【作者】 苑国良；

【导师】 亓宝秀；

【作者基本信息】 山东农业大学 ， 发育生物学， 2009， 博士

【摘要】 异三聚体G蛋白是在从酵母到人的所有真核生物中都高度保守的信号转导蛋白。它与偶联的膜受体蛋白和效应器蛋白合作,接受外界信息,并经过调整、集合与放大,准确无误地传递到细胞内,从而调控基本的生命过程,是细胞与环境信息交换必不可少的分子开关。异三聚体G蛋白属于GTPase,由α,β,γ三个亚基组成。研究表明,异三聚体G蛋白与含有七次跨膜结构域的质膜受体密切偶联(GPCRs)。目前哺乳动物中已发现大于20个Gα亚基,多于5个Gβ亚基,至少20个Gγ亚基。与哺乳动物相反,植物异三聚体G蛋白含有一个Gα亚基(GPA1),一个Gβ亚基(AGB1)和两个Gγ亚基(AGG1和AGG2)。研究表明,植物G蛋白参与许多激素、发育及环境信号的应答,从而调控植物生长、器官发育及病害及逆境抗性等。对于拟南芥和水稻研究表明,异三聚体G蛋白能够直接地,间接地组织特异性地影响生长素、ABA、GA、油菜素内酯(brassinosteroid)、鞘脂(sphingolipid )、D-葡萄糖信号、感受蓝光和病原菌信号传导等。虽然植物异三聚体G蛋白涉及广泛的信号转导过程,但已知的与Gα亚基或Gβγ二聚体相互作用的下游效应器还很少。但传统的方法,包括生物数据分析、遗传学与药理学等方法,对于筛选、鉴定植物G蛋白信号转导途径中的其它效应因子并不十分有效,因而寻找新的改良方法是必须的。在本项研究中,作者构建了拟南芥cDNA文库,借助酵母反向Ras恢复系统(reverse Ras Recruitment system,rRRS)来筛选、鉴定与拟南芥GPA1相互作用的蛋白因子,如ADL1C,ACC1,AtXB31等,并以AtXB31作为进一步的研究对象,通过Pull down,BiFC和CoIP等体外与体内蛋白结合实验证明了AtXB31同GPA1的互作。生物化学分析与异位过量表达,病原菌接种等遗传学研究进一步验证它们之间的相互作用。分离GPA1相互作用蛋白的突变体以及与包括gpa1在内的其它信号蛋白突变基因构成的双突变体。通过鉴定这些突变体对已知G蛋白介导的信号转导事件产生的效应与表型性状,揭示G蛋白信号转导途径的机理。取得的结果如下:1.提取拟南芥2星期和三星期幼苗,成熟的叶片、茎、花的总RNA,以随即引物为反转录引物,表达载体为pUra-Ras,构建了cDNA文库,1×105个克隆。2.构建pMet-WtGPA1,pMet-Q222L两种诱饵,借助反向Ras拯救恢复系统,筛选得到若干GPA1推论的互作因子,如:ADL1C,ACC1,AtXB31等。3.通过酵母双杂交,Pull down,BiFC,CoIP等体内体外蛋白互作实验,进一步证明AtXB31同GPA1的互作。其中酵母双杂交的互作发现,AtXB31的N-末端膜定位序列对于二者的互作是必需的。4.对于AtXB31结构分析表明,该基因具有三个保守domain,分别为:N-末端的膜定位序列,将蛋白定位在质膜上;锚定重复序列(ankyrin repeats),参与蛋白互作;C3HC4的环形锌指结构,具有潜在的E3连接酶的活性。生物信息学预测该蛋白不具有跨膜结构。RT-PCR检测,该基因在植物的重要组织中均有表达,其中在花中表达较高。GFP亚细胞定位在质膜上。经ABA和病原菌处理之后,AtXB31表达量提高。5.AtGPA1同AtXB31有可能共同调控病原菌Xcc8004信号途径。通过对AtXB31的异位过表达和RNAi敲除转基因植株同野生型col-o形态学比较发现,基本没有差别。分别对GPA1的无义突变体gpa1-4和AtXB31过表达以及RNAi敲除植株接种病原菌,野生型作为对照。结果表明,gpa1-4和AtXB31RNAi敲除植株均出现了疑似感病症状。并且已经证明AtXB31在转录水平上响应该病原菌。因此推论AtGPA1和AtXB31很有可能参与Xcc8004病原菌的信号调控。黄单胞菌是可造成植物严重病害的细菌,如黑腐病。因此研究该病原菌同植物之间的信号传递途径,找出关键调控基因,对于防治该病原菌可提供重要理论依据。筛选、鉴定植物G蛋白相互作用因子,并剖析其生理功能和在G蛋白信号转导中的作用,从而揭示植物G蛋白信号转导过程中的分子运转机制,这不但具有深远的理论意义,而且具有现实的生产指导意义,为提作物高光合效率,改良作物品质,提高作物抗逆性等品种改良工作提供理论依据。更多还原

