,, 刘小萍, 薄凯亮, 苗晗, 董邵云, 顾兴芳,, 张圣平,中国农业科学院蔬菜花卉研究所/农业农村部园艺作物生物学与种质创制重点实验室,北京 100081Cloning and Analysis of Folate Synthesis Key Genes in Cucumber
ZHOU Qi,, LIU XiaoPing, BO KaiLiang, MIAO Han, DONG ShaoYun, GU XingFang,, ZHANG ShengPing,Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences/Key Laboratory of Horticultural Crop Biology and Germplasm Creation, Ministry of Agriculture and Rural Areas, Beijing 100081通讯作者:
责任编辑: 赵伶俐
收稿日期:2020-02-14接受日期:2020-05-20网络出版日期:2020-09-16
| 基金资助: |
Received:2020-02-14Accepted:2020-05-20Online:2020-09-16
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周琪, 刘小萍, 薄凯亮, 苗晗, 董邵云, 顾兴芳, 张圣平. 黄瓜叶酸合成关键基因克隆与分析[J]. 中国农业科学, 2020, 53(18): 3764-3776 doi:10.3864/j.issn.0578-1752.2020.18.012
ZHOU Qi, LIU XiaoPing, BO KaiLiang, MIAO Han, DONG ShaoYun, GU XingFang, ZHANG ShengPing.
0 引言
【研究意义】叶酸是人体生命活动必需的水溶性B族维生素,其分子由蝶啶、对氨基苯甲酸和谷氨酸残基三部分组成[1]。根据取代基的不同,叶酸分为不同的种类,目前在动植物中已发现的叶酸种类有50多种[2,3]。叶酸参与核苷酸的生物合成、氨基酸代谢和甲基化循环,是细胞内主要的甲基供体,对DNA的合成、甲基化和修复至关重要[4,5,6]。美国食品和营养委员会推荐叶酸的摄取量成年人为400 μg·d-1,孕妇为600 μg·d-1。叶酸缺乏会引发巨幼红细胞贫血和老年人认知功能障碍等疾病[7,8]。但人体无法自身合成叶酸,必须依赖饮食摄入。与水稻、谷子等作物相比,蔬菜中叶酸含量较高,成为人体摄入叶酸的主要来源[9,10]。由于烹饪加热过程会导致叶酸降解[11,12,13],黄瓜以生食为主,能使其含有的叶酸不经烹饪降解而最大限度地保留,被人体吸收利用,成为人体补充叶酸的最佳选择之一。黄瓜中叶酸合成代谢的分子生物学研究尚未见报道,研究黄瓜果实的叶酸合成代谢具有重要意义。【前人研究进展】植物中叶酸代谢途径分为喋啶分支和pABA分支[3]。GTP在细胞质中形成喋啶,ADC在叶绿体中裂解生成pABA,随后二者被转入线粒体中形成二氢蝶酸,经谷氨酸化和还原反应形成四氢叶酸,最后通过加尾作用形成多尾叶酸[14,15]。在蝶啶分支中,GTP环化水解酶（GTPCHI）是催化第一步反应的酶,也是该分支的限速酶,调控该酶的基因为GCHI [16,17,18]。在pABA分支中,氨基脱氧分支酸合酶（ADCS）是其限速酶[19],由基因ADCS调控合成。GTP环化水解酶与氨基脱氧分支酸合酶被认为是限制植物叶酸合成途径的两个关键酶[20]。黄瓜是我国的重要蔬菜作物,尤其在设施生产中占有举足轻重的地位,2017年收获面积为123.52万hm2,产量达到6 482.46万t[21]。我国黄瓜以鲜食为主,能使其含有的叶酸得到最大限度的保留而被人体吸收利用。【本研究切入点】模式植物拟南芥中叶酸合成代谢相关研究较为深入,关于黄瓜叶酸代谢调控以及叶酸合成关键酶基因的克隆与表达分析尚未见报道。【拟解决的关键问题】本研究参考拟南芥调控叶酸合成代谢基因的蛋白序列,BLAST得到黄瓜的同源基因,并完成其在染色体上的定位。利用荧光定量PCR技术分析这些基因在测序黄瓜果实发育不同时期以及果实叶酸含量高低差异显著的2份材料中的表达量。通过蛋白序列比对、结构域分析以及进化树构建,分析调控叶酸合成限速步骤的关键酶基因的序列保守性,并克隆这两个关键酶基因,分析其序列在不同材料中的差异。1 材料与方法
1.1 试验材料
本研究试验材料为完成全基因测序的黄瓜自交系9930,以及果实叶酸含量高低差异显著的2份自交系5G（低）和02245（高）。试验材料于2018年春、秋两季种植于中国农业科学院南口试验基地连栋大棚（北京）,前期正常管理,9930黄瓜果实发育不同时期叶酸含量测定：开花前2 d、开花当天、开花后7 d、开花后14 d、开花后21 d果实中的叶酸含量。65G和02245的果实叶酸含量测定在植株盛瓜期取样,且选取植株中部节位、大小合适的商品瓜。1.2 试验方法
1.2.1 黄瓜叶酸合成代谢相关基因鉴定 根据文献[8]和TAIR数据库（1.2.2 黄瓜材料取样方式及叶酸含量测定 每一份材料3个生物学重复,每个重复选取3条瓜,每个瓜上、中、下部位切取3个薄片,用液氮速冻。同一个生物学重复的样品混合均匀并研磨成粉末,利用双酶法提取叶酸,利用液相色谱三重四级杆质谱联用仪测定叶酸含量[22]。
1.2.3 总RNA的提取与cDNA第一链的合成 果实取样后用锡箔纸包裹于液氮中速冻,然后用研钵将其研磨成粉末。根据TaKaRa的RNA提取试剂盒提取RNA,利用Nanodrop2000核酸仪检测其浓度,并经凝胶电泳检测其完整性,质量合格的RNA可观察到两条清晰明亮的带型。之后取适量的RNA,同时使用反转录试剂盒合成cDNA第一链。
