【作者】 王增兰；

【导师】 赵彦修； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2004， 博士

【摘要】 盐分胁迫主要包括渗透胁迫及离子胁迫及其造成的一系列次级胁迫如氧化胁迫等，严重干扰植物体内业已存在的细胞及整株水平上的水分及离子稳态，造成植物细胞分子损伤，生长减缓甚至死亡。因而植物对抗盐胁迫的一个重要策略是在高盐环境下重建体内的稳态(homeostasis)，包括重建离子稳态(ionic homeostasis)和渗透稳态(osmotic homeostasis)。本实验以与拟南芥近缘的盐芥为材料，构建了一个经过200mmol／L NaCl处理的盐芥叶片的cDNA文库，并从中随机挑选克隆单向测序，获得EST库；比较了盐胁迫下盐芥和拟南芥生长及渗透调节的变化；分析了盐芥P5CS基因在不同胁迫下和不同组织中的表达特性，探讨了其表达与脯氨酸合成、渗透调节之间的关系；分析了各种胁迫对硫氧还蛋白h、肌醇半乳糖苷酶基因表达的影响；构建了硫氧还蛋白h-GST原核表达载体和硫氧还蛋白h双元植物表达载体、肌醇半乳糖苷酶基因双元植物表达载体、SOS3基因双元植物表达载体，并转化拟南芥；同时构建了SOS3基因沉默载体并转化盐芥；分析了过量表达肌醇半乳糖苷酶基因、SOS3基因对拟南芥耐盐性的影响，主要实验结果如下： 1．盐芥(T.halophila)ESTs库的构建 构建了一个经过200mmol／L NaCl处理的盐芥叶片的cDNA文库，并从中随机挑选克隆单向测序，获得了1699个EST序列，其中1178个EST序列与以前鉴定的基因序列具有很高的同源性。通过序列分析，鉴定了813个独立的EST序列，其中549个序列与以前鉴定的基因具有同源性，264个为未知基因。所有的EST数据都公布在GeneBank的dbEST中。盐芥和拟南芥cDNA序列的一致性在EST总数和非冗余的克隆中分别达到95.76％、95.36％。耐盐相关或受盐胁迫调控基因的EST划分为8个主要的类别，占EST总数的18.89％。 2．盐胁迫下盐芥和拟南芥(Arabidopsis thaliana)生长及渗透调节的比较 用不同浓度的NaCl处理生长四周的盐芥和拟南芥幼苗，测定盐芥的盐度阈值；比较盐胁迫下盐芥、拟南芥干重、鲜重、K~+、Na~+含量以及游离氨基酸、脯氨酸、有机酸、可溶性糖等有机渗透保护物质的变化；比较NaCl对渗透势、MDA含量的影响。盐芥存活率达50％的NaCl浓度(盐度阈值)为300mmol／L，在中、低盐度下(50—150mmol／L NaCl)，盐芥生长并没有受到抑制，只有在较高盐度下(250，300mmol／L NaCl)，盐芥的生长才受到抑制。而拟南芥生长一开始就随着盐浓度的升高而降低。盐荞(Thellungiella hal叩hila)耐盐生理及分子生物学墓础研究 盐胁迫下Na+、K+离子在盐芥、拟南芥中变化趋势不同。相同盐度下拟南芥K+离子含量的降低幅度要比盐芥大。盐芥根中Na+含量随着盐浓度的升高变化并不大:而拟南芥根中Na+含量一直随盐浓度的升高而升高。拟南芥比盐芥积累更多的脯氨酸和可溶性糖，渗透势降低幅度大于盐芥，可能50一300~ol几NaCI没有对盐芥造成明显的渗透胁迫。盐胁迫下盐芥叶中MDA含量几乎不变，而拟南芥MDA含量随盐浓度的升高明显增加。研究结果表明盐芥耐盐性远高于拟南芥。 3.盐芥角尸JCS基因克隆及在胁迫条件下的表达分析 利用EST随机挑取克隆测序的策略从盐芥叶片cDNA文库分离得到编码盐芥△‘一二氢毗咯一5一梭酸合成酶基因的部分cDNA序列。该cDNA片段长约824bP，它编码△’一二氢毗咯一5一梭酸合成酶近C一端的154个氨基酸。BLAST同源性分析表明，该cDNA与己报道的拟南芥尸歹C万基因同源性最高。 Northem分析表明NaCI、ABA、低温、PEG均诱导尸JC夕基因的表达，而且尸JCS基因的表达具有组织特异性和发育阶段特异性。 4.盐芥ThTRXh，角GOLS和介召以刃基因克隆及功能分析 利用EST随机挑取克隆测序的策略从盐芥叶片cDNA文库分别分离得到编码盐芥硫氧还蛋白h(几2火万h)、肌醇半乳糖昔合酶(ThGOLS)、钙感应蛋白(ThS以竹)的全长cDNA序列。盐芥硫氧还蛋白h cDNA序列全长663一bp，编码118个氨基酸残基，分子量约12.9 kDa。存在保守的活性中心序列“WCP/GPC”。N。汕em分析表明盐芥Th7天Xh基因表达受NaCI、ABA、PEG诱导，不受低温诱导，也没有组织特异性。ThTRXh一GST融合蛋白体外表达及活性实验表明，盐芥硫氧还蛋白h的确具有h型的活性，最大反应速度Vmax为0.025从650.min一’，Km值为0.437林m。比。同时ThTRXh也在拟南芥中过量表达，并得到转基因株系。 肌醇半乳糖昔合酶(几GOLS)cDNA序列全长1 322一bP，编码337个氨基酸残基。与其它物种来源的肌醇半乳糖昔合酶具有较高的氨基酸序列相似性。 ABA、NaCI均诱导了肌醇半乳糖昔合酶基因在叶中表达;在根中的表达与叶不同，明显受ABA诱导，而NaCI的诱导作用非常微弱;PEG、低温既能上调叶中几GOLS的表达，又能诱导根中八GOLS的表达，说明肌醇半乳糖昔合酶的表达存在组织差异和胁迫诱导的差异。 肌醇半乳糖合酶基因八GOLS在拟南芥中过量表达提高了转基因拟南芥种子在含NaCI、甘露醇培养基上的萌发率，根的相对伸长率以及对LICI的耐受能力也明显好于野生型拟南芥。在幼苗生长阶段转基因植株鲜重、干重均高于野生型，尤其是在NaCI胁迫下其干物质积累明显较野生型增多。 经NaCI处理的野生型和转基因拟南芥其净光合更多还原

