Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (18): 3764-3776.doi: 10.3864/j.issn.0578-1752.2020.18.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Analysis of Folate Synthesis Key Genes in Cucumber

ZHOU Qi(),LIU XiaoPing,BO KaiLiang,MIAO Han,DONG ShaoYun,GU XingFang(),ZHANG ShengPing()   

  1. 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
  • Received:2020-02-14 Accepted:2020-05-20 Online:2020-09-16 Published:2020-09-25
  • Contact: XingFang GU,ShengPing ZHANG E-mail:15901563056@163.com;guxingfang@caas.cn;zhangshengping@caas.cn

RichHTML

23

PDF

594

Abstract:

【Objective】This study analyzed the quantity, location and expression pattern of folic acid metabolization-related genes in cucumber, cloned and made bioinformatics analysis of the key enzyme genes, aiming to lay a foundation for the study on the regulation mode of folic acid synthesis in cucumber. 【Method】The reported folic acid metabolism related genes in Arabidopsis thaliana was blasted in the cucumber genome database 9930 _V3 to obtain the folic acid metabolism related genes in cucumber. These genes were mapped onto the cucumber chromosome by using Mapchart, and their expression pattern was examined in different materials and at different developmental stages. Bioinformatics analysis of key enzyme genes was conducted by MEGA, Web LOGO, and ExPASy. Key enzyme genes were cloned by PCR amplification, and sequence differences were analyzed. 【Result】A total of 19 genes related to folate metabolism were blasted in cucumber, which were distributed non-uniformly on seven chromosomes, mostly on Chr.4 and Chr.5. The expression levels of 11 folic acid synthetic genes in fruits of sequenced material 9930, inbred line with low folic acid 65G, and inbred line with high folic acid 02245 at different developmental stages were analyzed. It was found that the expression pattern of CsFPGS, CsHPPK/CsDHPS, and CsDHNA were consistent with the changes of folic acid content, and there were significant differences in the expression levels of CsADCS, CsADCL, CsDHNA, CsHPPK/CsDHFS, CsFPGS and CsDHFS between 65G and 02245. CsGCHI and CsADCS were two key enzymes regulate folate synthesis in rate-limiting steps, and then their amino acid sequences and protein domains were analyzed. The result showed that it turned out that CsGCHI homologs all had two GTP_cyclohydroI domains, and CsADCS homologs all had two GATase domains, including one Anth_synt_I_N domain, and one Chorismate_bind domain. The domains were highly conserved in different species, evolutionary tree analysis clustered the proteins of closely related species together. The GCHI and ADCS gene were cloned from 65G and 02245, respectively. Sequence analysis showed that the full length of CsaV3_1G041250 was 3012 bp, the length of CDS sequence was 1 413 bp, and the mutations in the three SNP sites led to the variation of amino acid sequence. The full length of CsaV3_7G026240 was 3 047 bp, and the CDS length was 1 407 bp with no sequence variation. The total length of CsaV3_5G036360 was 7 941 bp, and the length of CDS sequence was 2 706 bp. 【Conclusion】It was identified that 19 genes were related to folate metabolism in cucumber. These genes were distributed unequally on seven chromosomes. CsFPGS, CsHPPK/CsDHPS, CsDHNA and CsADCS affected folic acid content and trend in cucumber fruit mostly, while CsGCHI and CsADCS were the Key enzyme genes regulating rate-limiting steps in folic acid synthesis, which was relatively conservative in function, and 3 SNP mutations led to variations in protein sequences in CsGCHI between 65G and 02245.

Key words: cucumber, folate, key enzyme, homology gene, cloning

Table 1

Folate metabolism related genes in cucumber"

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

Fig. 1

The 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"

Fig. 2

The expression level of folate synthetic genes in cucumber fruit at different stages"

Fig. 3

The 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"

Fig. 4

Protein 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"

Fig. 5

Protein 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"

Fig. 6

The sequence difference of CsaV3_1G041250 between 65G and 02245"