1.2.4 黄瓜叶酸合成基因在果实发育不同时期及不同材料中的表达量分析 以反转录cDNA为模板,以黄瓜中稳定表达的基因（Csa_2G301530）为内参基因,针对基因功能序列分别设计引物（附表2）,尽量保证引物无二聚体、发夹结构等。利用TaKaRa SYBR?Premix Ex Taq?II试剂盒（北京六合通经贸有限公司）对9930果实发育不同时期以及果实叶酸含量高低差异显著的65G和02245中的基因进行实时荧光定量分析,采用F=2-△△Ct法分析其表达量[23]。
1.2.5 黄瓜叶酸合成关键酶基因的蛋白序列比对及系统进化树构建 从NCBI上搜索各物种叶酸合成关键酶GTPCHI及ADCS同源基因的蛋白序列,利用MEGA软件对序列进行比对,构建系统进化树。利用在线软件（http://weblogo.berkeley.edu/logo.cgi）对5个结构域氨基酸保守性进行分析。
1.2.6 黄瓜GCHI及ADCS的克隆与序列分析 根据黄瓜基因组网站信息（
扩增产物均用1%的琼脂糖凝胶电泳进行检测,送至上海生物工程有限公司测序。测序结果拼接后,比对分析同一基因在不同材料中的序列差异。
1.2.7 基因的生物信息学分析 利用在线软件Expasy（
2 结果
2.1 黄瓜叶酸合成相关基因
拟南芥中已报道的与叶酸代谢相关的酶共14个,其中大部分酶参与叶酸合成过程,其余为叶酸转运子或与叶酸降解相关（表1）。其中羟甲基二氢蝶呤焦磷酸激酶（HPPK）/二氢蝶呤合成酶（DHPS）是一个双功能酶,催化两步连续的反应[10]。通过基因组数据库获取拟南芥中调控这些酶的基因序列,经BLAST比对寻找黄瓜中的同源基因。得到19个黄瓜中与叶酸代谢相关的基因,其中有11个（标*）参与合成途径,包含了叶酸从头合成的全部反应。Table 1
表1
表1黄瓜叶酸合成相关基因
Table 1
| 序号Number | 酶Enzyme | 英文缩写Abbreviation | 基因 Gene |
|---|---|---|---|
| 1 | GTP环化水解酶 I 1 | GTPCHI1 | CsaV3_1G041250* |
| 2 | GTP环化水解酶 I 2 | GTPCHI2 | CsaV3_7G026240* |
| 3 | 二氢新蝶呤醛缩酶 | DHNA | CsaV3_4G004540* |
| 4 | 羟甲基二氢喋呤焦磷酸激酶/二氢蝶酸合酶 | HPPK/DHPS | CsaV3_5G008630* |
| 5 | 二氢叶酸合酶 | DHFS | CsaV3_3G021950* |
| 6 | 二氢叶酸还原酶1 | DHFR1 | CsaV3_3G040860* |
| 7 | 二氢叶酸还原酶2 | DHFR2 | CsaV3_2G014940* |
| 8 | 叶酰聚谷氨酸合酶 | FPGS | CsaV3_5G037300* |
| 9 | 氨基脱氧分支酸合酶 | ADCS | CsaV3_5G036360* |
| 10 | 氨基脱氧分支酸裂解酶1 | ADCL1 | CsaV3_4G033410* |
| 11 | 氨基脱氧分支酸裂解酶2 | ADCL2 | CsaV3_3G048670* |
| 12 | 10-甲酰四氢叶酸合酶 | THFS | CsaV3_1G042270 |
| 13 | 叶酸转运子 | FOLT | CsaV3_6G046730 |
| 14 | 5-甲酰四氢叶酸环连接酶1 | 5-FCL1 | CsaV3_6G005120 |
| 15 | 5-甲酰四氢叶酸环连接酶2 | 5-FCL2 | CsaV3_1G001940 |
| 16 | γ-谷氨酰水解酶 | GGH | CsaV3_4G033040 |
| 17 | 亚甲基四氢叶酸脱氢酶/环水解酶 | MTHFD | CsaV3_4G000560 |
| 18 | 亚甲基四氢叶酸还原酶1 | MTHFR1 | CsaV3_2G011840 |
| 19 | 亚甲基四氢叶酸还原酶2 | MTHFR2 | CsaV3_5G038920 |
新窗口打开|下载CSV
2.2 基因的染色体定位
根据各基因在染色体上的物理位置,利用MapChart软件绘制黄瓜染色体物理图谱并进行基因定位（图1）。结果发现,这19个基因不均匀分布在黄瓜的7条染色体上,其中,4号和5号染色体上最多（4个）,1号和3号染色体次之（3个）,2、6号染色体较少（2个）,7号染色体最少（1个）。除基因CsaV3_2G011840（D）、CsaV3_2G014940（E）、CsaV3_3G021950（F）外,其余大多分布在染色体长、短臂的两端。图1
新窗口打开|下载原图ZIP|生成PPT图1黄瓜中叶酸合成相关基因在染色体上的位置
Fig. 1The position on the chromosome of genes involving in folate synthesis in cucumber
A: CsaV3_1G001940; B: CsaV3_1G041250; C: CsaV3_1G042270; D: CsaV3_2G011840; E: CsaV3_2G014940; F: CsaV3_3G021950; G: CsaV3_3G040860; H: CsaV3_3G048670; I: CsaV3_4G000560; J: CsaV3_4G004540; K: CsaV3_4G033040; L: CsaV3_4G033410; M: CsaV3_5G008630; N: CsaV3_5G036360; O: CsaV3_5G037300; P: CsaV3_5G038920; Q: CsaV3_6G005120; R: CsaV3_6G046730S:CsaV3_7G026240
2.3 9930果实发育不同时期叶酸含量及对应时期的叶酸合成基因的表达量