【Abstract】 High salt stress disrupts homeostasis in water potential and ion distribution. This disorder of homeostasis occurs at both the cellular and the whole plant levels. Drastic changes in ion and water homeostasis lead to molecular damage, growth inhibition and even death. Therefore, one important strategy for plants to survive under salt stress is to re-establish the homeostasis in high salt environment. Both ionic and osmotic homeostasis must be restored. With salt cress(T. halophila), a halophytic model system, we constructed a NaCl-treated cDNA library and sequenced randomly selected clones and constructed a EST Database; compared the growth and osmotic adjustment of salt cress with Arabidopsis; analyzed the expression of ThP5CS under various stresses and in different plant tissues, and discussed the relation between the expression of ThP5CS with Proline synthesis and osmotic adjustment; analyzed the effects of various stresses on the expression of ThTRX h and ThGOLS; constructed ThTRX h-GST fusion protein and expressed in E. Coli., and completed the constructions of binary Ti vector with ThTRX h, ThGOLS and ThSOS3, and transformed into Arabidopsis, respectively; and also completed the construction of gene silencing vector with ThSOS3 and transformed into salt cress. The transgenic Arabidopsis plants overexpressing ThTRX h, ThGOLS and ThSOS3 were also been analyzed to detect salt tolerance of the transgenic plants, respectively. The results were shown as follows: 1. Construction of EST Database from Salt CressWe constructed a NaCl-treated cDNA library of T. halophila and sequenced randomly selected clones. 1699 ESTs were generated. Among them, 1178 ESTs had significantly homology to previously identified genes. By sequence analysis, 813 unique clones were identified:549 shoewd homology to previous identified genes, 264 sequences matched uncharacterized genes. All our EST Data were submitted to dbEST in GenBank and are available on the internet now. The identity between T. halophila and Arabidopsis thaliana cDNA sequences in our EST collection are 95.76% in total ESTs and 95.36% in non-redundant clones. At least eight classes of genes were related to the salt tolerance, which accounted for about 18.89% of total sequenced ESTs.2. Comparation of Growth and Osmotic Adjustment of Salt Cress(T. halophila) and Arabidopsis under NaCl stressFour-week-old seedlings of salt cress and Arabidopsis were treated with different concentrations of NaCl, respectively. Salinity threshold for salt cress growth was determined. The changes in dry and fresh weights, K+ and Na+ contents, free amino acid, Proline, organic acid, soluble sugars were compared between salt cress and Arabidopsis. And the effects of NaCl on osmotic potential and MDA content were also compared between the two plants.The results showed that NaCl 300mmol/L was a salinity threshold for salt cress growth which decreased the survival rate by 50% relative to the medium without salt. Low and moderate salinity(50-150mmol/L NaCl) did not inhibit the growth of salt cress and only the high salinity(up to 250-300mmol/L NaCl) could inhibit its growth, while Arabidopsis growth always decreased with the increase in NaCl concentration.Under NaCl stress, Changes in Na+ and K+ content showed different tendency between salt cress and Arabidopsis.The decrease in K+content of Arabidopsis leaves was more than that of salt cress. The Na+ content in salt cress roots increased slightly with the increase in salinity, while the Na+ content in Arabidopsis roots had more increase with salinity increase. Our data also showed that Arabidopsis could accumulated more Proline and soluble sugars and result in more decrease in osmotic potential than salt cress. The MDA content in salt cress leaves almost had no changes under salt stress, while that of Arabidopsis leaves increased significantly with the increase of NaCl concentration. All these data demonstrated that salt cress had more salt tolerance than Arabidopsis. 3.Cloning and Expression Analysis of ThP5CS Gene under Various StressesPartia更多还原