[1] HANSON A D, GREGORY J F. Folate biosynthesis, turnover, and transport in plants. Annual Review of Plant Biology, 2011,62(1):105-125.
doi: 10.1146/annurev-arplant-042110-103819
[2] BASSET G J C, QUINLIVAN E P, GREGORY J F, HANSON A D. Folate synthesis and metabolism in plants and prospects for biofortification. Crop Science, 2005,45(2):449-453.
doi: 10.2135/cropsci2005.0449
[3] DE LEPELEIRE J, STROBBE S, VERSTRAETE J, BLANCQUAERT D, VISSER R G F, STOVE C, WAN DER STRAITEN D. Folate biofortification of potato by tuber-specific expression of four folate biosynthesis genes. Molecular Plant, 2018,11(1):175-188.
doi: 10.1016/j.molp.2017.12.008 pmid: 29277427
[4] NADERI N, HOUSE J D. Recent developments in folate nutrition. Advances in food and nutrition research, 2018,83.
doi: 10.1016/bs.afnr.2017.11.001 pmid: 29477220
[5] PIETRZIK K, BAILEY L, SHANE B. Folic acid and L-5- methyltetrahydrofolate: Comparison of clinical pharmacokinetics and pharmacodynamics. Clinical Pharmacokinetics, 2010,49(8):535-548.
doi: 10.2165/11532990-000000000-00000 pmid: 20608755
[6] WATANABE S, OHTANI Y, TATSUKAMI Y, AOKI W, AMEMIYA T, SUKEKIYO Y, KUBOKAWA S, UEDA M. Folate biofortification in hydroponically cultivated spinach by the addition of phenylalanine. Journal of Agricultural and Food Chemistry, 2017. doi: 10.1021/acs.jafc.7b01375.
pmid: 32936632
[7] LUCOCK M. Folic Acid: Nutritional biochemistry, molecular biology, and role in disease processes. Molecular Genetics and Metabolism, 2000,71(1/2):121-138.
doi: 10.1006/mgme.2000.3027
[8] 董薇. 水稻籽粒叶酸含量QTL分析及生物强化[D]. 北京: 中国农业科学院, 2011.
DONG W. QTL analysis and biofortification of folate content in rice (Oryza sativa L.). Beijing: Beijing Chinese Academy of Agricultural Sciences. 2011. (in Chinese)
[9] 韩娟英, 何曦, 蒋宙蕾, 梅沙, 张宁, 吴殿星. 富含叶酸水稻研究进展. 中国稻米, 2017,23(6):10-15.
HAN J Y, HE X, JIANG Z L, MEI S, ZHANG N, WU D X. Progress on high folate content rice. China Rice, 2017,23(6):10-15. (in Chinese)
[10] 邵丽华, 王莉, 白文文, 刘雅娟. 山西谷子资源叶酸含量分析及评价. 中国农业科学, 2014,47(7):1265-1272.
doi: 10.3864/j.issn.0578-1752.2014.07.003
SHAO L H, WANG L, BAI W W, LIU Y J. Evaluation and analysis of folic acid content in millet from different ecological regions in Shanxi province. Scientia Agricultura Sinica, 2014,47(7):1265-1272. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2014.07.003
[11] MARTÍNEZ A B O, BERRUEZO G R, CAVA M J B, GRACIÁ C M, CASTÓN, J P. Folate and folic acid intake estimation and food enrichment requirements. Archivos Latinoamericanos De Nutricion, 2005,55(1):5-14.
pmid: 16187672
[12] SAINI R K, NILE S H, KEUM Y S. Folates: Chemistry, analysis, occurrence, biofortification and bioavailability. Food Research International, 2016,89(pt.1):1-13.
doi: 10.1016/j.foodres.2016.07.013 pmid: 28460896
[13] 梁颖, 张毅, 李艺, 丁莹, 刘贤金. 烹饪及贮藏对八种常见叶菜中叶酸含量的影响. 现代食品科技, 2018,34(3):173-177.
LIANG Y, ZHANG Y, LI Y, DING Y, LIU X J. Effects of cooking methods and storage on folates in leafy vegetables. Modern Food Science and Technology, 2018,34(3):173-177. (in Chinese)
[14] BASSET G J C, QUINLIVAN E P, GREGORY J F, HANSON A D. Folate synthesis and metabolism in plants and prospects for biofortification. Crop Science, 2005,45(2):449-453.
doi: 10.2135/cropsci2005.0449
[15] BASSET G J C, QUINLIVAN E P, ZIEMAK M J, DE LA GARZA R D, FISCHER M, SCHIFFMANN S, BACHER A, GREGORY J F, HANSON A D. Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proceedings of the National Academy of Sciences, 2002,99(19):12489-12494.
doi: 10.1073/pnas.192278499
[16] NUNES A C S, KALKMANN D C, ARAGO F J L. Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene. Transgenic Research, 2009,18(5):661-667.
doi: 10.1007/s11248-009-9256-1
