黄瓜果实维生素C合成关键基因克隆与分析

doi:10.3864/j.issn.0578-1752.2023.03.009

Abstract

Abstract:

【Objective】The aim of this study was to identify the location, quantity and expression characteristics of genes involved in regulating the synthesis of vitamin C (Vc) by L-galactose pathway in cucumber fruits, and to clone the key genes, so as to lay a foundation for the regulation of Vc synthesis in cucumber.【Method】According to the reported Vc-related genes within the L-galactose pathway in Arabidopsis, the encoded amino acid sequence was used for BLAST in Cucumber 9930_V2 reference genome database. TBtools software was used to map the gene position on cucumber chromosomes. The expression of these genes in two cucumber accessions with significant differences in fruit Vc content was analyzed by qRT-PCR. The homologous genes encoding rate limiting enzymes GDP-L-galactose phosphorylase (GGP) and GDP mannose-3'5'- epimerase (GME) were cloned by PCR amplification, and the sequence differences of these genes in cucumber with high Vc and low Vc were analyzed by sequencing. Phylogenetic tree was constructed to analyze the relatedness cucumber GME, GGP and homologs in other species.【Result】Twenty one homologous genes involved in the synthesis of Vc related enzymes, including PMI, PMM, GMPase, GME, GGP, GPP, GalLDH, and GalLDH in L-galactose pathway, were compared in cucumber and were obtained by BLAST, which were distributed on seven chromosomes, with the most numbers on chromosome 5 and chromosome 1. By analyzing the expression of these genes in R48 (with low Vc) and CG45 (with high Vc), it was found that the genes regulating PMI, PMM, GMPase, GME and GalLDH were significantly different between the two materials. The sequence analysis of Vc synthesis rate-limiting enzyme GGP and GME related genes showed that the full length of CsGME2 gene was 3 537 bp in R48 and 3 541 bp in CG45. There were multiple SNP sites and Indel difference between the two materials, among which one mutation site was located in the CDS region, and resulted in the amino acids changes. Through the analysis of the protein properties of rate limiting enzymes GME and GGP regulating vitamin C synthesis, it was found that the protein properties of GME and GGP in different species were not significantly different, which were hydrophilic proteins and their functions were relatively conservative. Evolutionary tree analysis found that the clusters with close genetic relationship among different species were highly conservative during evolution.【Conclusion】Twenty one L-galactose pathway related genes of cucumber Vc synthesis were identified, which were distributed on seven chromosomes. It was speculated that the key enzymes including PMI, PMM, GMPase, GME, GalLDH and GGP might affect the Vc content in cucumber fruits. The functions of key enzymes GME and GGP regulating the rate limiting step of Vc synthesis were relatively conservative. The SNP site on CsGME2 gene in the two materials of high Vc and low Vc resulted in changes in amino acid sequence.

Key words: cucumber, vitamin C, gene clone, expression analysis

WANG ZhuangZhuang, DONG ShaoYun, ZHOU Qi, MIAO Han, LIU XiaoPing, XU KuiPeng, GU XingFang, ZHANG ShengPing. Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit[J].Scientia Agricultura Sinica, 2023, 56(3): 508-518.

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References 35

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基因 Gene	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
Csa1G181570	TTATCTCAAGGACCATTATCCC	TCCATCAAGAGGATCAACG
Csa1G183590	ATTGGACCATTCGACACC	TAACAGCATCCATCACCG
Csa1G006280	CACCAGTTTGGAGAGTTGATT	TCGAAACACCTTGAATTGTG
Csa1G600810	CCTGAGGATGTTGGAACC	CTACAGCACCATTCCGTCT
Csa2G011460	TCTAGTGCTTGCATCTATCCTG	AGCTTCTCCAGACCGTAGG
Csa2G013230	TGAAAGGCAGGGAATGTATC	CCATGCCCAATAATCGAA
Csa2G249820	CAAGCAGCAGAGGAGTCTATG	CATCAATGGGATCTAAAACCC
Csa3G300600	AAGTCTGCTCTAAGGTTGGC	TTCCTTGGCTGTTAATTGC
Csa3G829100	GCTGATCCGGTCACTAATG	CCTTCCCGATGAATTGAG
Csa3G133160	ACCTGATAGCCGATTTGC	GATTAGCAGCACACCACG
Csa4G236360	AAGTGAGGAAGCCATACAGC	TCAAGGGCAAGTAGTTTATCG
Csa4G015100	CTCTATTATCAACATCGGATCG	TGGGATGAACTGCATGAG
Csa5G011690	TCGAAATGGGATGCTTAAC	CTCCCGAAGGATAGAAACC
Csa5G167200	TTGGCGAGATCCTTGAAC	AACTTCTCTGCATCAGCACA
Csa5G182060	TTAGAAGTTGCTACGATGAAGG	TTGCTAACAACACGACCG
Csa5G512920	CACTGGATCGAAGGATAACTTG	GGCAAGCAAGATGGTATATTTG
Csa5G272920	TGTATGGGCTTGACTGGAG	TCAGTGCCAACCAGGATT
Csa6G008750	GCGAGCCATTCTTTGTCC	CACACCGTATTTGGAAGGC
Csa6G511690	ATGGAGCTGACTGATGAGC	AACTGTTGGAACTTTGCCA
Csa7G067450	GGTGACAAGGAGCATTGAC	GGAATCGTCTCGTTCACAC
Csa7G219200	AGGTCCTCTTCCGGTTTG	CAATAGTGGCGCAACCAC
Actin	TCCACGAGACTACCTACAACTC	GCTCATACGGTCAGCGAT

