| 黄瓜果实维生素C合成关键基因克隆与分析 |
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| 引用本文： | 王壮壮,董邵云,周琪,苗晗,刘小萍,徐奎鹏,顾兴芳,张圣平.黄瓜果实维生素C合成关键基因克隆与分析[J].中国农业科学,2023,56(3):508-518. |
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| 作者姓名： | 王壮壮 董邵云 周琪 苗晗 刘小萍 徐奎鹏 顾兴芳 张圣平 |
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| 作者单位： | 1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/蔬菜生物育种全国重点实验室，北京 1000812青岛农业大学园艺学院，山东青岛 266000 |
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| 基金项目： | 国家现代农业产业技术体系(CARS-23);中国农业科学院创新工程(CAAS-ASTIP-2017-IVF);蔬菜生物育种全国重点实验室项目 |
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| 摘 要： | 【目的】鉴定调控黄瓜果实中L-半乳糖途径维生素C(Vc)合成相关基因的位置、数量及表达特征，同时对关键基因进行克隆分析，旨在为黄瓜果实中Vc合成调控研究奠定基础。【方法】根据已报道的拟南芥中L-半乳糖途径合成Vc相关基因，利用蛋白编码的氨基酸序列在黄瓜9930＿V2参考基因组数据库中进行BLAST比对，确定黄瓜中的同源基因，借助TBtools软件绘制基因在染色体上的位置。通过q RT-PCR分析上述基因在果实Vc含量差异显著的两份黄瓜材料中的表达量。利用PCR扩增对限速酶GDP-L-半乳糖磷酸化酶（GGP）及GDP-甘露糖-3′5′-差向酶（GME）同源基因进行克隆，测序分析这些基因在高Vc含量与低Vc含量黄瓜果实中的序列差异。构建系统进化树，分析黄瓜果实GME、GGP与其他物种中同源基因的亲缘关系。【结果】在黄瓜基因组中比对到21个参与L-半乳糖途径合成Vc相关酶PMI、PMM、GMPase、GME、GGP、GPP、Gal DH、Gal LDH的同源基因，7条染色体均有分布，在5号染色体和1号染色体上分布最多。通过对21个基因在两份果实Vc含量高低差异显著的两份材料CG45（高Vc含...
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| 关 键 词： | 黄瓜 Vc 基因克隆 表达分析 |
| 收稿时间： | 2022-04-13 |
Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit |
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| 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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| Authors: | WANG ZhuangZhuang DONG ShaoYun ZHOU Qi MIAO Han LIU XiaoPing XU KuiPeng GU XingFang ZHANG ShengPing |
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| Institution: | 1Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture and Rural Affairs/State Key Laboratory of Vegetable Biobreeding, Beijing 1000812College of Horticulture, Qingdao Agricultural University, Qingdao 266000, Shandong |
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| 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. |
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| Keywords: | cucumber vitamin C gene clone expression analysis |
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