关键词： AGG1   isoprenylation   signal transduction   AKT/PROTEIN KINASE-B   PHOSPHOLIPASE C-BETA   GTP-BINDING PROTEINS   COUPLED RECEPTORS   PLASMA-MEMBRANE   CELL-DIVISION   RESIDUES   GENE   MUTAGENESIS   RESISTANCE  

摘要： Heterotrimeric G proteins (G-proteins) are a diverse class of signal transducing proteins which have been implicated in a variety of important roles in plants. When G-proteins are activated, they dissociate into two functional subunits (a and the beta gamma, dinner) that effectively relay the signal to a multitude of effectors. In animal systems, the beta gamma dimer is anchored to the plasma membrane by a prenyl group present in the gamma subunit and membrane localization has proven vital for heterotrimer function. A semi-dominant negative strategy was designed aiming to disrupt heterotrimer function in Arabidopsis thaliana (ecotype Columbia) plants by over-expressing a truncated y subunit lacking the isoprenylation motif (gamma*). Northern analysis shows that the levels of expression of the mutant -y subunit in several transgenic lines (35S-gamma*) are orders of magnitude higher than that of the native subunits. In-depth characterization of the 35S-gamma* lines has been carried out, specifically focusing on a number of developmental characteristics and responses to several stimuli previously shown to be affected in alpha- and [beta-deficient mutants. In all cases, the transgenic lines expressing the mutant gamma subunit behave in the same way as the alpha- and/or the beta-deficient mutants, albeit with reduced severity of the phenotype. Our data indicates that signaling from both functional subunits, a and the beta/gamma dimer, is disrupted in the transgenic plants. Even though physical association of the subunits has been previously reported, our research provides evidence of the functional association of alpha and beta with the gamma subunits in Arabidopsis, while also suggesting that plasma membrane localization may be critical for function of plant heterotrimeric G proteins. (C) 2007 Elsevier B.V. All rights reserved.

关键词： BASIPETAL AUXIN TRANSPORT   LATERAL ROOT DEVELOPMENT   ALPHA-SUBUNIT   MOLECULAR-CLONING   PLASMA-MEMBRANE   COMPLEX TRANSLOCATION   INDOLEACETIC-ACID   PHOSPHOLIPASE-C   CELL-DIVISION   GENE  

摘要： The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical G alpha and G beta subunit genes and two G gamma subunit genes ( AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gg genes revealed specialized roles for each Gg subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that G beta gamma 1 and G beta gamma 2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gg subunit in this root response differs, with Gbg1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbg2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in G beta gamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both G gamma subunits form functional G beta gamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein - mediated signaling in plants.

关键词： 拟南芥   生长素   异三聚体G蛋白   生长发育   钙离子通道  

摘要： 本实验以拟南芥的野生型(ws)和异三聚体G蛋白α亚基基因GPA1缺失突变体(gpa1,gpa1-2)和超表达突变体(wGα,cGα)为材料,探索不同种类的生长素对拟南芥根系生长发育的影响,并对异三聚体G蛋白在生长素调控根生长发育的信号转导过程中的作用及其下游效应器进行了研究,为揭示生长素调控植物根系生长发育的作用机理提供理论依据。主要实验成果如下: 1.以野生型拟南芥(ws)为实验材料,通过施加不同浓度(0-0.2 mg·L)不同种类的生长素类物质(NAA,2,4-D,IAA,IBA,),对野生型拟南芥在不同种类的生长素作用下根生长发育状况进行了观测。结果表明:4种生长素均能抑制野生型拟南芥主根伸长而促进侧根生长;随着生长素浓度的升高,4种生长素对野生型拟南芥主根的抑制作用均逐渐增强,其中2,4-D的抑制作用最强,在浓度很低时(0.01 mg·L )主根生长就表现出受抑制;野生型拟南芥的侧根生长对于4种生长素的敏感性不同,其中2,4-D的促进作用最弱,低浓度(0.01 mg·L)促进侧根生长,随着浓度的升高,促进作用下降。NAA在时0.05mg·L的促进作用最强。 2.以拟南芥的野生型(ws)和异三聚体G蛋白α亚基基因GPA1缺失突变体(gpa1,gpa1-2)和超表达突变体(wGα,cGα)为材料,通过施加不同浓度梯度、不同种类的生长素类物质(NAA,IAA,IBA,2,4-D),对5种基因型拟南芥的根生长发育的一些形态指标进行了观测比较。结果表明:G蛋白α亚基未参与生长素诱导的拟南芥主根的生长发育过程而参与了NAA、2,4-D和IAA诱导的拟南芥侧根生长发育过程,且G蛋白在NAA诱导的拟南芥侧根生长发育过程中可能起正调节作用,而在2,4-D和IAA诱导的拟南芥侧根生长发育过程中可能起负调节作用,在IBA诱导的拟南芥侧根生长发育过程中可能不起作用。 3.在证实了NAA、2,4-D调控根生长发育的信号转导过程中有G蛋白参与后,而在G蛋白的下游必定有响应的效应器,这些效应器中有可能包含Ca通道,在上述试验的基础上施加钙离子通道有机抑制剂:异搏定,地而流卓(100μmol·L);钙离子通道无机抑制剂:氯化镧和氯化铝(10μmol·L、50μmol·L、100μmol·L)进行研究,观察其对于根生长发育的影响以及对生长素生物学效应的阻断作用。实验结果显示:细胞外钙离子与NAA诱导的拟南芥根生长发育关系密切;钙离子通道有机抑制剂的施加对NAA调控根生长发育过程无明显影响,而钙离子通道无机抑制剂的施加对NAA调控根生长发育过程有影响;表明在NAA诱导的拟南芥根生长发育过程中细胞外钙离子可能通过电压依赖型钙通道进入细胞内部参与侧根的生长发育,即在NAA诱导的拟南芥侧根生长发育过程中G蛋白的下游效应器可能是Ca通道。而细胞外钙对2,4-D调控根生长发育无明显影响,表明Ca可能未参与2,4-D诱导的拟南芥侧根生长发育的信号转导过程。

