【作者】 尹海波；

【导师】 赵彦修；

【作者基本信息】 山东师范大学 ， 发育生物学， 2003， 硕士

【摘要】 盐胁迫能够破坏植物细胞的水势均衡和离子均衡状态，引起高渗胁迫和离子胁迫，使植物生长延滞，甚至死亡。为了存活，植物在长期的进化过程中形成了一系列适应机制。一方面增加细胞内可溶性物质，另一方面则通过Na~+外排或Na~+区隔化机制来维持胞质内较低的Na~+浓度，以消除Na~+的毒害。Na~+和可溶性物质的次级转运依赖于由质子泵建立的质子电化学梯度。在植物中三类不同的质子泵产生跨膜质子电化学梯度：质膜H~+-ATPase，液泡膜H~+-ATPase和液泡膜H~+-PPase。液泡膜H~+-PPase在建立跨液泡膜的质子电化学梯度中起着重要作用。 环境胁迫诱导植物体内不同的生理生化和分子响应，包括基因的表达。这些响应是由胁迫刺激和胞内信号传递引起的。在真核生物中，MAP激酶信号转导途径是一种重要的传递外界刺激到细胞质或细胞核的方式。植物中MAP激酶信号转导途径参与多种胁迫信号的传递，包括渗透胁迫和氧化胁迫等。 本实验从400mmol／L NaCl处理的盐地碱蓬地上部分构建的λZap-cDNA文库中克隆了编码MAPKK的全长cDNA(SsMAPKK，AY093683)和编码液泡膜H~+-PPase的部分cDNA(SsVP)，据已知序列设计引物利用PCR方法获得编码盐地碱蓬液泡膜H~+-PPase的全长cDNA。对这两基因的序列特征、基因组结构和在盐胁迫下的表达特性做了详细的分析。结果表明：SsVP与已报道的红叶藜(Chenopodium rubrum)的液泡膜H~+-PPase基因(CVP)同源性最高，Clustalx软件对不同物种中液泡膜H~+-PPase基因所作的系统进化分析表明SsVP与CVP聚为一簇，均属于类型Ⅰ类液泡膜H~+-PPase；Southern杂交表明该基因在盐地碱蓬基因组中可能是多拷贝的；Northern杂交结果表明在盐胁迫和干旱胁迫下SsVP的转录都是上行调节的，400mmol／L NaCl处理24小时后，盐地碱蓬地上部分中SsVP的表达量增加，同时盐地碱蓬液泡膜H~+-PPase的酶活性在盐胁迫下也增加，干旱胁迫处理4天后盐地碱蓬地上部分中SsVP的表达量明显增加。SsMAPKK与已报道的烟草NPK2同源性最高，Southern杂交表明该基因在盐地碱蓬基因组中只有一个拷贝。以前的实验已证明SsMAPKK的转录水平受盐胁迫的诱导，由此推测SsVP和SsMAPKK都与盐地碱蓬对盐的反应有关。 将SsVP和SsMAPKK的全长cDNA分别克隆入植物表达载体pCAMBIA1300和pROKⅡ中，导入根瘤农杆菌GV3101后，由花浸泡法进行拟南芥遗传转化，转化SsVP 盐地碱蓬Ssop和SsMAPKK基因的克隆与功能鉴定的拟南芥在含潮霉素（25mg／）的MS培养基上筛选，获得T；代转基因植株。转化SSMAPKK的拟南芥在含卡那霉素自0mg／L）的 MS培养基上筛选，通过统计含卡那霉素MS培养基上的TZ植株中的抗性苗和非抗性苗的比值，初步确定外源基因在转基因拟南芥基因组中的整合位点数，转基因植株经分子生物学检测共得到19个独立的株系，PCR结果表明均有夕「源基因的插入，Northern杂交结果表明外源基因在转基因植株中能够正常转录表达。理论上，Ssop的过量表达可增加转基因植株细胞跨液泡膜的质子电化学梯度，为次级转运提供驱动力，从而增加可溶性物质和Na十向液泡内的转运，提高转基因植株的抗旱和抗盐性。而，．．lrmPKK的过量表达可激活其下游的抗性基因的转录表达，增加转基因植株对多种胁迫的耐受性。更多还原