测定黄瓜自交系9930果实发育不同时期的叶酸含量（图2-a）,结果表明,开花当天叶酸含量最高,为42.16 μg/100 g FW,开花21 d的叶酸含量最低,为21.76 μg/100 g FW。叶酸含量的规律表现为,在开花之前呈上升趋势,开花当天达到最高,开花之后呈下降趋势。图2
新窗口打开|下载原图ZIP|生成PPT图2黄瓜叶酸合成基因在果实发育不同时期的表达量
Fig. 2The expression level of folate synthetic genes in cucumber fruit at different stages
分析与叶酸合成相关的11个基因在9930果实发育不同时期的表达量,从表达量变化趋势来看,在同源基因中,CsGCHI1与CsGCHI2（图2-c、d）、CsADCL1与CsADCL2（图2-g、h）的趋势一致。CsDHFR1的表达量在整个发育过程中呈下降趋势,而CsDHFR2先上升后下降（图2-e、f）。在开花前,除基因CsDHFR1（图2-e）外的其他基因表达量均上升,与总叶酸含量呈正相关。在开花之后,基因CsDHFR1、CsFPGS、CsHPPK/CsDHPS、CsDHNA在果实发育过程中呈现下降的趋势（图2-b、e、j、l）。在整个发育过程中,基因CsFPGS、CsHPPK/CsDHPS、CsDHNA、CsADCS与叶酸含量变化趋势完全一致（图2-b、i、j、l）。
2.4 果实叶酸含量高低差异显著材料65G和02245中基因的表达量分析
分别在2018年春季和2018年秋季测定核心种质群体[24]叶酸含量,从中挑选出在两季中叶酸含量比较稳定的两份材料65G及02245,65G果实的平均叶酸含量为21.75 μg/100 g,02245果实的平均叶酸含量36.15 μg/100 g。差异显著性分析表明,两者总叶酸含量具有显著差异,且65G总低于02245（图3-a）。测定11个叶酸合成相关基因在65G及02245中的表达量,分析发现,基因CsADCL1、CsADCL2、CsDHNA、CsFPGS的表达量具有显著差异,基因CsADCS、CsDHFS、CsHPPK/CsDHPS表达量具有极显著差异（图3-b）,且这些基因均在02245中高表达,与叶酸含量正相关。图3
新窗口打开|下载原图ZIP|生成PPT图3不同材料叶酸含量及基因表达量分析
*表示显著差异,**表示极显著差异。a：65G、02245叶酸含量;b：65G、02245果实中基因表达量
Fig. 3The folate content in 65G and 02245 and the expression level of folate metabolism related genes
*represent significant differences, ** represent extremely significant difference. a: 65G、02245 folate content; b: 65G、02245 gene expression
2.5 黄瓜及其他物种GCHI蛋白质序列分析及进化树构建
在细胞质中,GTPCHI催化GTP合成甲酸和二氢蝶呤三磷酸,这一反应是真核生物合成叶酸喋啶分支的第一步反应,也是该分支的限速步骤[16,17,18]。黄瓜中控制GTPCHI合成的同源基因是CsaV3_1G041250和CsaV3_7G026240（表1）。这两个基因与拟南芥中该基因蛋白质序列的一致性分别为60%和61%（图4-a）。利用NCBI网站进行BLAST分析得到除黄瓜外8个物种中该基因的蛋白序列,利用MEGA6中的Clustal W对蛋白序列进行比对。结果表明,各物种中GCHI同源基因均含有两个GTP_cyclohydroI结构域,且无论是单子叶植物水稻、玉米还是双子叶植物拟南芥中,该蛋白的结构域高度保守,说明它们可能来自共同的祖先（图4-b）。图4
新窗口打开|下载原图ZIP|生成PPT图4黄瓜及其他物种GCHI蛋白序列比对以及进化树
a：蓝色框内为GTP_cyclohydroI结构域;b、c：GTP_cyclohydroI结构域保守性分析;d：GCHI系统进化树分析
Fig. 4Protein sequences alignment and evolutionary-tree generation of GCHI in cucumber and other species
a: GTP_cyclohydroI in the blue box; b, c: amino acid conservation analysis; d: phylogenetic tree of GCHI
对各物种GCHI的氨基酸序列进行系统进化树分析（图4-c）,发现同一物种或亲缘关系较近的物种聚在一起,黄瓜首先与其他葫芦科作物甜瓜、西瓜等聚在一起,说明该基因在亲缘关系较近的物种中进化是保守的,可能行使着相同或相近的功能。
2.6 黄瓜及其他物种ADCS蛋白质序列分析及进化树构建
在叶绿体中,ADCS催化叶酸前体4-氨基-4-脱氧络合物（ADC）的生物合成,该步骤是pABA分支的限速步骤[14],黄瓜中控制该酶合成的同源基因是CsaV3_5G036360。CsaV3_5G036360与拟南芥该基因蛋白序列的一致性为57%。同样通过BLAST找到除黄瓜外10个物种中该基因的同源蛋白序列,蛋白序列比对发现,ADCS含有4个结构域,包括2个GATase,1个Chorismate_bind和1个Anth_synt_I_N结构域（图5-a）,这4个结构域在不同物种中高度保守（图5-b—e）。图5
新窗口打开|下载原图ZIP|生成PPT图5黄瓜及其它物种ADCS蛋白序列比对以及进化树
a：红色框内为GATase结构域,蓝色框内为Anth_synt_I_N结构域,绿色框内为Chorismate_bind结构域;b、c：GATase结构域;d：Anth_synt_I_N结构域;e：Chorismate_bind结构域; f：ADCS系统进化树分析
