苯丙氨酸和谷氨酸联合处理对大豆芽菜中5-甲基四氢叶酸富集的影响
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摘要
[目的]本文旨在研究苯丙氨酸与谷氨酸对大豆芽菜中5-甲基四氢叶酸(5-CH_3-THF)富集的影响及其机制。[方法]通过单因素及全排列试验优化出苯丙氨酸与谷氨酸的最适处理浓度,采用HPLC法测定5-CH_3-THF和对氨基苯甲酸(pABA)含量,采用氨基酸自动分析仪测定氨基酸组成,并通过荧光定量PCR测定叶酸合成相关基因相对表达量。[结果]苯丙氨酸与谷氨酸处理均显著增加大豆芽菜中5-CH_3-THF含量,其含量随苯丙氨酸浓度的增加呈先升后降趋势,且苯丙氨酸浓度为3 mmol·L~(-1)时5-CH_3-THF含量最高;而当谷氨酸浓度大于0.5 mmol·L~(-1)时,谷氨酸各浓度处理之间差异不显著。二者联合处理结果表明:3 mmol·L~(-1)苯丙氨酸和1.0 mmol·L~(-1)谷氨酸为最佳联合施用浓度,在此条件下,大豆芽菜中5-CH_3-THF含量最高,比对照增加55.50%;pABA含量比对照增加134.49%;氨基酸代谢活跃,其中甲硫氨酸、甘氨酸及组氨酸等含量均增加;叶酸合成相关基因的相对表达量提高,羟甲基二氢蝶呤焦磷酸化-二氢喋酸合成酶(HPPK-DHPS)、二氢叶酸还原酶(DHFR)、多聚谷氨酸合成酶(FPGS)等基因的相对表达量分别为对照的4.91、5.58、3.59倍。[结论]苯丙氨酸与谷氨酸联合处理大豆芽菜可促进氨基酸代谢,增大叶酸的需要量,提高叶酸合成相关基因相对表达量,促进中间底物pABA的积累,从而使5-CH_3-THF得以富集。
[Objectives]The aim of this study was to investigate the effect of phenylalanine and glutamate treatment on the enrichment of 5-methyltetrahydrofolate(5-CH_3-THF)and its mechanism in soybean sprouts. [Methods]The concentration of phenylalanine and glutamate was optimized by the single-factor and permutation experiment. The content of 5-CH_3-THF and p-aminobenzoic acid(pABA)was determined by HPLC. The amino acid composition was determined by amino acid automatic analyzer and the relative expression levels of the folate synthesis-related genes were determined by real-time fluorescence quantitative PCR. [Results]Both phenylalanine and glutamate treatment significantly increased the 5-CH_3-THF content in soybean sprouts,the content of 5-CH_3-THF increased first and then decreased with the increase of phenylalanine concentration,and reached the maximum when the concentration of phenylalanine was 3 mmol·L~(-1). However,when the concentration of glutamate was greater than 0.5 mmol·L~(-1),the difference between the different treatments was not significant. The results showed that 3 mmol·L~(-1) phenylalanine combined with 1.0 mmol·L~(-1) glutamate was the best application concentration. Under this condition,the content of 5-CH_3-THF in soybean sprouts was the highest,which was 55.50% higher than that of the control,and the pABA content increased by 134.49%. The amino acid metabolism was active,and the contents of methionine,glycine and histidine increased. The relative expression level of folate synthesis-related genes increased,including hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase(HPPK-DHPS),dihydrofolate reductase(DHFR),and folypolyglutamate synthase(FPGS),which were 4.91,5.58,3.59 times of control individually. [Conclusions]The combined treatment with phenylalanine and glutamate on soybean sprouts could promote amino acid metabolism and increase the requirement of folate,and thereby increased the relative expression of folate synthesis-related genes,promoted the accumulation of intermediate substrate pABA,and finally enriched 5-CH_3-THF.
