【作者】 郭英慧；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 高盐和真菌胁迫是影响植物生长发育的重要影响因子。植物在长期的进化过程中形成了多种适应机制,其中包括胁迫条件下一些基因被诱导表达,并通过直接或间接的方式调控下游靶基因的表达,从而在提高植物抗逆性中发挥重要作用。因此,研究在适应胁迫过程中重要基因的表达调控及功能对了解植物在逆境条件下的响应和适应机制是十分必要的。锌指蛋白是一类生物体中广泛存在的具有典型锌指结构特征的超蛋白家族,几乎参与了生物体生长发育的各个阶段。根据锌指蛋白的结构及功能特点,锌指蛋白主要分为九大类:C2H2,C8,C6,C3HC4,C2HC,C2HC5,C4,C4HC3,CCCH(C,H分别代表半胱氨酸和组氨酸),一些与逆境相关的植物锌指蛋白相继被人们发现和研究。但迄今为止,与逆境胁迫相关的植物CCCH型锌指蛋白尚未有报道。本研究以世界上重要经济作物棉花为材料,构建了盐诱导棉花幼苗叶片cDNA文库,并用反向Northern差异筛选法获得一个与盐胁迫相关的棉花锌指蛋白基因GhZFP1。进一步序列比较及同源性分析表明,GhZFP1编码了一个CCCH型锌指蛋白,目前对该类型锌指蛋白的研究相对较少。因此本论文通过对其序列、表达及生物学功能的研究,探索了GhZFP1与植物抗逆性之间的关系,并利用酵母双杂交的方法,从棉花中筛选到9个与其相互作用的蛋白,从而进一步对其信号响应及分子作用机制有了更深层次的了解。主要研究结果如下:1、利用Clontech SmartTM cDNA Library ConstructionKit构建盐胁迫诱导棉花幼苗叶片cDNA文库,通过反向Northern差异筛选法获得一个与盐胁迫相关的锌指蛋白基因GhZFP1 5’端。设计特异引物进行3’-RACE克隆到GhZFP1基因的3’端,拼接后用特异引物扩增到1017bp的GhZFP1全长cDNA,编码一个339氨基酸的蛋白,推测其分子量为38.4kDa。2、同源序列比较表明,GhZFP1编码了一个CCCH型锌指蛋白,属于一个研究较少的CCCH型锌指蛋白家族。氨基酸序列分析表明GhZFP1有两个比较典型的CCCH型锌指(CX8CX5CX3H,CX5CX4CX3H),在其N端和C端分别有一个核定位信号序列(NLS)和一个可能的核输出信号序列(NES),跨膜结构分析表明在其190-213氨基酸之间存在一个跨膜结构域(TM),因此推测GhZFP1可能作为一种重要调控因子起作用。GhZFP1是棉花中首次被克隆的CCCH型锌指蛋白基因,对其表达及功能探索将大大促进整个CCCH型锌指蛋白家族的研究。3、Northern表达分析结果表明GhZFP1受NaCl、PEG和SA诱导表达,在一定时间范围内其表达水平随诱导时间的延长而增加,但不受CuSO4、ABA、4?C、CaCl2和乙烯诱导,说明GhZFP1对盐胁迫的非专一性,可以响应多种生物与非生物胁迫。在NaCl处理下,GhZFP1在茎和叶中均有表达,以茎中最高,在根中没有表达,并且在中棉所19号(ZMS19)中的表达量要显著高于在中棉所17号(ZMS17)中的表达,说明GhZFP1表达不仅具有一定的组织特异性而且与不同棉花品种的耐盐性也有关。Southern杂交结果表明GhZFP1在棉花基因组中是以单拷贝的形式存在的。4、聚类分析表明GhZFP1与拟南芥中一小类CCCH型锌指蛋白在整个系统进化树上形成独特的一枝。序列分析表明该分枝的每个成员除了具有保守的两个锌指外,还有一些其它共同特征,在其第一个锌指前都有一个由38个氨基酸组成的保守区,在其C端都有一个NES序列,基因内部都没有内含子。用SubLoc v1.0亚细胞定位分析软件预测它们都定位于细胞核内,但均未预测到可能的NLS序列。最近的拟南芥芯片研究和GhZFP1诱导表达分析也表明,该分枝成员的表达都受到不同生物和非生物胁迫诱导,因此命名其为与SRZFP亚家族(stress-related zinc finger protein subfamily)。本研究是在植物中首次发现与胁迫相关的CCCH型锌指蛋白家族,可能也是高等植物特有的CCCH型锌指蛋白家族。5、将GhZFP1-GFP融合蛋白在洋葱表皮细胞内瞬时表达,在洋葱表皮细胞的细胞核内观察到绿色荧光,表明GhZFP1编码了一个核定位蛋白。6、在烟草中超表达GhZFP1基因,用GhZFP1特异探针标记的Northern杂交结果表明GhZFP1只在转基因植株中表达。同野生型相比,转基因烟草能在200mM,250mM NaCl条件下正常生长,生长势、根长及鲜重都要明显好于野生型。经过200mM NaCl处理后,转基因植株Fv/Fm和K+/Na+比要明显高于野生型植株,尤其是体内积累的Na+离子含量要小于野生型植株,转基因植株地上部K+/Na+比要远高于野生型植株。这些结果说明GhZFP1的过量表达能够提高转基因烟草的耐盐能力。用立枯菌(Rhizoctonia solani)对烟草植株进行活体接菌实验,结果表明转基因植株较野生型抗病能力明显增强。7、从GhZFP1中选取N端大约185氨基酸的亲水区域构建原核表达载体pET30a-GhZFP1-185,并在大肠杆菌BL21(DE3)中表达融合蛋白,将该融合蛋白进行纯化获得抗原,免疫家兔,其抗血清效价为1:1000。8、用BD MatchmakerTM Library Construction & Screening Kits构建酵母双杂交文库,以GhZFP1 N端1-237氨基酸(m1)为诱饵蛋白对文库进行筛选,获得9个与其相互作用的靶蛋白。Northern杂交结果表明,其中的一个靶蛋白基因GZIRD21A的表达受PEG和SA诱导,而另一个靶蛋白基因GZIPR5的表达受NaCl和SA诱导,它们的诱导表达模式与GhZFP1的表达模式十分相似,初步推测GZIRD21A和GZIPR5可能在响应不同的生物和非生物胁迫中,与GhZFP1形成复杂的蛋白复合物,从而在不同的信号传递途径中发挥着重要作用。9、GhZFP1与GZIRD21A和GZIPR5的相互作用结构域的鉴定实验表明,GhZFP1与GZIRD21A和GZIPR5的相互作用具有一定的特异性,要进一步确定相互作用的最小范围,还需构建更多的GhZFP1缺失体。更多还原

【Abstract】 High salinity and fungal pathogen attack are major stresses that are commonly encountered by plants during their developments. To adapt to the changes of environmental stresses, a series of physiological and biochemical reactions occur to eliminate or alleviate the physiological damage induced by improper surroundings. Upon exposure to the stresses, many genes are induced and their products either directly protect plants against stresses or further control the expression of other target genes. To understand the adaptive mechanisms of development of plants and their response to environmental stresses, it is imperative to know the function of crucial genes and their regulation during different stress response in plants。