Fig. 5Protein sequences alignment and evolutionary-tree generation of ADCS in cucumber and other species
a: GATase in red box, Anth_synt_I_N in blue box, Chorismate_bind in green box; b, c: GATase; d: Anth_synt_I_N; e: Chorismate_bind; f: Phylogenetic tree of ADCS
从ADCS同源蛋白构建的进化树中可以看到,这个基因在进化的过程中十分保守,遵循单子叶植物同源蛋白聚类到一起,双子叶植物同源蛋白聚类到一起的进化原则。黄瓜与同属葫芦科的甜瓜等聚类到一起（图5-f）。表明这个基因在不同植物中的功能比较保守,都催化叶酸前体的合成。
2.7 黄瓜GCHI与ADCS基因全长的克隆与序列分析
分别扩增果实叶酸含量高低差异显著的65G和02245中调控叶酸合成限速步骤的关键酶基因GCHI和ADCS的序列。结果表明,在两份材料中,CsaV3_1G041250全长均为3 012 bp,CDS长度均为1 413 bp（附图1）。在两份材料中有多个SNP差异位点,有7处变异发生在内含子区域,CDS区有3处突变未引起蛋白质序列的改变,有3处SNP的突变导致了氨基酸序列的改变,第2 474 bp处,65G与02245编辑的氨基酸分别为天冬酰胺和苏氨酸,第2 669 bp处分别为丝氨酸和酪氨酸,第3 018 bp处分别为丙氨酸和缬氨酸（图6）。在两份材料中,CsaV3_7G026240全长均为3 047 bp,CDS长度均为1 407 bp,在内含子区有一个变异,氨基酸序列无差异（附图2）;Csa5G623430全长均为7 941 bp,CDS长度均为2 706 bp,变异均发生在内含子区域,氨基酸序列无差异（附图3）。图6
新窗口打开|下载原图ZIP|生成PPT图6黄瓜CsaV3_1G041250基因在65G、02245中的序列差异
Fig. 6The sequence difference of CsaV3_1G041250 between 65G and 02245
2.8 黄瓜GCHI与ADCS基因的生物信息学分析
黄瓜CsGCHI及CsADCS编码蛋白的等电点（pI）及分子量预测结果表明CsGCHI等电点理论值为6.38,编码468个氨基酸,估计分子量51.621 kD,总平均亲水性为-0.350,说明该蛋白为亲水性蛋白。CsGCHI蛋白分子不稳定参数为37.90,小于40,因此该蛋白是稳定蛋白。CsADCS等电点理论值为5.88,编码901个氨基酸,估计分子量101.17 kD,总平均亲水性为-0.350,该蛋白为亲水性蛋白。CsADCS蛋白分子不稳定参数为44.31,大于40,因此该蛋白是不稳定蛋白。亚细胞定位情况预测结果表明,CsGCHI蛋白最可能定位于高尔基体, 而CsADCS蛋白定位于细胞质的可能性最大。3 讨论
拟南芥中已报道的与叶酸代谢相关的酶有14个,分别由26个基因调控[8]。****们利用同源比对来寻找其他作物中与叶酸代谢相关的基因,其中水稻中14个,番茄中10个。本研究获得19个黄瓜叶酸代谢相关基因,其中11个参与叶酸合成,包括了叶酸从头合成的全部反应过程。这19个基因在黄瓜7条染色体上均有分布,且大多位于染色体长、短臂两端。对番茄果实叶酸合成基因表达模式的研究发现,基因GCHI、ADCS 和 ADCL随着果实成熟,其表达量逐渐降低,基因DHFR和DHFS的表达在果实发育后期显著升高,而其他基因在不同发育阶段变化不明显[25]。在小麦种子发育过程中,基因GCHI、ADCS的表达量逐步下降,基因FPGS在种子5—30 d的表达量维持恒定,而后迅速降低[20]。在水稻中,GCHI、ADCS和HPPK均在开花期表达最高（不同时期表达变化5—6倍）,在乳熟期（5—10 DPA）至种子成熟时期,这3个基因的表达又逐步回升。FPGS在抽穗期表达量最高而后逐步下降[26]。本研究中,在开花之前,CsDHFR1>CsDHFR2;在开花之后,CsDHFR1<CsDHFR2。推测CsDHFR1在开花之前起主要作用,CsDHFR2在开花之后起主要作用。在黄瓜中,基因CsADCS、CsGCHI、CsHPPK/CsDHFS与水稻中这些基因的表达趋势类似,在开花当天表达量最高;基因CsFPGS也与小麦和水稻中类似,在果实发育过程中呈下降趋势,推测它们可能受相同或相近的模式调控。尤为重要的是,黄瓜中存在与果实叶酸含量变化趋势完全一致的基因CsDHNA、CsHPPK/ CsDHFS、CsFPGS以及CsADCS,推测它们对不同时期叶酸含量变化趋势起主要作用。叶酸合成分为pABA分支和喋啶分支,pABA分支包括两步反应,分支酸和谷氨酰胺在氨基脱氧分支酸合成酶（ADCS）的催化下生成氨基脱氧分支酸,其产物在氨基脱氧分支酸裂解酶（ADCL）的催化下合成对氨基苯甲酸（pABA）[27]。黄瓜中调控这两步反应的基因CsADCS、CsADCL表达量在65G与02245中均具有显著差异。喋啶分支包括4步反应,GTP在GTPCHI的催化下形成二氢新蝶呤三磷酸（DHN-P3）,DHN-P3经两步去磷酸化反应生成二氢新蝶呤（DHN）,DHN的侧链在二氢新蝶呤醛缩酶（DHNA）的作用下被切除,释放出二氢蝶啶（HMPHP）[28]。在黄瓜中,调控这4步反应的基因仅CsDHNA在65G与02245中具有显著差异。