关键词： GCN5-RELATED N-ACETYLTRANSFERASE   HISTONE ACETYLTRANSFERASE   MACROMOLECULAR STRUCTURES   AUTOINDUCTION MEDIUM   CATALYTIC MECHANISM   CRYSTAL-STRUCTURE   PROGRAM   SUPERFAMILY   DATABASE   COMPLEX  

摘要： We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 angstrom with an R factor of 14.9% (R-free = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with N-15 and C-13 + N-15. The X-ray structure and NMR ensemble were closely similar with rmsd 1.4 angstrom for residues 8-93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in N-15-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540-CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys(76) and His(41) residues of this family.

关键词： 异三聚体G蛋白   生长素   赤霉素   细胞分裂素   信号转导   植物激素  

摘要： 本论文以拟南芥野生型(ws生态型)、异三聚体G蛋白α亚基基因缺失突变体利超表达株系五种材料作为试验材料，采用生长素(NAA)、细胞分裂素(6-BA)和赤霉素对拟南芥的幼苗和生长发育的植株进行处理，对激素处理前后根系生长状况、植株高度、叶片形状、抽苔时间等参数进行了测量统计。并比较了不同基因型幼苗之间生理指标在植物激素处理前后变化情况的差异，试图寻找异三聚体G蛋白参与植物激素信号转导过程的证据。\n 试验结果显示，在生长素处理之后，拟南芥根系生长受到抑制、不定根数量有明显增加，生长素对不定根的诱导与异三聚体G蛋白表达量之间存在明显正相关。生长素能够明显促进植株高度的增加，增加幅度与异三聚体G蛋白有一定关联。在生长素刺激后，叶片的生长受到一定程度抑制，该效应也与异三聚体G蛋白有关。推测在生长素信号转导过程中，可能有异三聚体G蛋白的参与。\n 细胞分裂素刺激之后，能够抑制拟南芥根系生长、促进植株增高，但是对叶片生长的影响不大，不同基因型对细胞分裂素刺激的反应基本一致，推测异三聚体G蛋白可能参与了细胞分裂素抑制根生长的信号转导过程。\n 赤霉素处理显著增加了植株高度，同时促使抽苔时间提前，野生型和超表达株系的反应程度明显比缺失突变体高，而在GA促进抽苔的生理反应中，缺失突变体对GA处理没有任何反应。结果表明异三聚体G蛋白参与了GA信号转导过程。\n 总体上看，除了在某些反应中处于绝对必需的位置(如GA诱导抽苔的反应)外，在大多数条件下异三聚体G蛋白都只是起到了加强作用，即有G蛋白的条件下，某些激素调节的生理反应程度明显加强，而在没有异三聚体G蛋白的条件下，植物激素可以通过某些不依赖于G蛋白的信号转导途径启动与生长发育有关的生理生化反应。

