通过构建DHN1-mGFP4融合蛋白表达载体, 研究了渗透胁迫条件下脱水蛋白DHN1的表达及其亚细胞分布的动态变化. 采用PCR方法在脱水蛋白基因dhn1两端引入XbaⅠ和BamHⅠ限制性内切酶位点, 克隆到质粒pBIN-35S mGFP4, 构建DHN1-mGFP4融合蛋白表达载体, 并采用基因枪转化猕猴桃(A. delicisoa)悬浮细胞. 培养10 h后观察到高效表达的GFP绿色荧光, 绿色荧光只出现在细胞核内. 提高培养介质渗透势后, 可以诱导脱水蛋白向细胞质分布(主要集中在质膜周围), 并且增加介质渗透势可以明显缩短细胞质出现绿色荧光的时间. 蛋白合成抑制剂环己亚胺能够抑制细胞质绿色荧光的出现, 暗示细胞质出现的脱水蛋白是诱导产生的结果. ABA可以明显促进细胞质绿色荧光的出现, 并且随着介质渗透势的升高迅速缩短细胞质绿色荧光的出现时间.     

植物细胞质膜氧化还原系统电子传递活性,组成及生理功能

植物细胞质膜氧化还原系统电子传递活性、组成及生理功能邱全胜梁厚果（兰州大学生物系,兰州７３０００１）ＴＨＥＥＬＥＣＴＲＯＮＴＲＡＮＳＰＯＲＴＡＣＴＩＶＩＴＹ,ＣＯＮＳＴＩＴＵＥＮＴＳＡＮＤＰＨＹＳＩＯＬＯＧＩＣＡＬＦＵＮＣＴＩＯＮＳＯＦＴＨＥＲＥＤ...     

双组分系统——细胞识别渗透胁迫信号的感应器

双组分系统是广泛存在于原核和真核细胞中的信号转导系统.主要由组氨酸蛋白激酶(HPK)和响应调节蛋白(RR)两个组分组成. 双组分系统信号通路一般包括信号的输入、HPK自身磷酸化、RR磷酸化、信号输出等环节.对双组分系统信号转导机制及其在渗透胁迫信号识别和传导中的作用进行了综述.     

植物质膜钾离子转运体研究进展

近年,随着分子生物学技术的不断发展和广泛应用,有关植物质膜钾离子转运体的研究取得重要进展。目前已经克隆到多种质膜钾离子转运体基因并对钾离子转运体生化特性以及结构功能进行了广泛研究。研究认为,质膜钾离子转运体可分为钾离子载体和钾离子通道。钾离子通道又可分为内向性K+通道α亚基、K+通道β亚基及外向性K+通道等三类。本文对上述质膜钾离子转运体的生化特性以及结构功能研究的进展进行了综述。     

小麦根质膜H^＋—ATPase的部分纯化

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）根为材料,采用不连续蔗糖密度梯度离心法制备高纯度质膜微囊。质膜经ＴｒｉｔｏｎＸ１００和ＫＣｌ处理后,再用Ｚｗｉｔｅｒｇｅｎｔ３１４增溶Ｈ＋ＡＴＰａｓｅ,最后用硫酸铵沉淀得到部分纯化的质膜Ｈ＋ＡＴＰａｓｅ。ＳＤＳＰＡＧＥ结果表明,经过上述步骤纯化,分子量为９４ｋＤ的膜蛋白组分得到富集;与质膜相比,其含量提高１５．７倍。部分纯化的质膜Ｈ＋ＡＴＰａｓｅ可以水解ＡＴＰ,受Ｋ＋刺激,并被Ｎ,Ｎ′ｄｉｃｙｃｌｏｈｅｘｙｌｃａｒｂｏｄｉｍｉｄｅ（ＤＣＣＤ）抑制;ＡＴＰ水解活力被Ｎａ３ＶＯ４抑制９５％,但不被ＮａＮ３、ＮａＮＯ３和Ｎａ２ＭｏＯ４抑制。     