随后,pABA分支和喋啶分支的产物转入线粒体中合成四氢叶酸。首先二氢蝶啶在二氢蝶啶焦磷酸激酶（HPPK）的催化下生成具有活性的焦磷酸盐化合物,然后与pABA在二氢蝶酸合酶（DHPS）的催化作用下形成二氢蝶酸（DHP）。随后在二氢叶酸合成酶（DHFS）的催化下形成单尾形式的二氢叶酸。二氢叶酸分别在二氢叶酸还原酶（DHFR）和叶酰多谷氨酰合成酶（FPGS）的催化下形成多尾形式的四氢叶酸[29]。黄瓜中调控这几步反应的基因中,CsHPPK/CsDHPS、CsDHFS、CsFPGS的表达量在65G与02245中具有显著差异。在整个叶酸合成通路中有6个基因在65G与02245具有显著差异,且在叶酸含量高的材料中,差异基因的表达量也高,基因的表达量与叶酸含量呈正相关。
叶酸的强化主要依靠过量表达叶酸合成过程中的酶实现,GTPCHI和ADCS分别是叶酸合成过程中喋啶分支和PABA分支的限速酶[20]。国内外的****将控制这两个酶的基因在拟南芥[30]、番茄[31]、水稻[32]中过量表达,叶酸含量均得到了显著提高。因此,在植物中过量表达控制这两个酶的基因,是利用转基因技术提高植物中叶酸含量的有效途径。本研究中,黄瓜CsGCHI两个同源基因氨基酸序列与拟南芥中该酶氨基酸序列的同源性较高。氨基酸序列比对以及结构域分析表明,GTPCHI的结构在整个进化过程中得到了很好的保存。扩增两份不同材料中CsGCHI基因序列,在CDS区找到6个SNP位点的差异,其中有3个导致了氨基酸序列的改变,且都发生在具有GTPCHI催化活性的保守结构域内,但是该基因的表达量没有差异。推测该基因可能通过氨基酸序列的改变行使功能,并进一步影响叶酸含量。黄瓜CsADCS氨基酸序列与拟南芥中该酶氨基酸序列同源性较高,各物种中该基因的蛋白序列高度保守且都具有4个结构域,可能行使相同或相似的功能。
4 结论
本研究成功鉴定出19个不均匀分布在7条染色体上的黄瓜叶酸代谢相关基因,基因CsFPGS、CsHPPK/CsDHPS、CsDHNA和CsADCS是影响黄瓜果实叶酸含量变化,导致叶酸含量高低显著差异的关键基因。调控叶酸合成限速步骤的关键酶基因CsGCHI及CsADCS功能相对保守,CsGCHI在黄瓜65G、02245中有3个SNP位点的突变导致了氨基酸序列的差异。本研究明确了相关基因在黄瓜果实发育不同时期以及不同材料中的表达量差异,分析了关键酶基因的序列差异,为揭示黄瓜叶酸合成代谢调控网络奠定了基础。致谢：
感谢中国农业科学院生物技术研究所公共实验室韩丽妲老师及实验室成员万幸、杨敏等对黄瓜果实叶酸含量测定提供的帮助！参考文献 原文顺序
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被引期刊影响因子
DOI:10.1146/annurev-arplant-042110-103819URL [本文引用: 1]
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DOI:10.1016/j.molp.2017.12.008URLPMID:29277427 [本文引用: 2]
Insufficient dietary intake of micronutrients, known as
DOI:10.1016/bs.afnr.2017.11.001URLPMID:29477220 [本文引用: 1]
Starting with a brief history of beriberi and the discovery that thiamin deficiency is its cause, the symptoms and signs are reviewed. None are pathognomonic. The disease has a low mortality and a long morbidity. The appearance of the patient can be deceptive, often being mistaken for psychosomatic disease in the early stages. The chemistry of thiamin and the laboratory methodology for depicting its deficiency are outlined. The diseases associated with thiamin deficiency, apart from malnutrition, include a number of genetically determined conditions where mutations, either in the cofactor relationship or a transporter, provide the etiology. It is emphasized that such mutations are often epigenetically responsive to megadoses of thiamin or one of its derivatives. The use of thiamin in clinical practice requires a high index of suspicion on the part of the clinician since it has a part to play in eating disorders, diabetes, neurodegenerative disease, and cancer. A high rate of critical illness and postsurgery thiamin deficiency have been reported, particularly those associated with gastrointestinal