[17] DE LA GARZA R D, QUINLIVAN E P, KLAUS S M J, BASSET G J C, GREGORY J F, HANSON A D. Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proceedings of the National Academy of Sciences of the United States of America, 2004,101(38):13720-13725.
doi: 10.1073/pnas.0404208101 pmid: 15365185
[18] RAVANEL S, QUINLIVAN E P, WHITE R, GIOVANNONI J, REBEILLE F, NICHOLS B, SHINOZAKI K, SEKI M, GREGORY J HANSON A D, Folate synthesis in plants: The last step of the p -aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. The Plant Journal, 2004,40(4):453-461.
doi: 10.1111/j.1365-313X.2004.02231.x pmid: 15500462
[19] HOSSAIN T, ROSENBER G I, SELHUB J, KISHORE G. Enhancement of folate in plants through metabolic engineering. Proceedings of the National Academy of Sciences, 2004,101(14):5158-5163.
[20] BEKAERT S, STOROZHENKO S, MEHRSHAHI P, BENNETT M J, LAMBERT W, GREGORY J F, SCHUBERT K, HUGENHOLTZ J, WAN SER STRAETEN D, HANSON A D. Folate biofortification in food plants. Trends in Plant Science, 2008,13(1):28-35.
doi: 10.1016/j.tplants.2007.11.001
[21] 张圣平, 顾兴芳. 黄瓜重要农艺性状的分子生物学. 中国农业科学, 2020,53(1):117-121.
doi: 10.3864/j.issn.0578-1752.2020.01.011
ZHANG S P, GU X F. Molecular biology of important agronomic traits in cucumber. Scientia Agricultura Sinica, 2020,53(1):117-121. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2020.01.011
[22] WAN X, HAN L D, YANG M, ZHANG H Y, ZHAN C Y, HU P. Simultaneous extraction and determination of mono-/polyglutamyl folates using high-performance liquid chromatography-tandem mass spectrometry and its applications in starchy crops. Analytical and Bioanalytical Chemistry, 2019,411(13):2891-2904.
doi: 10.1007/s00216-019-01742-0 pmid: 30888468
[23] WANG M, JIANG B, PENG Q W, LIU W R, HE X M, LIANG Z J, LIN Y E. Transcriptome analyses in different cucumber cultivars provide novel insights into drought stress responses. International Journal of Molecular Sciences, 2018,19(7):2067.
[24] 刘盼娜, 顾兴芳, 苗晗, 黄三文, 张忠华, 崔金莹, 王烨, 张圣平. 黄瓜核心种质遗传多样性的苗期和初花期形态标记分析. 植物遗传资源学报, 2015,16(3):472-478.
LIU P N, GU X F, MIAO H, HUANG S W, ZHANG Z H, CUI J Y, WANG Y, ZHANG S P. Genetic diversity analysis of seeding and early flowering stage morphological marker in cucumber core germplasm. Acta plantarum genetic resources, 2015,16(3):472-478. (in Chinese)
[25] WALLER J C, AKHTAR T A, LARA-NÚÑEZ A, GREGORY J F, MCQUINN R P, GIOVANNONI J J, HANSON A D. Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit. Molecular Plant, 2010,3(1):66-77.
doi: 10.1093/mp/ssp057 pmid: 20085893
[26] GUSSIN G N. Activation of transcription initiation and regulation of tryptophan biosynthesis in fluorescent pseudomonad// pseudomonas. Springer, Boston, MA, 2004: 293-322.
[27] ANUKUL N, RAMOS R A, MEHRSHAHI P, CASTELAZO A S, PARGER H, DIEVART A, LANAU N, MIEULET D, TUCKER G, GUIDERDONI E, BARRETT D A, BENNETT M J. Folate polyglutamylation is required for rice seed development. Rice, 2010,3(2/3):181-193.
doi: 10.1007/s12284-010-9040-0
[28] 姚琳. 大豆GmGCHI和GmADCS基因共表达对拟南芥叶酸含量的影响[D]. 武汉: 华中农业大学 2013.
YAO L. The effect of co-expression of Glycine max GmGCHI and GmADCS genes on the folate content of Arabidopsis thaliana[D]. Wuhan: Huazhong Agricultural University, 2013. (in Chinese)
[29] BLANCQUAERT D, VAN DAELE J, STOROZHENKO S, STOVE C, LAMBERT W, WAN DER STRAETEN D. Rice folate enhancement through metabolic engineering has an impact on rice seed metabolism, but does not affect the expression of the endogenous folate biosynthesis genes. Plant Molecular Biology, 2013,83(4/5):329-349.
doi: 10.1007/s11103-013-0091-7
[30] 梁业红. 过量表达细菌的FolC和FolP基因对提高拟南芥叶酸含量的研究[D]. 北京: 中国农业科学院, 2005.
