关键词: 黄瓜, Vc, 基因克隆, 表达分析

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