玉米5-甲酰四氢叶酸环化连接酶基因的克隆及其在叶酸代谢调控中的功能研究
发布时间：2018-07-27 16:44
【摘要】：叶酸作为介导一碳代谢的重要辅因子,参与生物体内的多种代谢活动。5-甲酰四氢叶酸环化连接酶(5FCL)被报道催化贮存形式的5-甲酰四氢叶酸(5-CHO-THF)向代谢活性形式的叶酸转化,本文对玉米中的5-甲酰四氢叶酸环化连接酶(5FCL)进行了克隆和功能分析。通过体外酶活分析及在拟南芥中过表达实验验证了其蛋白功能,分析了玉米中Zm5FCL的表达模式和亚细胞定位,并对玉米胚和胚乳中叶酸的分布及叶酸合成、代谢基因的表达模式进行了分析,取得的主要研究结果如下:1、基于拟南芥At5FCL的蛋白序列,我们从玉米中克隆了其同源蛋白(GI:GRMZM5G807835),命名为Zm5FCL。与番茄和拟南芥5FCL蛋白序列比对,结果显示序列相似度分别达到53.07%和52.38%,与人类、酵母、细菌的相似度分别达到22%-28%。Zm5FCL蛋白中包含5-formyltetrahydrofolate cycloligase结构域,推测其可能存在功能的保守性。2、Zm5FCL体外蛋白酶活测定及其在拟南芥5fcl突变体和野生型中的过表达分析结果显示:体外酶活体系中Zm5FCL蛋白能够将5-CHO-THF转化为5,10-次甲基四氢叶酸(5,10-CH=THF);在5fcl突变体中过表达Zm5FCL后,突变体叶片中5-CHO-THF含量恢复到野生型水平,说明Zm5FCL能够互补At5FCL的功能;在野生型拟南芥中过表达Zm5FCL时,叶片中的5-CHO-THF显著降低。3、利用玉米原生质体瞬时表达分析显示,初步判定Zm5FCL::GFP融合蛋白定位于细胞质中。在玉米籽粒发育过程中,Zm5FCL基因在胚中的表达量明显高于胚乳。叶酸水平检测分析显示,5-甲基四氢叶酸(5-CH3-THF)含量在胚和胚乳之间存在存在显著差异,胚中明显高于胚乳,而胚和胚乳中的5-CHO-THF含量没有明显差异。4、通过对胚和胚乳中叶酸合成及一碳代谢相关基因的转录水平进行分析,发现ZmGTPCHI-2,ZmGDCH-1,ZmGDCT,ZmDHC5,Zm10-FDF,Zm5FCL六个基因在胚中表达水平明显高于胚乳,ZmDHNA1,ZmHPPK,ZmADCL2,Zm DHFR-1,ZmDHFR-2,ZmDHFR-4,ZmGDCH-4,ZmGDCP,ZmDHC1,ZmDHC4,ZmMTHFR1,ZmMTHFR2在胚乳中的表达水平明显高于胚,这些结果说明玉米胚和胚乳中叶酸的合成和分布存在着不同的调控机制。
[Abstract]:Folic acid, as an important cofactor mediating a carbon metabolism, is involved in a variety of metabolic activities. 5-formyltetrahydrofolate cyclization ligase (5FCL) has been reported to catalyze the conversion of 5-formyltetrahydrofolic acid (5-CHO-THF) into the metabolic active form of folic acid. In this paper, 5-formyl tetrahydrofolate cyclic ligase (5FCL) from maize was cloned and its function was analyzed. The protein function was verified by enzyme activity analysis in vitro and overexpression in Arabidopsis thaliana. The expression pattern and subcellular localization of Zm5FCL in maize were analyzed, and the distribution of folic acid and folic acid synthesis in maize embryo and endosperm were analyzed. The main results are as follows: 1. Based on the protein sequence of Arabidopsis thaliana At5FCL, we cloned its homologous protein (GI:GRMZM5G807835) from maize, named Zm5FCL. Compared with tomato and Arabidopsis 5FCL protein sequence, the sequence similarity was 53.07% and 52.38%, respectively. The similarity with human, yeast and bacteria reached the 5-formyltetrahydrofolate cycloligase domain in 22%-28%.Zm5FCL protein, respectively. The in vitro protease activity of Zm5FCL and its overexpression in Arabidopsis thaliana 5fcl mutant and wild-type were conjectured. The results showed that Zm5FCL protein in the enzyme activity system of Arabidopsis thaliana could transform 5-CHO-THF into 5hm5FCL. Hydrofolic acid (5 ~ (10 ~ (-) CHT _ (THF); after overexpression of Zm5FCL in 5fcl mutants, 5-CHO-THF content in leaves of mutant returned to wild-type level, indicating that Zm5FCL could complement the function of At5FCL, and 5-CHO-THF in leaves decreased significantly when Zm5FCL was over-expressed in wild-type Arabidopsis thaliana. It was preliminarily determined that the Zm5FCL::GFP fusion protein was located in the cytoplasm. The expression of Zm5FCL gene in embryo was significantly higher than that in endosperm. The analysis of folic acid level showed that the content of 5-methyl-tetrahydrofolic acid (5-CH3-THF) was significantly different between embryo and endosperm, and the content of 5-CH3-THF in embryo was significantly higher than that in endosperm. However, there was no significant difference in 5-CHO-THF content between embryo and endosperm. The transcriptional level of folic acid synthesis and one carbon metabolism related gene in embryo and endosperm were analyzed. 