[Objectives]The aim of this study was to investigate the effect of phenylalanine and glutamate treatment on the enrichment of 5-methyltetrahydrofolate(5-CH_3-THF)and its mechanism in soybean sprouts. [Methods]The concentration of phenylalanine and glutamate was optimized by the single-factor and permutation experiment. The content of 5-CH_3-THF and p-aminobenzoic acid(pABA)was determined by HPLC. The amino acid composition was determined by amino acid automatic analyzer and the relative expression levels of the folate synthesis-related genes were determined by real-time fluorescence quantitative PCR. [Results]Both phenylalanine and glutamate treatment significantly increased the 5-CH_3-THF content in soybean sprouts,the content of 5-CH_3-THF increased first and then decreased with the increase of phenylalanine concentration,and reached the maximum when the concentration of phenylalanine was 3 mmol·L~(-1). However,when the concentration of glutamate was greater than 0.5 mmol·L~(-1),the difference between the different treatments was not significant. The results showed that 3 mmol·L~(-1) phenylalanine combined with 1.0 mmol·L~(-1) glutamate was the best application concentration. Under this condition,the content of 5-CH_3-THF in soybean sprouts was the highest,which was 55.50% higher than that of the control,and the pABA content increased by 134.49%. The amino acid metabolism was active,and the contents of methionine,glycine and histidine increased. The relative expression level of folate synthesis-related genes increased,including hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase(HPPK-DHPS),dihydrofolate reductase(DHFR),and folypolyglutamate synthase(FPGS),which were 4.91,5.58,3.59 times of control individually. [Conclusions]The combined treatment with phenylalanine and glutamate on soybean sprouts could promote amino acid metabolism and increase the requirement of folate,and thereby increased the relative expression of folate synthesis-related genes,promoted the accumulation of intermediate substrate pABA,and finally enriched 5-CH_3-THF.
引文
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[22] 李莎,姜凌,王崇英,等.叶酸在植物体内功能的研究进展[J].植物学报,2012,47(5):525-533.Li S,Jiang L,Wang C Y,et al.Research advances in the functions of plant folates[J].Chinese Bulletin of Botany,2012,47(5):525-533(in Chinese with English abstract).
[23] 于妍,宋万坤,刘春燕,等.植物天冬氨酸代谢途径关键酶基因研究进展[J].生物技术通报,2008(增刊):7-11,17.Yu Y,Song W K,Liu C Y,et al.Research development of key enzymes gene on aspartic acid metabolic pathway in plants[J].Biotechnology Bulletin,2008(Suppl):7-11,17(in Chinese with English abstract).
[24] Jabrin S,Ravanel S,Gambonnet B,et al.One-carbon metabolism in plants.Regulation of tetrahydrofolate synthesis during germination and seedling development[J].Plant Physiology,2003,131(3):1431-1439.
[25] Storozhenko S,de Brouwer V,Volckaert M,et al.Folate fortification of rice by metabolic engineering[J].Nature Biotechnology,2007,25(11):1277-1279.
[2] Blancquaert D,Storozhenko S,Loizeau K,et al.Folates and folic acid:from fundamental research toward sustainable health[J].Critical Reviews in Plant Sciences,2010,29(1):14-35.
[3] Rader J I,Schneeman B O.Prevalence of neural tube defects,folate status,and folate fortification of enriched cereal-grain products in the United States[J].Pediatrics,2006,117(4):1394-1399.
[4] Scott J,Rébeillé F,Fletcher J.Folic acid and folates:the feasibility for nutritional enhancement in plant foods[J].Journal of the Science of Food and Agriculture,2000,80(7):795-824.
[5] Strobbe S,van der Straeten D.Folate biofortification in food crops[J].Current Opinion in Biotechnology,2017,44:202-211.
[6] D?ugosz-Grochowska O,Ko?ton A,Wojciechowska R.Modifying folate and polyphenol concentrations in Lamb’s lettuce by the use of LED supplemental lighting during cultivation in greenhouses[J].Journal of Functional Foods,2016,26:228-237.
[7] Hefni M,Witth?ft C M.Effect of germination and subsequent oven-drying on folate content in different wheat and rye cultivars[J].Journal of Cereal Science,2012,56(2):374-378.
[8] Hefni M,Witth?ft C M.Folate content in processed legume foods commonly consumed in Egypt[J].LWT-Food Science and Technology,2014,57(1):337-343.
[9] Shohag M J I,Wei Y Y,Yang X E.Changes of folate and other potential health-promoting phytochemicals in legume seeds as affected by germination[J].Journal of Agricultural and Food Chemistry,2012,60(36):9137-9143.
[10] Koehler P,Hartmann G,Wieser H,et al.Changes of folates,dietary fiber,and proteins in wheat as affected by germination[J].Journal of Agricultural and Food Chemistry,2007,55(12):4678-4683.
[11] Tzin V,Galili G.The biosynthetic pathways for shikimate and aromatic amino acids in Arabidopsis thaliana[J].The Arabidopsis Book,2010,8:e0132.
[12] Shohag M J,Wei Y Y,Yu N,et al.Natural variation of folate content and composition in spinach(Spinacia oleracea)germplasm[J].Journal of Agricultural and Food Chemistry,2011,59(23):12520-12526.
[13] Shohag J I.蔬菜叶酸:检测技术、提高途径和基因型差异[D].杭州:浙江大学,2013:131-133.Shohag J I.Folates in vegetables:determination,enhancement and genotypic variation[D].Hangzhou:Zhejiang University,2013:131-133(in Chinese with English abstract).
[14] Thaler C.Folate metabolism and human reproduction[J].Geburtshilfe und Frauenheilkunde,2014,74(9):845-851.
[15] 曹能,李璋.叶酸在人体内作用的研究进展[J].生物学通报,2003,38(2):20-22.Cao N,Li Z.Research progress of the role of folic acid in human body[J].Bulletin of Biology,2003,38(2):20-22(in Chinese).
[16] Fattal-Valevski A,Bassan H,Korman S H,et al.Methylenetetrahydrofolate reductase deficiency:importance of early diagnosis[J].Journal of Child Neurology,2000,15(8):539-543.
[17] Watanabe S,Ohtani Y,Tatsukami Y,et al.Folate biofortification in hydroponically cultivated spinach by the addition of phenylalanine[J].Journal of Agricultural and Food Chemistry,2017,65(23):4605-4610.
[18] Nair P M,Vining L C.Phenylalanine hydroxylase from spinach leaves[J].Phytochemistry,1965,4(3):401-411.
[19] Pribat A,Noiriel A,Morse A M,et al.Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts[J].The Plant Cell,2010,22(10):3410-3422.
[20] 阚静,李莉,许激扬.叶酸的生物合成及其代谢工程研究进展[J].中国生化药物杂志,2009,30(4):284-286.Kan J,Li L,Xu J Y.Research progress in folic acid biosynthesis and its metabolic engineering[J].Chinese Journal of Biochemical Pharmaceutic,2009,30(4):284-286(in Chinese).
[21] 李友勇,孙海丽,罗姗.氨基酸L-和D-异构体对离体小麦胚植株生长的影响[J].生物技术通报,2006(1):73-77.Li Y Y,Sun H L,Luo S.Influence of L- and D-isomers of amino acids on growth of wheat embryos seedling in vitro[J].Biotechnology Bulletin,2006(1):73-77(in Chinese with English abstract).
[22] 李莎,姜凌,王崇英,等.叶酸在植物体内功能的研究进展[J].植物学报,2012,47(5):525-533.Li S,Jiang L,Wang C Y,et al.Research advances in the functions of plant folates[J].Chinese Bulletin of Botany,2012,47(5):525-533(in Chinese with English abstract).
[23] 于妍,宋万坤,刘春燕,等.植物天冬氨酸代谢途径关键酶基因研究进展[J].生物技术通报,2008(增刊):7-11,17.Yu Y,Song W K,Liu C Y,et al.Research development of key enzymes gene on aspartic acid metabolic pathway in plants[J].Biotechnology Bulletin,2008(Suppl):7-11,17(in Chinese with English abstract).
[24] Jabrin S,Ravanel S,Gambonnet B,et al.One-carbon metabolism in plants.Regulation of tetrahydrofolate synthesis during germination and seedling development[J].Plant Physiology,2003,131(3):1431-1439.
[25] Storozhenko S,de Brouwer V,Volckaert M,et al.Folate fortification of rice by metabolic engineering[J].Nature Biotechnology,2007,25(11):1277-1279.