【作者】 孙虹；

【导师】 沈颂东；

【作者基本信息】 苏州大学 ， 细胞生物学， 2010， 硕士

【摘要】 在植物体内,叶酸(又称维生素B9)及其衍生物大多是以带多个谷氨酸尾的形式参与生物化学反应。在叶酸合成途径中,多聚谷氨酸加尾酶(polyglutamyl synthetase, FPGS)的作用是将谷氨酸残基连接到四氢叶酸形成多聚谷氨酸四氢叶酸。在拟南芥中有3个基因编码3个FPGS亚型,它们分别定位于细胞质(AtDFD)、线粒体(AtDFC)和叶绿体(AtDFB)中。本文主要研究线粒体定位的多聚谷氨酸加尾酶(AtDFC)的功能。其T-DNA插入突变体在氮限制条件下表现出较野生型更不适应的表型,突变体主根长度较野生型更短,因此我们试图探索AtDFC在氮限制条件下的作用机理以及叶酸代谢与氮代谢之间的关系。首先通过检测植株中单尾形式的5-CH3-THF和5-CHO-THF叶酸含量,发现这两者在突变体和野生型中没有明显差异。检测游离氨基酸含量,发现突变体中多种氨基酸含量均比野生型高,说明突变体中可能参与生长发育的氮源不足,需要内源蛋白质裂解释放出游离氨基酸以维持生长。接下来确定了突变体和野生型在氮限制条件下出现明显差异的NO3-浓度为0.3 mM,在0.3 mM氮限制条件下生长10天的突变体,其主根长度及植株地上部分生物量均约为野生型的一半,说明突变体更难以适应氮限制条件。对正常生长条件下和氮限制生长条件下的植株进行real-time RT-PCR分析,分析表明当AtDFC失去功能时,氮代谢、氨基酸代谢和光呼吸相关基因表达发生明显变化,说明叶酸代谢途径受到扰动会影响氮代谢和光呼吸反应,由此AtDFC将叶酸代谢与氮代谢、光呼吸反应联系起来。最后对突变体进行了的互补实验,互补的株系幼苗对氮限制条件的反应与野生型相同,说明突变体无法适应氮限制的表型是由于AtDFC基因功能缺失造成的。更多还原

【Abstract】 In plants, folate (or vitamin B9) and its derivatives are involved in many metabolic pathways as an important cofactor for one-carbon transfer reactions. In the folate biosynthesis pathway, the polyglutamyl synthetase (FPGS) catalyzes addition of polyglutamyl residues to theγsite of the first glutamate of tetrahydrofolate. AtDFB, AtDFC and AtDFD are folylpolyglutamate synthase (FPGS) localized in plastid, mitochondria and cytosol respectively in Arabidopsis. In this study, the function of mitochondria-targeted AtDFC was investigated using T-DNA insertion mutant. The mutant could not adapt to nitrogen limitation of 0.3 mM NO3-, mainly characterized by shorter primary root than the wild type. So we intended to uncover the molecular and physiological mechanisms underlying the inadaptability of the loss-of-function mutant Atdfc under nitrogen limitation. First, the content of the monoglutamate folate (5-CH3-THF and 5-CHO-THF) were analyzed and we found no significant difference between the wild type and the Atdfc. Then we measured the free amino acids, and found that the content of many amino acids are much higher in Atdfc than in wild type. This may indicate that the endogeneous nitrogen is not sufficient to satisfy Atdfc growth, thus the mutant needs to promote protein lysis into amino acids to maintain its development. It was found by real-time RT-PCR that the expression patterns of the genes involved in nitrogen metabolism, amino metabolism and photorespiration changed significantly in the mutant as compared to wild type when subjected to nitrogen limitation, thus AtDFC could be the connection point between the nitrogen metabolism and the photorespiration. At last, the transgenic complementation lines were obtained and the phenotype was recovered to wild type under nitrogen limitation, proving the phenotype of the mutant Atdfc was caused by the T-DNA insertion in the locus of AtDFC.更多还原