引物名称 Primer name	正向引物 Forward	反向引物 Reverse
CsGME1-1	TGTCTGAGTCCAAGAGAACG	GCATCTGACTCCTTCAAGCT
CsGME1-2	TGAGGAAGAAAGTACGCAAC	TGAGTTACGACCACGGACAC
CsGME1-3	AGACTTCCGTGAGCCAGTGA	ACGGTGGACCTCATAATATC
CsGME2-1	TTAGGCAGCCAATCGTCGGA	CTGTCCACTAGATGAGAGCCA
CsGME2-2	TCCTTGTGTGACAATCATC	CAGTCAATGGTTCGCTACTGC
CsGME2-3	ACATATGGACCGAGAATGA	AGTAGTGGCATGAGTCGAGA
CsGGP-1	CCCTAGAAGATTCGAAGTA	GCCAGATATCTTGTTCAGC
CsGGP-2	GCTTTCATTCCTAGTGGATA	ATCCGTTGCCCACAATCAGC
CsGGP-3	GATGGAGTGATCATCTCTGA	AGGACCATTAGTGATAGGTA

酶 Enzyme	登录号（拟南芥） Accession No. (Arabidopsis)	登录号（黄瓜） Accession No. (cucumber)	基因名称 Gene name	染色体位置 Chromosome	物理位置 Location (bp)
PMI	AT3G02570	Csa3G300600 Csa5G167200	CsPMI1 CsPMI2	Chr3 Chr5	17101685-17104783 (+) 6525799-6529720 (-)
PMM	AT2G45790	Csa5G011690	CsPMM	Chr5	410127-419862 (+)
GMPase	AT2G39770	Csa6G008750 Csa3G829100 Csa1G006280 Csa2G013230 Csa1G600810	CsGMPase1 CsGMPase2 CsGMPase3 CsGMPase4 CsGMPase5	Chr6 Chr3 Chr1 Chr2 Chr1	901131-904917 (+) 901131-904917 (+) 1169141-1173256 (+) 2269704-2282961 (-) 23233328-23240457 (+)
GME	AT5G28840	Csa2G011460 Csa5G182060 Csa5G512920	CsGME1 CsGME2 CsGME3	Chr2 Chr5 Chr5	2039207-2042353 (+) 8093540-8097076 (+) 17891804-17895624 (-)
GGP	AT4G26850 AT5G55120	Csa7G219200	CsGGP	Chr7	7788896-7792047 (+)
GPP	AT3G02870	Csa1G181570 Csa6G511690 Csa1G183590 Csa2G249820	CsGPP1 CsGPP2 CsGPP3 CsGPP4	Chr1 Chr6 Chr1 Chr2	11166020-11169584 (+) 26388577-26391259 (-) 11213505-11222521 (+) 12197999-12203006 (+)
GalDH	AT4G33670	Csa7G067450 Csa5G272920 Csa4G015100 Csa3G133160	CsGalDH1 CsGalDH2 CsGalDH3 CsGalDH4	Chr7 Chr5 Chr4 Chr3	4137998-4141177 (+) 11505791-11512104 (+) 1982420-1984762 (+) 8687749-8692048 (-)
GalLDH	AT3G47930	Csa4G236360	CsGalLDH	Chr4	10080624-10094987 (+)

物种 Species	氨基酸长度 Length (aa)	蛋白质量 Mw (kD)	等电点 pI	不稳定系数 II	总平均疏水性 GRAVY	序列号 Accession
黄瓜 Cucumis sativus	376	42.52	5.94	41.85	-0.411	XP_004139232
甜瓜 Cucumis melo var. makuwa	409	46.50	6.05	49.3	-0.429	TYK18415.1
冬瓜 Benincasa hispida	376	42.62	5.94	42.09	-0.432	XP_038890351
南瓜 Cucurbita moschata	376	42.54	6.05	41.96	-0.415	XP_022946492
苦瓜 Momordica charantia	376	42.51	5.81	39.45	-0.414	XP_022143153
银葫芦亚种 Cucurbita argyrosperma subsp. sororia	397	44.78	5.74	43.98	-0.409	KAG6586308
桑葚 Morus notabilis	379	42.87	6.21	40.5	-0.437	XP_024025147
向日葵 Helianthus annuus	376	42.60	5.84	43.02	-0.408	XP_022002729
开心果 Pistacia vera	376	42.48	5.93	39.46	-0.410	XP_031268788
石榴 Punica granatum	386	43.47	6.16	38.39	-0.383	OWM82038

物种 Species	氨基酸长度 Length (aa)	蛋白质量 Mw (kD)	等电点 pI	不稳定系数 II	总平均疏水性 GRAVY	序列号 Accession
黄瓜 Cucumis sativus	445	49.99	5.18	44.23	-0.224	XP_004139797.1
甜瓜 Cucumis melo var. makuwa	537	60.04	5.13	45.37	-0.174	TYK23075.1
冬瓜 Benincasa hispida	448	50.23	5.24	43.20	-0.163	XP_038897875.1
苦瓜 Momordica charantia	446	49.77	4.86	48.18	-0.206	XP_022139724.1
南瓜 Cucurbita moschata	445	49.58	5.14	49.30	-0.167	XP_022940636.1
银葫芦亚种 Cucurbita argyrosperma subsp. sororia	445	49.61	5.09	48.32	-0.165	KAG7037363.1
西葫芦 Cucurbita pepo subsp. pepo	445	49.57	5.10	48.71	-0.162	XP_023524925.1
开心果 Pistacia vera	449	50.05	4.77	48.15	-0.222	XP_031282295.1
核桃 Juglans regia	449	49.83	5.16	46.50	-0.180	XP_018812019.2
桑葚 Morus notabilis	443	49.28	5.07	40.97	-0.126	XP_010096115.1
棉花 Gossypium hirsutum	450	50.55	5.10	54.38	-0.230	XP_040942951.1

Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit

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