【作者】 薛彤彤；

【导师】 郑成超； 钟圆；

【作者基本信息】 山东农业大学 ， 发育生物学， 2009， 博士

【摘要】 非生物胁迫是影响植物生长发育和产量的重要环境因素,而其中又以干旱、低温和盐渍的影响最为严重。植物在长期的进化过程中形成了一套主动的防御机制,能够识别逆境信息并通过信号传递最终调节植物的生长发育,从而抵御不良环境的影响。植物在逆境下通常伴随大量活性氧（ROS）的产生。一方面活性氧自由基作为信号分子有效地传递逆境信号,另一方面活性氧自由基水平的升高又会对植物造成伤害。目前,关于活性氧自由基介导的基因调控的研究很少,活性氧自由基在植物胁迫中的功能有待于进一步的探索。金属硫蛋白（Metallothionein,MT）是一类低分子量（6000～9000Da）、高Cys含量、具有金属结合能力的多肽,它具有独特的氨基酸排列顺序,即多肽的N端和C端具有两个富含Cys的金属结合结构域,其中的Cys按CX_nC的方式排列。研究表明在动物和真菌中MT在很多生理过程中发挥作用,包括细胞内激素平衡、氧化胁迫、离子伤害。近来,不断有研究证明植物MT也具有相似的功能。棉花是一种古老的纤维用经济作物,它具有广泛的应用价值和研究价值。本文以耐盐棉花品种中棉19（ZM19）为试验材料,通过反向northern差异筛选法从盐诱导棉花幼苗叶片cDNA文库中分离得到了一个与胁迫相关的克隆,通过序列比对发现该基因编码一种金属硫蛋白,命名为GhMT3a。并对该基因的功能和分子机制进行了探索。蛋白磷酸酯酶2C（PP2C）是蛋白磷酸酯酶中的一大类,广泛参与逆境信号的传递过程。迄今为止,对植物PP2C蛋白的研究只是局限于个别基因的鉴定与功能分析,没有研究从基因组水平对此基因家族进行分析。拟南芥和水稻的基因组测序的完成为全面的分析和对比双子叶和单子叶植物基因家族提供了条件。本研究中以已知PP2C基因的序列作为检索条件,利用BLAST查找其同源序列,检索了模式植物拟南芥和水稻的基因组,获得编码PP2C蛋白的基因。得到的结果用SMART或者Pfam检测序列中的PP2C元件,最后从拟南芥和水稻基因组中分别鉴定到80个和78个PP2C基因。在此基础上,我们做了基因家族的分类、染色体的定位、表达模式分析以及基因上游调控序列等方面的研究。主要结果如下:1.通过反向northern差异筛选盐胁迫诱导棉花幼苗叶片cDNA文库获得的一个编码63个氨基酸的基因。该基因编码蛋白与植物中金属硫蛋白具有很高的同源性,具有典型的植物3类金属硫蛋白序列特点,命名GhMT3a。2.Northern blot表达分析表明GhMT3a能被NaCl、低温、干旱、CuSO₄、ZnSO₄、百草枯（PQ）、过氧化氢（H₂O₂）、ABA和乙烯等多种胁迫及激素信号诱导表达,而抗氧化剂N-乙酰基-L-半胱氨酸（N-Acetyl-L-cysteine,NAC）能够有效的抑制NaCl、低温、干旱胁迫对GhMT3a诱导。对过氧化氢相对水平的测定结果表明NAC能够有效地降低NaCl、低温、干旱胁迫条件下棉花幼苗中ROS水平。这些结果说明GhMT3a具有金属硫蛋白受重金属诱导的功能,ROS是非生物胁迫诱导GhMT3a表达的重要信号物质。3.同野生型相比,过量表达GhMT3a的转基因烟草能在200、300 mM NaCl,4℃,25%PEG条件下更好的生长。而胁迫条件下,转基因烟草中的H₂O₂水平远低于野生型烟草。过量表达GhMT3a能够有效的提高转基因酵母对PQ和H₂O₂的抗性。这些结果说明GhMT3a能够在真核细胞内有效地清除ROS,维持ROS平衡从而提高植物对非生物胁迫的抗性。4.对GhMT3a体外结合Zn²⁺能力的测定表明每个GhMT3a蛋白最多能结合5个锌离子,说明GhMT3a具有金属硫蛋白家族结合金属离子的特性。5.利用BLAST同源检索分别从模式植物拟南芥和水稻中鉴定到80和78个PP2C基因。neighbor-joining法分别构建拟南芥和水稻中PP2C基因家族的系统发生树,根据分支上的解靴带值将拟南芥与水稻的PP2C家族分别分为13与11个亚家族,基因结构和蛋白质元件分析结果都支持这一分类。对拟南芥和水稻中PP2C基因的对比分析表明拟南芥的PP2C 13个亚家族在水稻PP2C家族中都能找到同源基因,说明植物中PP2C各个亚家族在单子叶和双子叶植物分化之前已经形成。6.利用网络数据库分别将拟南芥和水稻中的PP2C基因在染色体上进行了定位。通过对复制区域的PP2C基因的分析与对比发现,基因组加倍事件对单子叶和双子叶PP2C基因家族的形成都发挥了重大的作用。但是,基因的串联复制对水稻中的PP2C基因家族的形成的影响更为明显。7.利用MEME元件分析工具对拟南芥和水稻中的PP2C基因家族分析获得11个保守的元件序列。大部分元件广泛分布PP2C基因家族成员中。而个别元件仅仅存在于少数几个亚家族中。这种差异为研究不同亚家族之间功能的差别提供了线索。8.利用Genevestigator,MPSS和EST的公共数据库资源统计了拟南芥中PP2C基因的组织表达模式。根据表达模式的差异可以将PP2C基因分为三类,第一类的基因在四种检测的组织中都有表达,这类基因在拟南芥中有49个,第二类包括拟南芥中的28个基因,这些基因只在部分组织中表达;第三类的基因是组织特异表达的基因,包括3个拟南芥中的PP2C基因。同样,根据表达模式的差异,水稻中的PP2C基因也可以分为三类,分别包含27、45、6个PP2C成员。这些结果说明多数PP2C家族成员在植物发育的不同阶段发挥功能。9.通过Genevestigator上的芯片数据分析拟南芥PP2C基因家族所有成员在ABA及数种胁迫信号条件下的表达模式。与其他亚家族不同,亚家族A所有成员都能够被ABA和数种胁迫信号诱导表达。RT-PCR实验表明ABA和胁迫处理条件下水稻亚家族A成员的表达也受到了明显的诱导。这些结果说明单子叶和双子叶植物中PP2C亚家族A的成员在ABA介导的胁迫信号传导中发挥着重要的作用。10.利用网络数据库以及矩阵频率对拟南芥和水稻亚家族A中19个基因转录起始位点上游1000bp调控序列进行了详细的分析。结果表明它们都含有ABA响应元件（ABRE）,为这些基因在ABA信号途径中发挥功能提供了证据。其他调控元件,比如茉莉酸酮酯响应元件（TGACG motif）,在水稻和拟南芥中的分布存在显著差异,暗示单子叶和双子叶植物PP2C基因家族存在不同的调控模式。更多还原

【Abstract】 Abiotic stresses such as low temperature,drought and high salt,influence plant growth, productivity and development.To survive these challenges,plants have developed elaborate mechanisms to perceive external signals and to manifest adaptive responses with the proper physiological and morphological changes.One of the most common and crucial consequences is the generation of ROS in plants,which can elicit a potentially damaging oxidative burden on cellular constituents and/or act as signals for engaging mechanisms that ameliorate oxidative stress.Although the deleterious effects of ROS have long been known,knowledge on the molecular mechanisms of ROS-mediated gene regulations is limited and whether they play different roles in plant stress response have remained largely unexplored.Cotton is one of the most important economic crops in our country.People are always interested in identifying and developing new cotton breed to make it can be widely cultivated in saline-alkali