bypass. Emphasis is placed on thiamin deficiency as a major cause of asymmetric dysautonomia, because of the high degree of sensitivity to thiamin deficiency in the brainstem, cerebellum, and hypothalamus. The relationship of thiamin with regional pain syndrome, eosinophilic esophagitis, its analgesic capacity, and its preventive use in obstetrics is raised as a potential issue. The role of thiamin in SIDS and autism is outlined. It is emphasized that megadose thiamin is being used as a drug, either in stimulating the damaged cofactor/enzyme combination, or mitochondria.
DOI:10.2165/11532990-000000000-00000URLPMID:20608755 [本文引用: 1]
There is a large body of evidence to suggest that improving periconceptional folate status reduces the risk of neonatal neural tube defects. Thus increased folate intake is now recommended before and during the early stages of pregnancy, through folic acid supplements or fortified foods. Furthermore, there is growing evidence that folic acid may have a role in the prevention of other diseases, including dementia and certain types of cancer. Folic acid is a synthetic form of the vitamin, which is only found in fortified foods, supplements and pharmaceuticals. It lacks coenzyme activity and must be reduced to the metabolically active tetrahydrofolate form within the cell. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the predominant form of dietary folate and the only species normally found in the circulation, and hence it is the folate that is normally transported into peripheral tissues to be used for cellular metabolism. L-5-methyl-THF is also available commercially as a crystalline form of the calcium salt (Metafolin(R)), which has the stability required for use as a supplement. Studies comparing L-5-methyl-THF and folic acid have found that the two compounds have comparable physiological activity, bioavailability and absorption at equimolar doses. Bioavailability studies have provided strong evidence that L-5-methyl-THF is at least as effective as folic acid in improving folate status, as measured by blood concentrations of folate and by functional indicators of folate status, such as plasma homocysteine. Intake of L-5-methyl-THF may have advantages over intake of folic acid. First, the potential for masking the haematological symptoms of vitamin B(12) deficiency may be reduced with L-5-methyl-THF. Second, L-5-methyl-THF may be associated with a reduced interaction with drugs that inhibit dihydrofolate reductase.
