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盐角草钠/铵胁迫适应及铵转运基因家族功能研究
肖薪龙
学位类型博士
导师姚银安
2016
学位授予单位中国科学院大学
学位授予地点北京
学位专业生态学
关键词盐胁迫 铵毒害 离子转运 氮代谢 Unigene再组装 铵转运蛋白
摘要铵是植物重要的氮源之一,适宜浓度的NH4+促进植物生长,高浓度的NH4+对植物产生铵毒胁迫。盐胁迫往往对植物的氮代谢及生长产生不利影响。盐角草做为最耐盐的一种盐生植物常生长在盐湖边等土壤湿润的厌氧环境,不仅被NaCl促进生长,还具有强大的NH4+吸收能力和耐铵胁迫能力。然而,盐生环境下盐角草的氮代谢特征、转运NH4+的分子机理及NaCl增强盐角草耐铵能力的机制尚不清楚。本研究以生长于新疆的盐角草为研究对象,研究盐角草对钠/铵离子转运特征及耐逆性,克隆并分析了6个铵转运(Ammonium transporter, AMT)基因表达特征及其功能。在此过程中,筛选了盐角草的RT-qPCR内参基因,发现一种简便的对转录组目的unigene再拼接的方法。主要结果如下: (1)Na+积累及氮同化酶活力提高是NaCl促进盐角草生长的原因。低于200 mM NaCl处理下的盐角草细胞长得更大,可能是Na+积累后渗透调节产生的膨压所致。NaCl处理还能提高氮/铵同化关键酶(如酶谷氨酰胺合成酶和谷氨酸脱氢酶)的基因表达水平和酶活力,降低了根部游离NH4+浓度。试验结果表明NaCl还具有增强盐角草耐铵毒害能力。 (2)通过统计最近十年关于筛选植物内参基因的研究数据,再结合盐角草的转录组表达谱数据,发现不存在普遍稳定的内参基因,基因家族成员表达稳定性差异很大可能是已发表研究结果不一致的因素之一。因此,需要针对物种和试验条件筛选稳定的内参基因。通过试验筛选到盐角草在盐、氮/铵、干旱、冷、热胁迫及不同组织和不同生长时期各个条件下稳定表达的内参基因,这将有助于利用RT-qPCR技术对盐角草进行准确的基因定量分析。 (3)为研究盐角草对NH4+转运的分子机理,利用一种对转录组目的unigene再拼接的方法获得更长的序列,克隆了6个SeAMT基因。这些SeAMT基因在盐角草的不同部位表达。SeAMT1.2a、SeAMT1.2b和SeAMT3.1主要在根部表达,负责将根际外的铵态氮转运到根部细胞。SeAMT1.1和SeAMT1.3主要在地上部表达,负责地上部铵的转运和储存。SeAMT1.4的表达量较低,在根部和地上部都能检测到微量表达,可能起到铵转运辅助的作用。缺氮处理下,SeAMT1.2a和SeAMT1.2b会受到诱导且随时间不断提高表达量,以适应氮饥饿逆境。6个SeAMT基因在高浓度NH4+处理下先诱导上调,然后反馈抑制,SeAMT基因也会受到NaCl调控。通过GFP亚细胞定位分析发现SeAMT蛋白几乎都定位在细胞膜上,支持SeAMT做为NH4+膜转运蛋白的结论。通过酵母31019b和拟南芥qko的功能互补研究,发现6个SeAMT有不同程度的NH4+转运功能。SeAMT1亚家族几乎承担全部的铵转运任务,SeAMT3.1虽然在根部表达,但在异源表达系统没有表现铵转运功能。SeAMT1.2a和SeAMT1.2b序列相似性较高,铵转运能力也比其他SeAMT基因显著。它们在根部表达且受氮饥饿诱导上调,可能是铵转运及氮代谢调控过程中两个最为重要的基因。此外,SeAMT1.2a和SeAMT1.2b基因具有提高耐盐性的作用。酵母CY162转化试验发现SeAMT1.2a在低K+情况下具有一定的K+转运功能,这可能是该基因提高转基因酵母和拟南芥耐盐性的原因之一。
其他摘要Ammonium is one of the most important nitrogen sources for plants. An appropriate concentration of NH4+ promotes plant growth, while causes toxicity with excess ammonium. Salt stress often imposes an adverse effect on nitrogen metabolism and plant growth. Salicornia europaea, one of the most salt-tolerant halophyte, often grows at salt lake side where soil is saline, humid and anaerobic. Not only the growth of this species is promoted by NaCl, it also has strong ability in NH4+ absorption and ammonium tolerance. However, it is still unclear about its nitrogen metabolism characteristics under saline environment, the molecular mechanism of NH4+ transport, and why NaCl can enhance its ammonium tolerance. In this study, S. europaea was tested under various Na+/NH4+ treatment to uncover the characteristics of ions transport and stress tolerance. Six members of ammonium transporter (AMT) gene family were cloned, and analyzed with gene expression characteristics, and gene functions were identified. In addition, we selected suitable RT-qPCR reference genes for S. europaea under various conditions and found a simple method for further assembly of targeted unigenes in a transcriptome. The main results were as follows: (1) Na+ accumulation and improvement of nitrogen assimilation enzyme activity could be the main reasons for NaCl facilitated S. europaea growth. Cell size became larger under NaCl (< 200 mM) treatment, which could be the cause that Na+ accumulation improved turgor pressure by osmotic regulation. Furthermore, NaCl promoted corresponding genes expression levels and enzymes activity for nitrogen or ammonium assimilation, such as glutamine synthetase (GS) and glutamate dehydrogenase (GDH). NH4+ concentration was decreased and toxicity reduced by NaCl treatment in root under ammonium stress, which indicate NaCl also improved S. europaea ammonium tolerance. (2) Through data statistics of researches on screening plant reference genes in the last ten years, and combining the transcriptome expression profile data of S. europaea, we found that none of the analyzed genes can guarantee universally stable expressions. The tested genes belonging to multigene family could be one reason of variations in the published studies. Therefore, we validated suitable reference genes for gene expression analysis in S. europaea under various conditions (salt stress, nitrogen/ ammonium stress, drought stress, cold stress, heat stress, various tissues, various age) by RT-qPCR, which will benefit future accurate gene quantitative in S. europaea. (3) In order to find the molecular mechanism of NH4+ transport, we cloned six SeAMT genes from S. europaea after obtain longer sequence using a method for further assembly of AMT unigenes in transcriptome. These genes were expressed in different parts of the plant, to complete the NH4+ absorption from rhizosphere and NH4+ transport in the body. SeAMT1.2a, SeAMT1.2b and SeAMT3.1 were mainly expressed in the root, in charge of the NH4+ absorption from rhizosphere root cells. SeAMT1.1 and SeAMT1.3 mainly were expressed in shoot for NH4+ transport and storage in overground part of plant. The expression quantity of SeAMT1.4 was very low, which could be detected both in root and shoot. Confronted with nitrogen deficiency, expression of SeAMT1.2a and SeAMT1.2b increased with time to adapt to the nitrogen starvation adversity. Six SeAMT genes were all induced up-regulation by high concentration of NH4+, and then feedback inhibition. SeAMT genes were also regulated by NaCl. The analysis of GFP subcellular localization indicated that nearly all SeAMT protein orientation on the cell membrane, supporting the conclusion that SeAMTs were membrane transporters of NH4+. Through complementary function test in yeast 31019b and Arabidopsis thaliana qko, we found that six SeAMT have different ability for NH4+ transport. Members of SeAMT1 subfamily undertook almost all tasks for ammonium transport. Although SeAMT3.1 was expressed in root, it did not showed any ability of ammonium transport in heterologous expression systems. SeAMT1.2a and SeAMT1.2b, with high sequence similarity, had more powerful ability for NH4+ transport than other SeAMT genes. They were expressed in root and were induced up-regulation by nitrogen starvation. It indicated that SeAMT1.2a and SeAMT1.2b were two important genes for ammonium transport and nitrogen metabolism regulation. In addition, we found the two genes also improved salt resistance of yeast and A. thaliana. According to the result of gene transformation in yeast CY162, SeAMT1.2a promoted K+ transport under a low K+ condition. This could be one of reasons that the gene improve salt resistance of genetically modified organisms
学科领域生态学
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14762
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
推荐引用方式
GB/T 7714
肖薪龙. 盐角草钠/铵胁迫适应及铵转运基因家族功能研究[D]. 北京. 中国科学院大学,2016.
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