【作者】 胡晓丽；

【导师】 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2008， 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。大量研究表明,在逆境条件下植物体内积累渗透调节物质如脯氨酸、可溶性糖、甜菜碱等,同时,许多基因被诱导表达。植物体内存在复杂的信号传递途径,由蛋白激酶和蛋白磷酸酶催化的蛋白质可逆磷酸化过程是细胞信号转导的重要组成部分,是细胞内重要的调节机制,广泛参与植物的生长、发育、增殖、分化等。蛋白质的可逆磷酸化反应对于信号的快速、精确传递起着无可替代的作用。蛋白磷酸酶,通过逆转蛋白激酶的作用,调节可逆磷酸化过程。生物化学和分子遗传学研究表明,PP2C广泛参与逆境信号的传递过程,可能在许多信号转导途径中作为负调控因子,在调节由环境胁迫如冷害、干旱、损伤等激活的信号通路中起作用。但关于逆境信号传递途径的研究还很有限,PP2C参与逆境信号途径的具体机制尚不清楚。本研究从玉米根系中分离到PP2C类蛋白磷酸酶基因ZmPP2C2,并对该基因的表达特性和功能进行了分析。结果表明,该基因在根、茎、叶和种子中都有表达,在种子表达高;不同逆境胁迫及信号分子处理后ZmPP2C2的表达有所不同,ZmPP2C2在转录水平和翻译水平响应低温逆境;过量表达该基因可提高转基因烟草植株对干旱、低温和盐渍的抗性。主要结果如下:1.利用同源序列设计简并引物,通过RT-PCR的方法从玉米根系中克隆到PP2C类蛋白磷酸酶基因的中间片段,通过5’-RACE和3’-RACE分别克隆到5’和3’片段,拼接后设计特异引物扩增到全长cDNA。命名为ZmPP2C2 (GenBank注册号:AY830123)。该基因全长为1399 bp,ORF为855 bp,编码284个氨基酸,分子量约为30.9 kDa。同源序列比较发现,ZmPP2C2的序列与拟南芥、水稻、苜蓿、玉米的ZmPP2C类基因的序列同源性较高。结构同源性分析表明,ZmPP2C2有11个序列保守的结构域和6个保守的参与金属离子与磷酸基团相互作用的氨基酸。跨膜结构分析表明,ZmPP2C是一个亲水蛋白,存在一个明显的内向超螺旋结构和两个潜在的外向超螺旋。2、Southern杂交结果显示,ZmPP2C2在玉米基因组中以单拷贝形式存在。Northern杂交结果表明ZmPP2C2在根、茎、叶、种子中均有表达,在种子中表达量高。低温、PEG、NaCl、ABA均能诱导ZmPP2C2的表达,并且Ca2+在ABA诱导的ZmPP2C2表达过程中起负调控作用。ZmPP2C2在转录和翻译水平都表现出对低温逆境的响应,表达量随时间变化有所不同。3、将pBI121-ZmPP2C2-GFP融合蛋白在洋葱表皮细胞表达。在荧光显微镜下观察到细胞核内有GFP激发的绿色荧光,说明ZmPP2C2的基因产物定位于细胞核。系统进化树分析表明,ZmPP2C2蛋白与拟南芥、苜蓿中的PP2C蛋白高度同源。4、构建了原核表达载体pET-ZmPP2C2,并在大肠杆菌BL21中表达融合蛋白,诱导纯化后免疫小白鼠,制备抗体。Western杂交表明,转基因植株中ZmPP2C2基因已在蛋白水平过量表达。5、构建pBI-ZmPP2C2正义表达载体,利用农杆菌介导的叶盘法转化烟草。用PCR、Northern杂交及Western杂交的方法,对带卡那抗性的转基因烟草植株进一步检测,结果证明成功地获得了超表达ZmPP2C2基因的转基因烟草植株。6、在逆境胁迫(低温、高盐、干旱)处理条件下,过量表达ZmPP2C2基因的转基因烟草种子比野生型烟草种子萌发率高。7、盐渍、干旱处理过程中野生型和转基因烟草叶片的净光合速率(Pn)都呈下降趋势,但野生型的降低较明显。胁迫对两种类型烟草叶片的细胞间隙CO2浓度(Ci)和气孔导度(Gs)的影响无明显差异。盐胁迫下,转基因烟草Fv/Fm降幅较小,PSⅡ受伤害较轻。8、逆境胁迫(低温、高盐、干旱)下,两种类型烟草叶片的相对电导率、MDA的含量都呈上升趋势,膜透性增大,电解质外渗量增多,膜脂过氧化作用增强。但转基因烟草相对电导率和MDA含量较野生型烟草低,说明转基因烟草膜脂过氧化程度低于野生型烟草,膜伤害程度较轻。9、在低温胁迫下,两种类型烟草叶片的SOD、CAT活性都呈上升趋势,而POD活性呈先升高后降低的趋势;在高盐胁迫下,两种类型烟草叶片的SOD活性呈先升高后降低的趋势,而POD、CAT活性都呈上升趋势;在干旱胁迫下,野生型和转基因烟草叶片的SOD、POD、CAT活性都呈现先升高后降低的趋势;但在三种胁迫下转基因烟草SOD、POD、CAT活性始终高于野生型烟草。10、逆境胁迫(低温、高盐、干旱)下,转基因烟草相容性物质脯氨酸、可溶性糖和可溶性蛋白的积累明显高于野生型烟草。更多还原

【Abstract】 Abiotic stresses such as low temperature, drought and high salt, influence plant growth, productivity and development. To cope with unpredictable environmental changes, plants evolve signal transduction mechanisms by which they receive stress signals and regulate their stress-tolerant reactions. Molecular and cellular responses to these stresses have been analyzed extensively at the biochemical level: various kinds of proteins and smaller molecules, including sugars, proline, and glycine betaine, accumulate, in addition, many genes are induced by stresses. These observations suggest the existence of several cellular signal transduction pathways between the perception of stress signals and gene expression. Yet the mechanisms underlying the perception and transduction of stress signals manifested in development of stress tolerance are just being revealed.Reversible protein phosphorylation, mediated by protein kinases and protein phosphatases, is the predominant regulatory mechanism in biology, modulating cellular processes such as signaling, division, growth and development. Protein phosphatases, by opposing the action of protein kinases, provide modulation and reversibility of the phosphoregulatory mechanism. Biochemical and molecular genetic studies have identified PP2Cs as regulators of stress. However, knowledge of the transduction pathways, especially in stress responses, is still incomplete. The involvement of PP2Cs in stresses acclimation is also far from clear.In this study, we isolated a PP2C gene (ZmPP2C2) from Zea mays roots and characterized its expression patterns in different tissues and in response to abiotic stresses and signal substances. We show that ZmPP2C2 was basically expressed in the roots, stems, leaves and seeds, especially in seeds. Moreover, we found that ZmPP2C2 exhibited different responses to diverse abiotic stresses and signal substances, and revealed a quick and transient response to low temperature. Then, to study the contribution of PP2Cs to stress signaling we generated 35S::ZmPP2C2 transgenic tobacco. The transgenic plants showed enhanced tolerance to cold, drought and salt than wild type tobacco. The main results are as follows:1. In Southern blot analysis, hybridization of the ZmPP2C2 probe with genomic DNA of Zea mays reveals that the ZmPP2C2 gene is present in the Zea mays genome as a single copy. Northern blot analysis revealed that this clone was expressed in roots, stems , leaves and seeds, and the transcript were relatively more abundant in seeds than in other organs. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from Zea mays according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 5’and 3’fragment of the cDNA was isolated by 5’and 3’RACE. The clone, which named ZmPP2C2 (Acession Numeber:AY830123), contains 1399 bp nucleotides with an open reading frame (ORF) of 855 bp comprising 284 amino acid residues with the predicted molecular mass of 30.9 kDa. The deduced amino acid sequence showed high identities with PP2C from Arabidopsis, Oryza sativa, Medicago and Zea mays. Amino acid sequence alignment revealed that six of the seven residues that are putatively involved in the coordination of the phosphate and metal ions were present in the sequence, and the eleven conserved motifs presented in all Ser/Thr PP2Cs were found in the catalytic domain. Software analysis showed that ZmPP2C2 protein was a hydrophilic protein, which contained one typical superhelix from outside to inside and two potential superhelixes from inside to outside.2. ABA and low temperature significantly increased the transcript levels of ZmPP2C, and Ca2+ acts as a negative regulator in ABA-induced ZmPP2C2 transcription. Besides, ZmPP2C2 response to low temperature at both transcription and translation level.3. The pBI121- ZmPP2C2-GFP fusion protein is clearly targeted to the nucleus in a transient transfection assay. Construction of a dendrogram based on the homologous full-length amino acid sequences revealed the close imilarity between ZmPP2C2 and the PP2C from Arabidopsis and Medicago.4. A recombinant of prokaryotic expression vector pET- ZmPP2C2 was constructed and expressed in E.Coli. BL21. The strong induced fusion protein were purified and used to immunize white mice to obtain antiserum. Western hybridization revealed the presence of the strong positive protein signals corresponding to ZmPP2C2 in transgenic tobacco.5. The full-length ZmPP2C2 cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR , Northern blot and Western blot. It was indicated that the ZmPP2C2 gene had been recombined into tobacco genome and transgenic tobacco plants were obtained.6. Expression of the ZmPP2C2 gene attenuated the delay in germination at low temperature, salt, and drought stress, and improved the germination rate in transgenic tobacco.7. Under salt and drought stress, Pn in both wild type tobacco and transgenic tobacco decreased, but Pn decreased less in transgenic tobacco compared with wild type tobacco. There was no obvious difference between wild-type and transgenic plants about the change of Ci and Gs. However, the level of Fv/Fm in transgenic tobacco decreased less compared with wild type tobacco under salt stress. It indicated that PSⅡof transgenic tobacco plants suffered less injury compared with wild type tobacco.8. Under environmental stress (low temperature, salt and drought), the relative electrical conductivity and MDA content in both wild type and transgenic tobacco plants increased, which resulted in the increase of membrane permeability and electrolyte leakage, and the structure and function of cell membrane was damaged . However the degree of increase in transgenic tobacco plants was lower than that in wild-type tobacco plants, which indicated that the extent of the membrance lipid perioxidation in transgenic tobacco was lighter than that in wild type tobacco. It is favorable to maintain the function of cell membrane.9. Under cold stress , the activities of SOD and CAT increased in both wild types and transgenic tobacco plants, while the activitie of POD increased first and then decreased in both wild types and transgenic tobacco plants. However ,Under salt stress , the activitie of SOD increased first and then decreased in both wild types and transgenic tobacco plants, and the activities of POD and CAT increased in both wild types and transgenic tobacco plants. Interestedly, the activities of SOD , POD and CAT increased first and then decreased in both wild types and transgenic tobacco plants. Furthermore, in all stresses the transgenic tobacco plants always sustained higher activities of both SOD, POD and CAT.10. Under environmental stress (low temperature, salt and drought), the transgenic tobacco plants accumulated more proline , soluble sugars and soluble protein compared with wild type tobacco plants.更多还原