红肉火龙果果糖激酶基因HpFRK1克隆、表达与酶学活性分析  

Cloning,expression,and enzymatic activity analysis of the fructokinase gene HpFRK1 in red pitaya(Hylocereus polyrhizus)

作　　者：解璞[1] 晏霜 王红林 郑乾明 XIE Pu;YAN Shuang;WANG Honglin;ZHENG Qianming(Guizhou Institute of Pomology Science,Guizhou Academy of Agricultural Science,Guiyang 550006,China;Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Region,Guizhou Academy of Agricultural Science,Guiyang 550006,China)

机构地区：[1]贵州省农业科学院贵州省果树科学研究所,贵阳550006 [2]贵州省农业科学院农业农村部喀斯特山区作物基因资源与种质创新重点实验室,贵阳550006

出　　处：《西北植物学报》2024年第10期1589-1596,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金项目(32060674);贵州省高层次创新型人才项目(黔科合平台人才-GCC[2022]025-1);贵州省科技计划项目(黔科合中引地[2023]033)。

摘　　要：【目的】克隆红肉火龙果果糖激酶基因,检测其表达和酶活性,解析其对果实发育可溶性糖积累的生理功能。【方法】从‘紫红龙’果肉克隆HpFRK1基因,进行生物信息学和亚细胞定位分析,利用qRT-PCR检测基因表达,通过大肠杆菌诱导表达获得重组蛋白并检测酶活性。【结果】HpFRK1的开放阅读框长度为993bp,编码330个氨基酸,具有磷酸果糖激酶B家族结构域。HpFRK1与甜菜BvFRK亲缘关系最近,均属于细胞质定位的FRKs。HpFRK1在成熟茎和不同发育时期果实均表达,在花后20d的果实表达量最高,随果实发育逐渐下调,花后30d(果实成熟)表达量最低。亚细胞定位检测表明HpFRK1主要定位于细胞核和细胞质。诱导获得的Hp-FRK1重组蛋白特异性催化果糖磷酸化(Km为11.01mmol/L)。【结论】HpFRK1在细胞质特异性催化果糖磷酸化,在红肉火龙果果实发育期间负调控果实可溶性糖积累。[Objective]The study aims to clone the fructokinase(FRK)gene of red pitaya(Hylocereus polyrhizus),detect its expression and enzyme activity,and analyze its physiological function in soluble sugar accumulation during fruit development.[Methods]HpFRK1 gene was cloned from‘Zihonglong’fruits,and bioinformatics and subcellular localization analysis were carried out.Gene expression was detected by qRT-PCR.Recombinant protein was obtained by E.coli induction and enzyme activity was detected.[Results]The open reading frame(ORF)of HpFRK1 was 993 bp in length,encoding 330 amino acids with a phosphofructokinase type B(pfkB)domain.HpFRK1 had the closest genetic relationship with sugar beet BvFRK,both of which belonged to cytoplasm localized FRKs.HpFRK1 was expressed in dif-ferent developmental stages of mature stems and fruits.The expression level of HpFRK1 was the highest at 20 days after flowering and the lowest at 30 days after flowering(fruit ripening),and was gradually de-creased with fruit development.Subcellular localization assay showed that HpFRK1 was mainly localized in the nucleus and cytoplasm.HpFRK1 recombinant protein specifically catalyzed fructose phosphorylation(K m=11.01 mmol/L).[Conclusion]HpFRK1 specifically catalyzes fructose phosphorylation in the cyto-plasm and negatively regulates fruit soluble sugar accumulation during the development of red-fleshed pita-ya fruits.

关 键 词：红肉火龙果 果糖激酶 基因克隆 表达分析 原核表达 

分 类 号：Q78[生物学—分子生物学] S667.9[农业科学—果树学]