Molecular Biological Analysis of the biosynthesis of glycine betaine in Amaranthus

1. 甜菜碱在甜菜碱积累植物中的生物合成途径及其限制步骤。盐胁迫下，外源胆碱增加了苋菜离体叶片GB积累水平。这表明内源性胆碱（胆碱单加氧酶的底物）的供应至少在盐胁迫的初始阶段是有限的。因此，进一步研究影响GB积累的因素。有报道称，从植物中通过乙醇胺由丝氨酸合成胆碱。光呼吸作用可能在光合组织丝氨酸生物合成中起重要作用，因此研究了CO_2浓度对GB积累的影响。在高浓度CO_2条件下，盐胁迫条件下的GB含量显著降低。盐胁迫下叶绿素积累可能依赖于光呼吸合成丝氨酸。胆碱单加氧酶（CMO）在盐胁迫后1 d内在真叶和下胚轴中积累。另一方面，CMO在子叶和根的积累相对缓慢。不同植物组织对盐胁迫的响应存在差异。当苋菜幼苗转移到黑暗环境时，即使在盐胁迫下也没有发生CMO的积累。说明CMO的积累除了需要盐胁迫外，还需要光照。分离了CMO基因CMOUpstream区域的基因表达调控，构建了CMO启动子：：GUS构建体。利用该构建体对苋菜成熟叶片进行了瞬时检测，并对转基因拟南芥进行了表达分析。鉴定了DNA中的抑制区和增强区。

1. Biosynthetic pathway of glycine betaine (GB) and its limiting step in GB accumulating plant.Accumulation level of GB in excised leaves of Amaranthus under salt stress was increased by exogenous supply of choline. It suggests that endogenous supply of choline (a substrate for choline monooxygenase) is limiting at least in the initial phase of salt stress.So the factors affecting GB accumulation was further investigated. It has been reported that choline is synthesized from serine via ethanolamine in plants. Photorespiration may have an important role in serine biosynthesis in photosynthetic tissue, so the effect of concentration of CO_2 on the accumulation of GB was studied. Under high concentration of CO_2, significant decrease in GB content was observed under salt-stressed condition. GB accumulation may depend on the synthesis of serine by photorespiration under salt stress.2. Induction mechanism of GB synthesis in GB accumulating plantAccumulation of choline monooxygenase (CMO) was observed within I day in real leaves and hypocotyls after transfer to salt stress. On the other hand, accumulation of CMO in cotyledons and roots relatively slowly occurred. Response to salt stress was different among plant tissues. When Amaranthus seedlings were transferred to darkness, accumulation of CMO did not occur even under salt stress. It shows the accumulation of CMO need light besides salt stress.3. Regulation of gene expression of CMOUpstream region of CMO gene was isolated and the CMO-promoter::GUS construct was made. By using this construct, transient assay was done with mature leaves of Amaranthus The expression analysis in transgenic Arabidopsis was also done with this construct. The suppressor and enhancer region in the DNA were identified.