LIANG Y H. Elevation of the folate content of Arabidopsis plants by overexpression of the bacteria FolC and FolP genes[D]. Beijing: Chinese Academy of Agricultural Sciences, 2005. (in Chinese)
[31] DE LA GARZA R I D, GREGORY J F, HANSON A D. Folate biofortification of tomato fruit. Proceedings of the National Academy of Sciences. 2007,104(10):4218-4222.
doi: 10.1073/pnas.0700409104
[32] STOROZHENKO S, DE BROUWER V, VOLCKAERT M, NAVARRETE O, BLANCQUAERT D, ZHANG G F, LAMBERT W, WAN DER STRAETEN D. Folate fortification of rice by metabolic engineering. Nature Biotechnology, 2007,25(11):1277-1279.
doi: 10.1038/nbt1351 pmid: 17934451
[1] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[2] LI QingLin,ZHANG WenTao,XU Hui,SUN JingJing. Metabolites Changes of Cucumber Xylem and Phloem Sap Under Low Phosphorus Stress [J]. Scientia Agricultura Sinica, 2022, 55(8): 1617-1629.
[3] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[4] KANG Chen,ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing. Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus [J]. Scientia Agricultura Sinica, 2022, 55(19): 3751-3766.
[5] DUAN YaRu,GAO MeiLing,GUO Yu,LIANG XiaoXue,LIU XiuJie,XU HongGuo,LIU JiXiu,GAO Yue,LUAN Feishi. Map-Based Cloning and Molecular Marker Development of Watermelon Fruit Shape Gene [J]. Scientia Agricultura Sinica, 2022, 55(14): 2812-2824.
[6] QU Cheng,WANG Ran,LI FengQi,LUO Chen. Cloning and Expression Profiling of Gustatory Receptor Genes BtabGR1 and BtabGR2 in Bemisia tabaci [J]. Scientia Agricultura Sinica, 2022, 55(13): 2552-2561.
[7] ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.
[8] CHEN Xi,LIU YingJie,DONG YongHao,LIU JinYan,LI Wei,XU PengJun,ZANG Yun,REN GuangWei. Effects of CMV-Infected Tobacco on the Performance, Feeding and Host Selection Behavior of Myzus persicae [J]. Scientia Agricultura Sinica, 2021, 54(8): 1673-1683.
[9] ZHANG Lu,ZONG YaQi,XU WeiHua,HAN Lei,SUN ZhenYu,CHEN ZhaoHui,CHEN SongLi,ZHANG Kai,CHENG JieShan,TANG MeiLing,ZHANG HongXia,SONG ZhiZhong. Identification, Cloning, and Expression Characteristics Analysis of Fe-S Cluster Assembly Genes in Grape [J]. Scientia Agricultura Sinica, 2021, 54(23): 5068-5082.
[10] TAN YongAn,JIANG YiPing,ZHAO Jing,XIAO LiuBin. Expression Profile of G Protein-Coupled Receptor Kinase 2 Gene (AlGRK2) and Its Function in the Development of Apolygus lucorum [J]. Scientia Agricultura Sinica, 2021, 54(22): 4813-4825.
[11] LI ZiTeng,CAO YuHan,LI Nan,MENG XiangLong,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang. Molecular Variation and Phylogenetic Relationship of Apple Scar Skin Viroid in Seven Cultivars of Apple [J]. Scientia Agricultura Sinica, 2021, 54(20): 4326-4336.
[12] WANG Na,ZHAO ZiBo,GAO Qiong,HE ShouPu,MA ChenHui,PENG Zhen,DU XiongMing. Cloning and Functional Analysis of Salt Stress Response Gene GhPEAMT1 in Upland Cotton [J]. Scientia Agricultura Sinica, 2021, 54(2): 248-260.
[13] WANG JunZheng,ZHANG Qi,GAO ZiXing,MA XueQiang,QU Feng,HU XiaoHui. Effects of Two Microbial Agents on Yield, Quality and Rhizosphere Environment of Autumn Cucumber Cultured in Organic Substrate [J]. Scientia Agricultura Sinica, 2021, 54(14): 3077-3087.
[14] TAN YongAn,ZHAO XuDong,JIANG YiPing,ZHAO Jing,XIAO LiuBin,HAO DeJun. Cloning, Preparation of Antibody and Response Induced by 20-Hydroxyecdysone of Target of Rapamycin in Apolygus lucorum [J]. Scientia Agricultura Sinica, 2021, 54(10): 2118-2131.
[15] KunNeng ZHOU,JiaFa XIA,Peng YUN,YuanLei WANG,TingChen MA,CaiJuan ZHANG,ZeFu LI. Transcriptome Research of Erect and Short Panicle Mutant esp in Rice [J]. Scientia Agricultura Sinica, 2020, 53(6): 1081-1094.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd