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

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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"

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