soil.Metallothioneins（MTs） are defined as a family of proteins with the characteristics of low molecular weight,high cysteine（Cys） residue content,and metal-binding ability.Plant MT proteins are distinguishable on the basis of the distribution of cysteine residues in their amino acid sequences.In animals,MTs are ubiquitous proteins associated with numerous cellular functions,including the regulation of metal homeostasis in cells and the response to metal toxicity and oxidative stress.In fungi,MTs have been proposed to be primarily involved in the response to metal toxicity or as general stress proteins.Recently,increasing numbers of reports have indicated that plant MTs may play important roles as they do in animals and fungi.In this research,a cDNA library was constructed by using mRNA isolated from salt-induced cotton seedlings and screened by differential hybridization.A cDNA clone,GhMT3a,which encodes a 64-amino acid type 3 plant MT,was isolated and characterized.Protein phosphatases（PPs）,by reversing the action of protein kinases,provide modulations of protein phosphoregulation.The protein phosphatase 2Cs（PP2Cs） from various organisms have been implicated involved in diverse environmental stress responses and developmental processes.To date,a genome-wide overview of the PP2C gene family in plants is not yet available.In this study,we performed multiple BLAST algorithms to search against the corresponding data set using the known PP2C proteins got from several database as our query.Then,we eliminated the proteins that did not contain the PP2C domains after detecting by SMART and Pfam.We further compared the rice and Arabidopsis PP2C genes to identify both shared and specific subfamilies.Following the analysis of gene structure and protein motifs,we traced gene duplication events that likely contributed to the expansion of the PP2C family.The expression patterns of the PP2Cs from rice and Arabidopsis were analyzed and compared.The upstream regulatory regions of the subfamily A members were finally analyzed in order to provide insight into the transcriptional regulation machineries that define the temporal and spatial expression and the stimuli responses.The main results are as follows:1.A cDNA clone,was isolated from a NaCl-induced cotton cotyledon cDNA library by differential hybridization screening.This clone showed high homology with type3 metallothein-like genes from various plants species.Our results indicated that this gene encodes a type3 MT protein,which was designated as GhMT3a.2.Northern blot analysis indicated that the mRNA accumulation of GhMT3a was induced by salt stress（NaCl）,CuSO₄,ZnSO₄,low temperature（4℃）,drought（25%PEG）,ABA（abscisic acid）,ethylene and H₂O₂ and paraquat（PQ）.Interestingly,N-Acetyl-L-cysteine（NAC） as a kind of antioxidant,could effectively reduce not only ROS accumulation but also the GhMT3a transcript level induced by salinity,drought,and cold stresses.These results suggested that GhMT3a might be regulated directly by ROS production under abiotic stress conditions.3.The transgenic tobacco plants overexpressing GhMT3a exhibited enhanced tolerance against high salinity（200 and 300mM NaCl）,low temperature（4℃） and drought（25%PEG） compared with WT plants.Moreover,H₂O₂ levels in transgenic tobacco plants were only half of those in WT plants under such stress conditions.The GhMT3a-overexpressing yeast cells were less sensitive to oxidants such as PQ and H₂O₂ than the control cells.These results suggested that GhMT3a could scavenge ROS effectively in eukaryotie cells and improved stress tolerance might be due to the keep of ROS balance in tobacco by overexpressing GhMT3a.4.A recombinant GST-GhMT3a fusion