关键词： 拟南芥   生长索   异三聚体G蛋白   突变体  

摘要： 以拟南芥的野生型（ws）、异三聚体G蛋白α亚基基因GPA1缺失突变体（gpal-1，gpal-2）和超表达突变体（wGa，cGa）为材料，通过施加不同浓度（0～0．2mg／L）的NAA处理，对拟南芥根生长发育的一些形态指标进行了观测比较。结果表明：（1）随着培养基中NAA浓度的不断升高，5种基因型主根的伸长生长均受到抑制，且抑制作用随浓度升高而增强；4种突变体和野生型主根的生长在相同浓度NAA处理下，无明显差异；（2）NAA在一定浓度范围内，对拟南芥侧根的生长发育起促进作用；在NAA诱导的侧根生长中，G蛋白超表达突变体比野生型更敏感，缺失突变体则不敏感。初步证明G蛋白不参与主根生长发育的调节，而在侧根生长发育中可能起正调节作用。

关键词： ABA   Arabidopsis thaliana   AtRGS1 protein   drought tolerance   RGS1 overexpression   HETEROTRIMERIC G-PROTEIN   ABSCISIC-ACID BIOSYNTHESIS   ALPHA-SUBUNIT GPA1   CELL-PROLIFERATION   SEED-GERMINATION   SYNTHASE GENE   GAMMA-SUBUNIT   RGS PROTEINS   BETA-SUBUNIT   THALIANA  

摘要： Regulator of G-protein signalling (RGS) proteins identified recently in Arabidopsis have been involved in the regulation of several physiological processes, but largely nothing is known about their roles at both the physiological and the molecular level. In the experiments reported here, the overexpression approach was used to present evidence that RGS1 protein plays critical roles in plant development and in modulating abscisic acid (ABA) and drought stress signal transduction. RGS1 affected the shapes of leaves, the development of floral buds, the elongation of stems, siliques, and hypocotyls, and the time of flowering. Post-germination growth was inhibited by 1 mu M ABA, and root growth was hypersensitive to ABA for 35S-RGS1 transgenic plants. RGS1 overexpression conferred more drought tolerance to transgenic plants, as compared with the wild type (Columbia). Reverse transcription-PCR (RT-PCR) results indicated that RGS1 overexpresssion significantly stimulated the expression of NCED and ABA2, that encode two key enzymes catalysing ABA biosynthesis. Furthermore, the expression of several stress-regulated genes was either up- or down-regulated in RGS1-overexpressing transgenic plants. Combining the results above with previous results, it is suggested that RGS1 exerted its effects on plant responsiveness to ABA and drought tolerance largely through changing the expression either of genes responsible for ABA biosynthesis, which leads to changes in endogenous ABA levels, or of stress-responsive genes.

关键词： 拟南芥   异三聚体G蛋白   生长素   根系发育   信号转导  

摘要： 植物异三聚体G蛋白对生长发育过程的调控作用,与其对外源或内源的生长调节信号物如生长素、赤霉素、脱落酸、乙烯、油菜素内酯及蔗糖等的转导密切相关,本文以拟南芥异三聚体G蛋白α亚基缺失突变体agb 1-2,β亚基缺失突变体gpa 1-3 ,二者的双突变体αβ36以及对应的生态型Col为研究材料,利用分子生物学、生物化学、植物生理学手段从不同角度分析了α、β亚基在根系发育中的作用;初步探讨了异三聚体G蛋白在生长素调节侧根发生发育过程中的作用路径,主要结果如下: 1、侧根发生开始于萌发后的第二天,与野生型相比,萌发后的1-2天内gpa1-3植株的侧根原基发生较多,第三天开始原基增加变慢、侧根发生进程滞后,其侧根发育主要集中于3-5时期,agb1-2与αβ36植株中侧根原基细胞发生较快,两者的侧根总数均显著高于gpa1-3及Col,以上结果说明,异三聚体G蛋白直接或间接地影响了拟南芥的侧根原基发生。 2、不同浓度的外源生长素、低浓度生长素运输抑制剂TIBA(10-7M)处理,各基因型无菌苗主根的平均每日增长速率无显著性差异,暗示G蛋白对主根伸长的影响较小。 3、外源IAA、NAA处理下各基因型的侧根发生均增多,随外源生长素处理浓度的加大,各突变体间表现出对生长素浓度变化的响应。10-7M TIBA处理显著抑制侧根发生,且处理后各基因型间侧根发生的差异消除。 4、ELISA法测得四种基因型根系中内源生长素含量由高到低依次为:gpa1-3,Col ,αβ36,agb1-2;萌发4天时去顶处理, agb1-2的生长素含量显著增加, gpa1-3去顶前后生长素含量未发生显著变化。 5、RT-PCR结果表明,根中agb1-2的生长素受体TIR1的表达量最低,细胞分裂周期蛋白CDC2a的表达量最高。根据以上结果并结合前人报道,表明异三聚体G蛋白调节生长素介导的侧根发育及拟南芥其他发育过程,且G蛋白不同亚基的作用不同。GPA1通过影响生长素极性运输的途径影响侧根发生,突变体gpa1-3对生长素运输的敏感性下降;Gβ亚基在G蛋白调节生长素对侧根发生的作用中起着负调节作用,且此调节作用与生长素的极性运输有关,与生长素进入细胞的途径没有关系。

