黄瓜果实维生素C合成关键基因克隆与分析

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

【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qRT-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、GalDH、GalLDH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含量)和R48(低Vc含量)的表达量分析,发现调控PMI、PMM、GMPase、GME、GalLDH这5个酶的基因在CG45和R48中有极显著的表达差异。对Vc合成限速酶GGP和GME相关基因在CG45和R48两份材料中进行克隆发现,CsGME2在R48中基因全长为3537 bp,在CG45中基因全长为3541 bp,该基因在两份材料存在多个SNP位点差异和Indel差异,有一个突变位点位于CDS区,且导致了氨基酸序列的改变。通过对调控Vc合成限速酶GME、GGP蛋白性质分析,发现限速酶GME、GGP在不同物种的蛋白性质差异不大,均为亲水性蛋白,功能相对保守。进化树分析发现不同物种亲缘关系较近的聚类在一起,进化过程高度保守。【结论】鉴定出21个分布于7条染色体上的黄瓜果实Vc合成的L-半乳糖途径相关基因,推测关键酶PMI、PMM、GMPase、GME、GalLDH、GGP可能影响黄瓜果实中Vc含量变化,调控Vc合成限速步骤关键酶GME、GGP功能相对保守,Vc合成限速步骤关键酶GME基因CsGME2在高Vc和低Vc两份材料中的一个SNP位点变异导致氨基酸序列的变化。

【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 3537 bp in R48 and 3541 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.