热带作物学报2025，Vol.46Issue(7)：1546-1553,8.DOI:10.3969/j.issn.1000-2561.2025.07.002
红肉火龙果果糖激酶基因HpFRK2的克隆、表达与酶活性分析
Cloning,Expression and Enzymatic Activity Analysis of the Fructo-kinase Gene HpFRK2 in Red Pitaya(Hylocereus polyrhizus)
摘要
Abstract
Plant fructokinase(FRK)specifically catalyzes fructose phosphorylation into glycolysis pathway,which is a key enzyme for fructose metabolism and soluble sugar accumulation.This study elucidated the expression pattern and enzyme characteristics of red pitaya HpFRK2 gene,which would provide a theoretical basis for understanding the mo-lecular mechanism of fruit soluble sugar accumulation and improving fruit quality.HpFRK2 was cloned from red pitaya'Zihonglong',and its expression pattern was analyzed by real-time fluorescence quantitative PCR.Subcellular localiza-tion was performed by transiently expressing in tobacco mesophyll cells,and the recombinant protein was obtained by prokaryotic expression and its enzyme activity was detected.The open reading frame(ORF)was 1026 bp,encoding 341 amino acids.The relative molecular weight of HpFRK2 was 36.95 kDa,and the theoretical isoelectric point was 5.94.Phylogenetic analysis showed that HpFRK2 was closely related to cassava MeFRK2,tomato S1FRK1,apple MdFRK1 and Arabidopsis AtFRK1.HpFRK2 contained a conserved domain of the phosphofructo kinase type B(pfkB)family.The expression level of HpFRK2 was the highest at 20 days of fruit development,gradually decreased with fruit develop-ment,and the expression level was the lowest at 30 days.In addition,the expression of HpFRK2 in stems was signifi-cantly lower than that in fruits.Subcellular localization results showed that HpFRK2 was mainly located in the cyto-plasm.A prokaryotic expression vector was constructed and expressed in Escherichia coli,and recombinant protein was successfully induced.The results of enzyme activity characteristics showed that HpFRK2 specifically catalyzed fructose phosphorylation,fructose did not have the substrate inhibition on its enzyme activity,and the Km value for fructose phosphorylation was 1.84 mmol/L.The results showed that HpFRK2 was located in the cytoplasm,specifically cata-lyzed fructose phosphorylation,was mainly expressed during the veraison period(20-23 d after flowering)of red pitaya fruit,and may negatively regulate fructose accumulation in fruit.关键词
红肉火龙果/果糖激酶/基因克隆/蛋白表达/酶活Key words
red pitaya/fructokinase/gene cloning/protein expression/enzyme activity分类
农业科技引用本文复制引用
罗冬兰,巴良杰,王红林,郑乾明..红肉火龙果果糖激酶基因HpFRK2的克隆、表达与酶活性分析[J].热带作物学报,2025,46(7):1546-1553,8.基金项目
国家自然科学基金项目(No.32302624,No.32160595,No.32060674). （No.32302624,No.32160595,No.32060674）
