【摘要】：葉酸作為介導一碳代謝的重要輔因子,參與生物體內的多種代謝活動。5-甲酰四氫葉酸環(huán)化連接酶(5FCL)被報道催化貯存形式的5-甲酰四氫葉酸(5-CHO-THF)向代謝活性形式的葉酸轉化,本文對玉米中的5-甲酰四氫葉酸環(huán)化連接酶(5FCL)進行了克隆和功能分析。通過體外酶活分析及在擬南芥中過表達實驗驗證了其蛋白功能,分析了玉米中Zm5FCL的表達模式和亞細胞定位,并對玉米胚和胚乳中葉酸的分布及葉酸合成、代謝基因的表達模式進行了分析,取得的主要研究結果如下:1、基于擬南芥At5FCL的蛋白序列,我們從玉米中克隆了其同源蛋白(GI:GRMZM5G807835),命名為Zm5FCL。與番茄和擬南芥5FCL蛋白序列比對,結果顯示序列相似度分別達到53.07%和52.38%,與人類、酵母、細菌的相似度分別達到22%-28%。Zm5FCL蛋白中包含5-formyltetrahydrofolate cycloligase結構域,推測其可能存在功能的保守性。2、Zm5FCL體外蛋白酶活測定及其在擬南芥5fcl突變體和野生型中的過表達分析結果顯示:體外酶活體系中Zm5FCL蛋白能夠將5-CHO-THF轉化為5,10-次甲基四氫葉酸(5,10-CH=THF);在5fcl突變體中過表達Zm5FCL后,突變體葉片中5-CHO-THF含量恢復到野生型水平,說明Zm5FCL能夠互補At5FCL的功能;在野生型擬南芥中過表達Zm5FCL時,葉片中的5-CHO-THF顯著降低。3、利用玉米原生質體瞬時表達分析顯示,初步判定Zm5FCL::GFP融合蛋白定位于細胞質中。在玉米籽粒發(fā)育過程中,Zm5FCL基因在胚中的表達量明顯高于胚乳。葉酸水平檢測分析顯示,5-甲基四氫葉酸(5-CH3-THF)含量在胚和胚乳之間存在存在顯著差異,胚中明顯高于胚乳,而胚和胚乳中的5-CHO-THF含量沒有明顯差異。4、通過對胚和胚乳中葉酸合成及一碳代謝相關基因的轉錄水平進行分析,發(fā)現ZmGTPCHI-2,ZmGDCH-1,ZmGDCT,ZmDHC5,Zm10-FDF,Zm5FCL六個基因在胚中表達水平明顯高于胚乳,ZmDHNA1,ZmHPPK,ZmADCL2,Zm DHFR-1,ZmDHFR-2,ZmDHFR-4,ZmGDCH-4,ZmGDCP,ZmDHC1,ZmDHC4,ZmMTHFR1,ZmMTHFR2在胚乳中的表達水平明顯高于胚,這些結果說明玉米胚和胚乳中葉酸的合成和分布存在著不同的調控機制。
[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〃杈炬按騫蟲槑鏄鵑珮浜庤儦涔，

本文編號：2148458