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DOI:10.3864/j.issn.0578-1752.2014.07.003URL [本文引用: 2]
【目的】通过对山西谷子资源叶酸含量的测定与评价，了解谷子叶酸含量的变异及其与地理分布的关系，为谷子种质营养含量和育种提供依据。【方法】分别在谷子的研究基地长治、汾阳和太原采集目前山西育种和种植中常用品种245个，记录谷子颜色后于60℃下烘干，采用常规方法研磨脱壳去糠，记录米粒颜色后研磨米粒，全部过100目筛子，测定其叶酸含量。叶酸用磷酸二氢钾溶液恒温水浴浸提，加苯胺处理过的活性炭吸附，用3%氨—70%乙醇洗脱，采用高锰酸钾氧化—间接荧光法测定。【结果】①山西省245份不同品种谷子叶酸含量平均为1.53 μg•g-1。谷子叶酸含量数值服从正态分布且为左偏态，说明谷子叶酸含量较多集中在平均值偏高水平。②不同地区谷子叶酸含量不同。同一品种在汾阳种植其叶酸含量显著低于太原和长治。日均温、日照时数和相对湿度对叶酸含量影响不显著，降雨量则显著影响叶酸含量。③谷粒颜色对叶酸含量影响不显著，小米颜色差异显著影响叶酸含量，从高到低依次为：褐色、绿色、黄色、鲜黄、浅黄和白色米粒品种。【结论】山西省谷子资源的叶酸含量存在较为丰富的遗传变异，变异范围0.37—2.37 μg•g-1，变异系数为26.2%。不同生态区谷子叶酸含量存在明显差异，春播晚熟区的叶酸含量显著高于春播中熟区。降雨量显著影响谷子叶酸含量。小米颜色差异对叶酸含量有显著影响。在鉴定评价基础上，以样品叶酸含量的平均数及其标准差（ ±s）为分类依据，筛选了一批高叶酸的谷子种质资源总计24份，占参试材料的9.8%。目前山西省农业生产常用谷子品种晋谷21，其叶酸含量约为2.0 μg•g-1，属于高叶酸含量品种。
DOI:10.3864/j.issn.0578-1752.2014.07.003URL [本文引用: 2]
【目的】通过对山西谷子资源叶酸含量的测定与评价，了解谷子叶酸含量的变异及其与地理分布的关系，为谷子种质营养含量和育种提供依据。【方法】分别在谷子的研究基地长治、汾阳和太原采集目前山西育种和种植中常用品种245个，记录谷子颜色后于60℃下烘干，采用常规方法研磨脱壳去糠，记录米粒颜色后研磨米粒，全部过100目筛子，测定其叶酸含量。叶酸用磷酸二氢钾溶液恒温水浴浸提，加苯胺处理过的活性炭吸附，用3%氨—70%乙醇洗脱，采用高锰酸钾氧化—间接荧光法测定。【结果】①山西省245份不同品种谷子叶酸含量平均为1.53 μg•g-1。谷子叶酸含量数值服从正态分布且为左偏态，说明谷子叶酸含量较多集中在平均值偏高水平。②不同地区谷子叶酸含量不同。同一品种在汾阳种植其叶酸含量显著低于太原和长治。日均温、日照时数和相对湿度对叶酸含量影响不显著，降雨量则显著影响叶酸含量。③谷粒颜色对叶酸含量影响不显著，小米颜色差异显著影响叶酸含量，从高到低依次为：褐色、绿色、黄色、鲜黄、浅黄和白色米粒品种。【结论】山西省谷子资源的叶酸含量存在较为丰富的遗传变异，变异范围0.37—2.37 μg•g-1，变异系数为26.2%。不同生态区谷子叶酸含量存在明显差异，春播晚熟区的叶酸含量显著高于春播中熟区。降雨量显著影响谷子叶酸含量。小米颜色差异对叶酸含量有显著影响。在鉴定评价基础上，以样品叶酸含量的平均数及其标准差（ ±s）为分类依据，筛选了一批高叶酸的谷子种质资源总计24份，占参试材料的9.8%。目前山西省农业生产常用谷子品种晋谷21，其叶酸含量约为2.0 μg•g-1，属于高叶酸含量品种。
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DOI:10.1016/j.foodres.2016.07.013URLPMID:28460896 [本文引用: 1]
Folates (Vitamin B9) include both naturally occurring folates and synthetic folic acid used in fortified foods and dietary supplements. Folate deficiency causes severe abnormalities in one-carbon metabolism can result chronic diseases and developmental disorders, including neural tube defects. Mammalian cells cannot synthesize folates de novo; therefore, diet and dietary supplements are the only way to attain daily folate requirements. In the last decade, significant advancements have been made to enhance the folate content of rice, tomato, common bean and lettuce by using genetic engineering approaches. Strategies have been developed to improve the stability of folate pool in plants. Folate deglutamylation through food processing and thermal treatment has the potential to enhance the bioavailability of folate. This review highlights the recent developments in biosynthesis, composition, bioavailability, enhanced production by elicitation and metabolic engineering, and methods of analysis of folate in food. Additionally, future perspectives in this context are identified. Detailed knowledge of folate biosynthesis, degradation and salvage are the prime requirements to efficiently engineer the plants for the enhancement of overall folate content. Similarly, consumption of a folate-rich diet with enhanced bioavailability is the best way to maintain optimum folate levels in the body.