Zinc finger proteins are defined as a superfamily with typical zinc finger domain that involved in many aspects of plant growth and development and have been widely found in many animals and plants. They have been classified into nine types according to the structural and functional diversities: C2H2, C8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3, CCCH (C, H represented Cysteine and Histidine, respectively). Until now, some zinc finger protein related to stress have been isolated and identified, but none of CCCH-type zinc finger proteins has been studied.In this experiment, a NaCl-induced cDNA library from cotton seedlings was constructed, and a new zinc finger protein gene GhZFP1 related to salt stress was identified by differential hybridization screening. Sequence and homology comparison revealed that GhZFP1 belonged to an unusual CCCH-type zinc finger proteins family. In this thesis, our studies are mainly focused on the sequence and expression analysis, function identification of the cotton gene GhZFP1. We used a yeast two-hybrid screen to identify proteins that interact with GhZFP1 expecting that such search might provide new insight into the function and molecular mechanism of GhZFP1 protein. The main results are as follows:1. A NaCl-induced cDNA library of cotton seedlings was constructed with Clontech SmartTM cDNA Library Construction Kit. We screened a salt-induced zinc finger protein gene GhZFP1 by differential hybridization from the cDNA library. The full length cDNA was further isolated by 3’-RACE and RT-PCR. The clone contains 1017 nucleotides and encodes 339 amino acid residues with the predicted molecular mass of 38.4 kDa.2. Sequence and homology comparison revealed that GhZFP1 encoded a putative CCCH-type zinc finger protein and belonged to a poorly studied CCCH-type zinc finger proteins family. Amino acid sequence analysis showed that GhZFP1 contained two typical zinc finger motifs (Cx8Cx5Cx3H, Cx5Cx4Cx3H). Moreover, a potential nuclear localization signal (NLS) and a putative leucine-rich nuclear export sequence (NES) were found in the N-terminal and C-terminal region of the protein, respectively。By the TMpred software, the deduced protein sequence contained one transmembrane (TM) segments at 190-213 amino acid residues, suggesting that it maybe act as an important regulator in responsing to stresses. GhZFP1 is the first CCCH-type zinc finger protein that had been identified and functionally characterized in cotton, and so a series of studies of it will accelerate the study of the whole CCCH-type zinc finger protein family.3. Northern blot results showed that the mRNA accumulation of GhZFP1 was induced by NaCl, PEG and SA treatments and kept increasing in a time-dependent course, but there was no significant GhZFP1 mRNA accumulation under 4?C, CuSO4, CaCl2, abscisic acid (ABA) and ethephon treatments, suggesting that GhZFP1 was not induced by specfic salt stress but induced by different biotic and abiotic stresses. Northern blot results also showed that GhZFP1 expression was up-regulated by NaCl stress in both leaves and stems, but not detected in roots. GhZFP1 mRNA was more abundant in ZMS19 than in ZMS17 under salt stress, indicating that GhZFP1 mRNA transcription levels were