关键词： 拟南芥   小G蛋白   脱落酸   信号转导   Rab类小G蛋白  

摘要： 小G蛋白是信号转导的重要分子开关，其进化非常保守，它与不同的调控因子和效应器分子相互作用，产生不同的功能。本研究以一个在衰老叶片中表达量显著升高的小G蛋白基因RabD2b为研究对象，通过分析各种胁迫处理条件下RabD2b基因的表达变化，观察T-DNA插入突变体、RabD2b过量表达植株和野生型植株表现型的观察初步研究了RabD2b基因的功能。主要结果如下： 1．表达谱分析检测表明，RabD2b基因在衰老叶片、花蕾和开放的花中表达量很高，特别是在衰老叶片和开放的花朵中，表达量大大升高。胁迫处理Northern杂交结果表明，RabD2b基因在干旱、盐、ABA和JA的胁迫处理下表达量上升。 2．对RabD2b T-DNA插入突变体系纯合突变体、CaMV 35S-RabD2b增强转化植株以及野生型的生长周期观测表明，该基因敲除可引起花期提早、生育期变短;增强表达该基因则使衰老变缓。 3．对RabD2b T-DNA插入突变体系纯合突变体、CaMV 35S-RabD2b增强转化植株以及野生型的ABA胁迫处理下种子萌发率的观测表明，该基因敲除后对ABA不敏感，而增强表达该基因则对ABA的敏感程度增强。表明该基因可能与ABA信号调控途径的正调控相关。 ***2bPro-GUS转基因植株T1代生长两周的植株GUS染色表明仅衰老子叶有GUS活性，而在ABA胁迫处理后，植株各组织器官都有GUS活性的表达，且GUS表达量上升。 本研究所发现的RabD2b基因对生长发育上的作用及对ABA的正调控作用机制为今后进一步研究植物ABA的调控表达途径提供了有价值的线索，对促进研究Rab类小G蛋白的功能也具有一定的意义。

关键词： 拟南芥   异三聚体G蛋白   分离纯化   凝胶过滤   离子交换层析   Western blotting   表达  

摘要： G蛋白偶联信号转导系统是一类重要的细胞信号转导途径,参与许多重要生命活动的调节。G蛋白于20世纪80年代首先在动物中被发现,并对其介导的信号转导机理进行了广泛而深入的研究。而在植物中的研究到80年代后期才开始,处于相对滞后阶段。拟南芥是一种模式植物,对拟南芥植物基因组序列的分析表明,在拟南芥中有1个编码α亚基(GPA1)、1个编码β亚基(AGB1)和2个编码γ亚基(AGG1、AGG2)的基因。最近对G蛋白偶联信号途径的研究显示:分析G蛋白Gα、Gβ和Gγ三个亚基间的相互作用对阐明其信号转导机理至关重要,而研究亚基间的相互作用必须首先分离纯化相应的蛋白质。 本文以拟南芥哥伦比亚(col)野生型悬浮培养细胞为材料,通过超声波破碎、硫酸铵沉淀、凝胶过滤、离子交换等纯化手段,结合SDS-PAGE、Native-PAGE、Western blotting等方法,从拟南芥细胞中分离纯化了G蛋白,并对分离纯化条件作了一些优化,为以后的深入研究提供了一些理论依据。同时通过对植株和细胞的不同处理(包括土壤干旱、PEG-8000和NaCl模拟干旱、ABA等激素、CTX以及温度等),研究了拟南芥G蛋白Gα的表达情况,证实了G蛋白参与了多种信号转导途径。具体研究结果为: 1.优化了G蛋白的分离纯化条件:从细胞的超声波破碎,研磨,离心,40-60%饱和度的硫酸铵分步沉淀,Sephadex G-25脱盐、DEAE-Sepharose Fast Flow离子交换、Sephadex G-200凝胶过滤,最后经过DEAE-Sepharose CL-6B得到纯化的目的蛋白,蛋白质收率为0.1052%。 2.纯化的蛋白质Native-PAGE显示为一条带,经过Western blotting确定能与Gα亚基的抗体发生免疫反应,证实为G蛋白。