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Folates are essential coenzymes involved in one-carbon metabolism. Folate deficiency is associated with a higher risk of newborns with neural tube defects, spina bifida, and anencephaly, and an increased risk of cardiovascular diseases, cancer, and impaired cognitive function in adults. In plants folates are synthesized in mitochondria from pterin precursors, which are synthesized from guanosine-5′-triphosphate (GTP) in the cytosol (pterin branch), and p-aminobenzoate (PABA), derived from chorismate in plastids (PABA branch). We generated transgenic lettuce lines expressing a synthetic codon-optimized GTP-cyclohydrolase I gene (gchI) based on native Gallus gallus gene. Immunoblotting analyses confirmed the presence of the gchI in transgenic lines. Twenty-nine transgenic lines were generated and 19 exhibited significant increase in the folate content, ranging from 2.1 to 8.5-fold higher when compared to non-transgenic lines. The folate content in enriched lettuce would provide 26% of the Dietary Reference Intakes for an adult, in a regular serving. Although the lettuce lines generated here exhibited high folate enhancement over the control, better folate enrichment could be further achieved by engineering simultaneously both PABA and pterin pathways.
DOI:10.1073/pnas.0404208101URLPMID:15365185 [本文引用: 2]
Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.
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In plants, the last step in the synthesis of p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a beta-lyase that converts 4-amino-4-deoxychorismate (ADC)--the reaction product of the PabA and PabB enzymes--to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast-targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiologic concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.
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DOI:10.1016/j.tplants.2007.11.001URL [本文引用: 3]
Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate biofortification of food crops is now a feasible complementary strategy to fight folate deficiency worldwide. The genes and enzymes of folate synthesis are sufficiently understood to enable metabolic engineering of the pathway, and results from pilot engineering studies in plants (and bacteria) are encouraging. Here, we review the current status of investigations in the field of folate enhancement on the eve of a new era in food fortification.
DOI:10.3864/j.issn.0578-1752.2020.01.011URL [本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2020.01.011URL [本文引用: 1]
DOI:10.1007/s00216-019-01742-0URLPMID:30888468 [本文引用: 1]
Folates are typically present in polyglutamyl form in organisms. In traditional extraction methods, polyglutamyl folates are hydrolyzed to monoglutamates, sacrificing valuable information. To advance folate metabolism research, we developed an accurate, sensitive, and reproducible extraction method for polyglutamyl folate species in maize, the main crop in most parts of the world. Twelve folates, including six polyglutamyl folates, were simultaneously determined in maize for the first time using high-performance liquid chromatography-tandem mass spectrometry. The glutamation states of the folates were protected by boiling, which inactivated the native conjugases. alpha-Amylase and protease were added to obtain better recoveries and decrease difficulties in centrifugation and filtration. The recoveries (n = 5) of six polyglutamyl folates were between 80.5 and 101%. All calibration curves showed good linear regression (r(2) >/= 0.994) within the working range. The instrumental limits of detection and quantitation ranged from 0.070 to 2.4 ng/mL and 0.22 to 8.0 ng/mL, respectively. Intra- and inter-day precision was below 7.81% and 11.9%, respectively (n = 5). Using this method, changes in poly- and monoglutamyl folates during maize germination were determined for the first time. The results suggest that folates were largely synthesized as germination initiated, and 5-methyltetrahydrofolate was the most abundant species. Tetraglutamyl 5-methyltetrahydrofolate contributed more than 50% of the 5-methyltetrahydrofolate species. Inverse changes in contents of 5,10-methenyltetrahydrofolate, and 10-formyl folic acid, monoglutamate, and diglutamate of 5-formyltetrahydrofolate were also observed, indicating potential regulation. Additionally, polyglutamyl folates in sweet potatoes were determined using this method, indicating its applications in starchy crops.
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DOI:10.1093/mp/ssp057URLPMID:20085893 [本文引用: 1]
Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed using wild-type tomato fruit and fruit engineered for high folate content. In wild-type fruit, the expression of genes specifying early steps in folate biosynthesis declined during development but that of other genes did not. In engineered fruit overexpressing foreign GTP cyclohydrolase I and aminodeoxychorismate synthase genes, the expression of the respective endogenous genes did not change, but that of three downstream pathway genes-aminodeoxychorismate lyase, dihydroneopterin aldolase, and mitochondrial folylpolyglutamate synthase-respectively increased by up to 7.8-, 2.8-, and 1.7-fold, apparently in response to the build-up of specific folate pathway metabolites. These results indicate that, in fruit, certain folate pathway genes are developmentally regulated and that certain others are subject to feedforward control by pathway intermediates. Microarray analysis showed that only 14 other transcripts (of 11 000 surveyed) increased in abundance by two-fold or more in high-folate fruit, demonstrating that the induction of folate pathway genes is relatively specific.
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DOI:10.1007/s12284-010-9040-0URL [本文引用: 1]
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DOI:10.1007/s11103-013-0091-7URL [本文引用: 1]
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DOI:10.1073/pnas.0700409104URL [本文引用: 1]
DOI:10.1038/nbt1351URLPMID:17934451 [本文引用: 1]
Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.