【Abstract】 Heterotrimeric G proteins are signal transducers which are conserved in eukaryotes ranging from yeast to humans. Heterotrimeric G proteins are GTPases, composed ofα,βandγsubunits. Studies with mammalian cells established that heterotrimeric G proteins associated with plasma membrane receptors which contain 7 transmembrane domains (GPCRs). In mammals, more than 20 Gα,5 distinct Gβand at least 12 Gγsubunits have been identified.In contrast, our understanding to the molecular mechanisms of activation, signaling and regulation of plant heterotrimeric G proteins is rudimentary. It is currently thought that plant heterotrimeric G proteins contain a single Gα-subunit (GPA1), a single Gβ(AGB1) and two Gγ-subunits (AGG1&2). Both GPA1 and AGB1 have been shown to be located at the plant plasma membrane. A wide range of biochemical, pharmacological, and genetic studies conducted over the last 20 years have shown that plant G proteins are involved in responses to a number of hormone, developmental and environmental signals. The involvement of heterotrimeric G proteins in plant hormone signaling is complex. In particular, recent genetic studies with Arabidopsis have revealed direct, indirect, tissue specific effects on auxin, ABA, GA, brassinosteroid, sphingolipid and D-glucose, Blue light, pathogen signaling. Although plant heterotrimeric G proteins are involved in this wide range of signaling events, there are only few known downstream effectors that physically interact with either the plant Gα, or the Gβγdimmer. Attempts to identify other components of plant G protein signaling by database mining, genetics and pharmacology have not been conclusive. Because conventional methods have not provided answers to these important questions, an innovative approach is required.In this subject, the yeast two-hybrid reverse Ras Recruitment system (rRRS) had been used to identify proteins that interact with GPA1. The putative ineracrors is ADLC1, ACC1, AtXB31 and so on. We focused on the AtXB31. Interactions had been confirmed by Pull down, BiFC, CoIP. AtXB31 mutants will be isolated and double mutants generated with gpa1. The phenotype and response of these to known G protein mediated events will be determined. The main results are as follow: 1. Constructing an Arabidopsis cDNA library of 2-week seedling, 3-weekseedling, leaf, stem, flower, which is 1×105 colonines.2. Constructing two kind of bait, which is pMet-WtGPA1 and pMet-Q222L, through reverse Ras Recruitment system, we got some putative interactors.3. The interaction is confirmed by yeast two hybridization, Pull down, BiFC, CoIP in vitro and in vivo. The N-Myristoylation site of AtXB31 is necessary for the interaction of GPA1 and AtXB31.4. The strcture analysis of AtXB31 suggests that, there is three conserved domain, such as N-Myristoylation site, ankyrin repeats, ring type Zinc figure of C3HC4. There is no transmembrane motif by Tmhmm software. The expression of AtXB31 can be detected in all major tissue of plant by RT-PCR.. Subcellular localization of the transiently expressed pBI121-AtXB31-GFP fusion protein in tobacco epidermal cells shows AtXB31-GFP fluorescence is concentrated to the intracellular membrane of cell. The expression of AtXB31 is enhanced treated with ABA and Xcc pathogen.5.AtXB31 and AtGPA1 may coregulate the signal transduction of Xcc8004 pathogens. From morphology, ectopic over-expression and RNA interference plants of AtXB31 showed no difference comparing with wild type. The mutant gpa1-4 and AtXB31RNAi is susceptible to the infection of Xcc8004. In other hand,we had proved AtXB31 response to the Xcc on transcription. So, AtXB31 and AtGPA1 may coregulate the signal transduction of Xcc8004 pathogens.As a bacterial pathogen, Xanthomonas campestris could cause serious disease to plant, such as black rot. Therefore, investigating the signaling pathways between palnt and pathogen and detecting the key regulary genes, will generate important theory against the pathogens.In conclusion, it would have tremendous potential for identifying components of plant GPA signaling pathways and, in combination with the use of biochemistry and the genetic screens, will generate novel information that will enhance our understanding of signal transduction mechanisms in plants. It would do a great good to upgrade of crops.更多还原