类谷氨酸亚胺甲基转移酶调控玉米籽粒叶酸含量的分子机理研究

5-formyltetrahydrofolate (5-formyl THF) is the main form of folates in maize kernels. Using genome-wide association analysis, a gene significantly associated with the content of 5-formyl THF in maize kernels was found. This gene encodes a folate binding protein, which could serve as a glutamate formiminotransferase (FT) , and thus is named as ZmFTL. Furthermore, it is reported that FT may have 5-formyltetrahydrofolate cycloligase (5-FCL) activity in some bacteria. Interestingly, three adjacent SNPs in ZmFTL lead to two-amino-acid residue difference in different maize inbreds, and result in significantly different 5-formyl THF content in maize kernels. This research is focused on the regulatory mechanism of ZmFTL underlying the content of 5-formyl THF in maize kernels. Using molecular simulation methods, the optimum substrate of the protein will be discovered, and the relationship between subtle structural differences caused by the natural variation of amino acids and the ability of folate binding will be investigated. Afterwards, the binding pocket of the protein will be found, and more critical amino acids for legand-binding will be confirmed. Finally, biochemical experiments will be performed in vivo and in vitro in order to validate our theoretical prediction. This study will provide a new directions for crop molecular design breeding.

5-甲酰四氢叶酸是玉米籽粒中叶酸的主要存在形式。在玉米籽粒中，我们发现了一个控制5-甲酰四氢叶酸含量的关键基因ZmFTL，可能具有谷氨酸亚胺甲基转移酶和/或5-甲酰四氢叶酸环化连接酶的功能。该基因存在三种等位变异形式，三个相邻的SNP位点导致了相邻两个氨基酸的突变。遗传学证据表明这两个氨基酸变异造成了不同玉米自交系籽粒中5-甲酰四氢叶酸含量的显著差异。本项目拟采用计算机分子模拟和实验验证相结合的方法探索ZmFTL基因功能，阐述该基因调控玉米籽粒叶酸含量的分子机理。首先用分子模拟方法预测此蛋白的最适底物和蛋白的活性位点，探究其对最适底物的催化机理，分析氨基酸自然变异导致的结构差异对蛋白催化能力的影响；然后突变关键氨基酸，用分子模拟手段研究人工突变蛋白的活性和稳定性；最后通过体外酶活检测和体内的转基因玉米籽粒叶酸含量检测进行实验验证。项目的实施将为作物分子设计育种提供新的思路。

5-甲酰四氢叶酸是玉米籽粒中叶酸的主要存在形式。在前期工作中，全基因组关联分析发现了一个可能调控玉米籽粒5-甲酰四氢叶酸含量的关键基因ZmGFT1(前称ZmFTL)，可能具有谷氨酸亚胺甲基转移酶和/或5-甲酰四氢叶酸环化连接酶的功能。该基因的三种等位变异形式导致了同一氨基酸位点的两种氨基酸形式，这种自然变异可造成不同玉米自交系籽粒中5-甲酰四氢叶酸含量的显著差异。本项目采用了计算机分子模拟和实验验证相结合的方法探索ZmGFT1基因功能，用分子模拟方法预测了蛋白的活性位点，发现了位于结合口袋附近的多态性位点造成的氨基酸变异会导致结合口袋空间结构变化，进而引起底物与蛋白结合活性的改变，从而影响其功能，阐述该基因调控玉米籽粒叶酸含量的可能机理：通过ZmGFT1蛋白与底物结合能的差异调控5-甲酰四氢叶酸含量。接下来的转基因拟南芥和转基因玉米籽粒叶酸含量检测验证了上述可能性。项目的实施为通过分子改造的方法提高作物叶酸含量提供了依据。