Molecular Biological Analysis of the biosynthesis of glycine betaine in Amaranthus

1.甜菜碱(Gb)积累植株中甜菜碱(Gb)的生物合成途径及其限制步骤外源供应胆碱提高了盐胁迫下籽粒扬离体叶中Gb的积累水平。这表明，至少在盐胁迫的初期，内源胆碱(胆碱单加氧酶的底物)的供应是有限的。因此，进一步研究了影响GB积累的因素。据报道，在植物中，胆碱是由丝氨酸通过乙醇胺合成的。光呼吸可能在光合作用组织中丝氨酸的生物合成中起重要作用，因此研究了CO2浓度对GB积累的影响。在高浓度CO_2下，盐胁迫条件下GB含量显著下降。Gb的积累可能依赖于盐胁迫下光呼吸合成丝氨酸。GB积累植株GB合成的诱导机制转移到盐胁迫后1d内观察到真叶和下胚轴中胆碱单加氧酶(CMO)的积累。另一方面，CMO在子叶和根中的积累相对较慢。不同植物组织对盐胁迫的反应不同。将籽粒苋苗转移到黑暗中，即使在盐胁迫下也不会发生CMO的积累。说明CMO的积累除了盐胁迫外，还需要光照。克隆了CMO基因上游区基因表达调控基因，构建了CMO启动子：：GUS载体。利用该构建物对籽粒扬成熟叶片进行了瞬时检测，并对其在转基因拟南芥中的表达进行了分析。对DNA中的抑制区和增强区进行了鉴定。

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.