【Abstract】 Salinity stress disrupts the homeostasis in water potential and ion distribution, results in osmotic and ionic stress in plants. It can arose growth arrest, and in some extreme conditions, even death of plants. To cope with salt stress, plants have developed a variety of adaptation mechanisms. One is the accumulation of the solute osmo-protectants, the other is the mechanisms of ion homeostasis including Na+ extrusion system and Na+ compartmentation into the vacuolar to reduce the toxic effects of this cation. The active transport of Na+ and solute molecules depends on the proton electrochemical gradients established by proton pumps. In plants, three distinct proton pumps generate proton electrochemical gradients across cell membranes: the P-type ATPase pumps, the vacuolar H+-ATPase and the vacuolar H+-pyrophosphatase. The vacuolar H+-PPase plays a role in establishing the H+ electrochemical gradient across the vacuolar membrane.Environmental stresses induce various biochemical, physiological and molecular responses, including gene expression. It is thought that these responses are promoted as a result of the perception of environmental stimuli and signal transduction in plant cells. In eukaryotes, it is very clearly that MAP kinase (mitogen-activated protein kinase) signal transduction pathways are important routes for the channelling of extracellular stimuli to cytoplasm or nucleus. In plants, MAPK module can be used for the transmission of multiple signals, including osmotic and oxidative stress.In this research, we isolated two cDNAs that may encode a MAP kinase kinase (SsMAPKK, AY093683) and a vacuolar H+-PPase (SsVP) from a X Zap-cDNA library constructed from a 400 mmol/L NaCl-treated Suaeda salsa aerial tissue. We analyzed their sequence characterizations, genomic structures and transcription levels under salinity stress. The results indicated that SsVP showed highest homology to the vacuolar H+-PPase (CVP) gene from Chenopodium rubrum and phylogenetic analysis indicated that Ss VP and CVP share a cluster, which showed that they might be more similar in evolution. Southern blot analysis showed that there was more than one copy of SsVP in the Suaeda salsa genome. Northern blot analysis indicated that the expression level of SsVP in S. salsa aerial tissue was significantly increased after 48h being treated with 400mmol/L NaCl. The activity of vacuolar H+-PPase of Suaeda salsa increased significantly in response to salt stress. The expression level of SsVP in S. salsa aerial tissue was also significantly increased after 4 days droughtstress. SsMAPKK showed highest homology to the NPK2 gene from Nicotiana tabacum . Genomic southern blot analysis suggested there was only one copy SsMAPKK gene in Suaeda salsa genome. Previously, we haved identified that SsMAPKK was induced in stems by salt stress.Then the SsVP ORF was integrated into the plant expression vector pCAMBIA1300 and SsMAPKK ORF was integrated into the plant expression vector pROKII. The integrated vector was introduced into Arabidopsis thaliana by in planta transformation method mediated by Agrobacterium tumefaciens GV3101. SsVP transformations were screened on media with hygromycin(25mg/L). we obtained TI plants. SsMAPKK transformations were screened on media with kanamycin(30mg/L). Nineteen individual kanamycin resistant plants were obtained. T2 plants were checked for integration of foreign gene by counting ratio of the number of tolerant plants to the number of non-tolerant plants on selection medium with kanamycin(30mg/L). PCR analysis indicated that all lines had been integrated of SsMAPKK. Northern analysis revealed the presence of expression of SsMAPKK mRNA in transgenic lines. In principle, SsVP overexpression can increase proton electrochemical gradients across the vacuolar membranes, which permit the secondary active transport of Na+ and solute molecules. SsMAPKK overexpression can active downstream stress-related gene to enhance transgenic plants tolerance to multiple environmental stress conditions.更多还原