鍙戠幇ZmGTPCHI-2,ZmGDCH-1,ZmGDCT,ZmDHC5,Zm10-FDF,Zm5FCL鍏釜鍩哄洜鍦ㄨ儦涓〃杈炬按骞虫槑鏄鹃珮浜庤儦涔，
本文编号：2148458
[Abstract]:Folic acid, as an important cofactor mediating a carbon metabolism, is involved in a variety of metabolic activities. 5-formyltetrahydrofolate cyclization ligase (5FCL) has been reported to catalyze the conversion of 5-formyltetrahydrofolic acid (5-CHO-THF) into the metabolic active form of folic acid. In this paper, 5-formyl tetrahydrofolate cyclic ligase (5FCL) from maize was cloned and its function was analyzed. The protein function was verified by enzyme activity analysis in vitro and overexpression in Arabidopsis thaliana. The expression pattern and subcellular localization of Zm5FCL in maize were analyzed, and the distribution of folic acid and folic acid synthesis in maize embryo and endosperm were analyzed. The main results are as follows: 1. Based on the protein sequence of Arabidopsis thaliana At5FCL, we cloned its homologous protein (GI:GRMZM5G807835) from maize, named Zm5FCL. Compared with tomato and Arabidopsis 5FCL protein sequence, the sequence similarity was 53.07% and 52.38%, respectively. The similarity with human, yeast and bacteria reached the 5-formyltetrahydrofolate cycloligase domain in 22%-28%.Zm5FCL protein, respectively. The in vitro protease activity of Zm5FCL and its overexpression in Arabidopsis thaliana 5fcl mutant and wild-type were conjectured. The results showed that Zm5FCL protein in the enzyme activity system of Arabidopsis thaliana could transform 5-CHO-THF into 5hm5FCL. Hydrofolic acid (5 ~ (10 ~ (-) CHT _ (THF); after overexpression of Zm5FCL in 5fcl mutants, 5-CHO-THF content in leaves of mutant returned to wild-type level, indicating that Zm5FCL could complement the function of At5FCL, and 5-CHO-THF in leaves decreased significantly when Zm5FCL was over-expressed in wild-type Arabidopsis thaliana. It was preliminarily determined that the Zm5FCL::GFP fusion protein was located in the cytoplasm. The expression of Zm5FCL gene in embryo was significantly higher than that in endosperm. The analysis of folic acid level showed that the content of 5-methyl-tetrahydrofolic acid (5-CH3-THF) was significantly different between embryo and endosperm, and the content of 5-CH3-THF in embryo was significantly higher than that in endosperm. However, there was no significant difference in 5-CHO-THF content between embryo and endosperm. The transcriptional level of folic acid synthesis and one carbon metabolism related gene in embryo and endosperm were analyzed. 鍙戠幇ZmGTPCHI-2,ZmGDCH-1,ZmGDCT,ZmDHC5,Zm10-FDF,Zm5FCL鍏釜鍩哄洜鍦ㄨ儦涓〃杈炬按骞虫槑鏄鹃珮浜庤儦涔，
本文编号：2148458
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