protein was constructed and expressed in E.coli. The purified GST,GhMT3a,and GST-GhMT3a fusion proteins were determined in vitro for their ability to bind metal ions.The results showed that the purified GhMT3a could bind Zn²⁺ ions in vitro and the binding capacity per GhMT3a molecule would be no more than five Zn²⁺.5.We used multiple BLAST algorithms to identify 80 and 78 PP2C family genes in Arabidopsis and rice,respectively.The phylogenetic trees of PP2C gene family were generated with the neighbor-joining method.Based on the statistical support of each branch, we divided the Arabidopsis and rice PP2C family into 13 and 11 subfamilies,respectively, which are supported by the analyses of gene structures and protein motifs.We further investigated the relationship between the Arabidopsis and rice PP2C protein families by the construction of a phylogenetic tree using the full-length protein sequences of 78 OsPP2Cs and 80 AtPP2Cs.The rice and Arabidopsis genes in each subfamily are thus more closely related to each other than to the PP2C genes of the same species from a different subfamily, suggesting that an ancestral set of PP2C genes defining each subfamily already existed before the monocot-eudicot divergence.6.Based on the chromosomal location information on Internet database,we localized Arabidopsis and rice PP2C genes in chromosomes.After analyzing the PP2C genes in duplicated segmental regions,we found whole genome and chromosomal segment duplications mainly contributed to the expansion of both OsPP2Cs and AtPP2Cs.But tandem or local duplication occurred more frequently in rice.7.Eleven motifs were identified shared among related proteins within the PP2C family using the MEME motif search tool.Some motifs are widespread among PP2C proteins and the others are specific to only one or two subfamilies.The different motif distribution in the respective protein sequences may provide an anchor to study the divergence of gene function in different subfamilies.8.We examined the expression of Arabidopsis PP2C genes in different tissure using Genevestigater,massively parallel signature sequencing（MPSS） and EST data.The expression of rice PP2C genes in different tissues were also summarized using MPSS and EST data. According to expression profiles,PP2C genes can be classified into three groups.The largest group is the genes that expressed in all tissues,including 49 genes in Arabidopsis and 27 genes in rice,and the second group contains 28 AtPP2Cs and 45 OsPP2Cs whose expression were detected in the majority,but not all,of the tissues.The third group includes 3 AtPP2Cs and 6 OsPP2Cs with tissue specific expression.9.The expression of all AtPP2Cs in response to ABA treatment and several environmental stimuli were examined using the Genevestigator microarray dataset.Unlike the PP2C genes from other subfamilies,all members from subfamily A were induced by several stimuli and ABA treatment.The expression pattern of the OsPP2C subfamily A genes using RT-PCR is in good agreement with the microarray data for Arabidopsis subfamily A members,suggesting that the members of this subfamily play foremost roles in ABA-mediated processes related to stress responses both in monocots and eudicots.10.The 1000-base-pair（bp） upstream promoter sequences of the 19 PP2C genes in subfamily A in Arabidopsis and rice were analyzed using the PlantCARE database and a frequency distribution approach.The abundance of ABA responsive elements in subfamily A PP2C gene promoters provides additional evidences for their role in ABA signaling.The different motif distributions in Arabidopsis and rice point to evolutionary divergent patterns of PP2C gene regulatory networks.更多还原