not only associated with tissue specificity but also associated with the salt tolerance of field-grown different cotton cultivars. Southern blot showed that it is a single-copy gene in cotton genome.4. The phylogenetic analysis of GhZFP1 indicated that several zinc finger proteins and GhZFP1 were most closely related to each other and significantly clustered together into a single branch in the tree, designated as SRZFP subfamily. Each member of this subfamily contained a conserved 38 amino acid sequence leading to the first putative zinc finger and a putative NES-like sequence at the C-terminal region, outside of these regions and zinc finger domains. Gene structural analysis revealed that each member of this subfamily was intronless gene. Expression profiles of SRZFP members in many microassay databases show that members of SRZFP subfamily might be involved in response to multiple stresses and play important roles in different signal transduction pathways.This is the first report showing that a CCCH-type zinc finger protein of SRZFP subfamily response to stresses in plants, indicating that SRZFP subfamily may be unique for higher plant species.5. GhZFP1-GFP fusion protein was constructed and transiently expressed in onion epidermal cells. The green fluorescence was detected in the nucleus of onion epidermal cells. These results are consistent with our observation that GhZFP1 encodes a nuclear-localized protein.6. GhZFP1 was introduced into tobacco plants. Northern blot analysis shows that GhZFP1 was expressed in plants of each transgenic line. The transgenic tobacco plants overexpressing GhZFP1 grew better than wild type tobacco in 200 mM,250 mM or 300 mM NaCl conditions, and then further verified by measuring changes in chlorophyll fluorescence (Fv/Fm) or endogenous Na+, K+ contents in shoots and roots , thereby validating the increased tolerance to salt stress. The enhanced resistance to Rhizoctonia solani in transgenic tobacco also demonstrated that disease resistance conferred by GhZFP1 was effective against fungal pathogens.7. A hydrophilic 5’fragment of GhZFP1 was selected to construct an E.coli expression vector pET30a-GhZFP1-185, and then it was induced by IPTG to express in E.coli strain BL21(DE3). The fusion proteins were purified and used to immune rabbits to obtain antiserum. The value of antibody reaches 1:1000.8. A yeast two-hybrid library was constructed with BD MatchmakerTM Library Construction & Screening Kits. By using the N-terminal 237 amino acids of GhZFP1 (m1) as bait, we screened and identified 9 positive clones that interacting with it. Northern blot analysis shows that expression of GZIRD21A was very rapidly induced by PEG and SA treatments, and GZIPR5 transcripts were induced by NaCl and SA treatments in cotton. These results are consisted with previous studies and resembled to the expression of GhZFP1, indicating that their interaction help to facilitates formation of higher order complexes and play important roles in transmitting different signal transduction pathway.9. Interaction regions analysis of GhZFP1 showed that it interacted with GZIRD21A and GZIPR5 by different amino acid sequences, To investigate the smallest binding sites of GZIRD21A and GZIPR5 in GhZFP1, a series deletions of GhZFP1 were need to be generated in the future study.更多还原