K^＋对大豆下胚轴质膜H^＋—ATPase水解与质子转运活力偶联的影响

以大豆（ Ｇｌｙｃｉｎｅ ｍａｘ Ｌ．） 下胚轴为材料, 采用二相法制得高纯度质膜微囊。实验发现,Ｋ＋ 对质膜Ｈ＋＿ＡＴＰａｓｅ水解活力和转运活力刺激差别显著,对转运活力刺激８５０％ , 对水解活力仅刺激２８ ．２％ 。动力学结果表明,有Ｋ＋时ＡＴＰ水解的Ｋｍ 值为０．７０ ｍｍｏｌ／Ｌ,Ｖｍａｘ 为３４４ ．８ ｎｍｏｌ Ｐｉ·ｍｇ－１ ｐｒｏｔｅｉｎ·ｍｉｎ－１ ; 无Ｋ＋ 时ＡＴＰ水解的Ｋｍ 值为１ ．１４ｍｍｏｌ／Ｌ, Ｖｍａｘ为２８５．７ ｎｍｏｌ Ｐｉ·ｍｇ－１ ｐｒｏｔｅｉｎ·ｍｉｎ－１ 。Ｋ＋ 对ＡＴＰ水解的最适ｐＨ 值也有影响,有Ｋ＋ 时为６ ．５ ,无Ｋ＋ 时降低到６．０ 。进一步实验发现,Ｋ＋ 对羟胺和钒酸钠的抑制作用影响较大,Ｋ＋ 可以提高质膜Ｈ＋＿ＡＴＰａｓｅ 对羟胺和钒酸钠的敏感性。结果表明,Ｋ＋ 可以调节大豆下胚轴质膜Ｈ＋＿ＡＴＰａｓｅ 水解与转运活力之间的偶联程度     

胰蛋白酶处理对质膜H~ -ATPase钒酸钠抑制效应的影响

采用经蔗糖密度梯度法纯化的大豆 (GlycinemaxL .)下胚轴质膜微囊为材料 ,分析了胰蛋白酶处理对质膜H ATPase钒酸钠抑制效应的影响。实验结果显示 ,温和胰蛋白酶处理显著提高H ATPase的ATP水解活力。并且发现酶切处理降低了钒酸钠对ATPase的抑制效应 ,当钒酸钠浓度为 2mmol/L时 ,ATPase活力仅被抑制 5 3.49% ,而未经酶切的对照组则被抑制 6 4.13%。ATP水解动力学分析表明 ,胰蛋白酶酶切处理既不影响ATP水解的Km 值也不影响钒酸钠的抑制类型 ,酶切前后的Km 值都等于 0 .34mmol/L ,并且都属于反竞争抑制。以上结果显示胰蛋白酶酶切处理可能改变了磷酸酶结构域的结构而影响了钒酸钠的抑制效应 ,暗示C_末端调节着磷酸酶结构域的结构和功能     

Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by microprojectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclohexylcarbodiimid, indicating that the cytoplasmic DHN1 was newly synthesized under osmotic stress. Furthermore, ABA promoted the presence of green fluorescence in the cytoplasm, and the GFP fluorescence was visualized within a shorter time under a higher osmotic potential.     

水分胁迫对小麦根细胞质膜氧化还原系统的影响

水分胁迫使小麦根质膜ＮＡＤＨ和ＮＡＤＰＨ的氧化速率及Ｆｅ（ＣＮ）６３－和ＥＤＴＡ－Ｆｅ３＋的还原速率明显降低。对照与胁迫处理的质膜氧化还原系统活性均不受鱼藤酮抗霉素Ａ和ＫＣＮ等呼吸链抑制剂的影响。在不加Ｆｅ（ＣＮ）６３－作为电子受体时,水杨基羟肘酸（ＳＨＷ）可明显刺激质膜ＮＡＤＨ的氧化和Ｏ２吸收速率。水分胁迫促使ＳＨＡＭ刺激的ＮＡＤＨ氧化明显降低,但却使Ｏ２吸收略有上升。