苹果L-半乳糖-1-磷酸磷酸酶cDNA克隆及其表达

cDNA Cloning and Expression Analysis of L-galactose-1-phosphate Phosphatase in Apple

【目的】了解苹果果实L-半乳糖-1-磷酸磷酸酶(L-galactose-1-phosphate phosphatase,GPP)基因的特性,探索苹果GPP基因表达特性及其与抗坏血酸(ascorbic acid,AsA)水平的关系。【方法】通过RT-PCR从苹果果实中克隆GPP全长cDNA,分析其序列特征,进行原核表达,制备GPP特异抗体,分析GPP mRNA及其蛋白表达水平与苹果不同组织AsA的关系。【结果】从‘嘎啦’苹果果实中克隆的GPP cDNA(GenBank登录号为FJ752240)包含的最大开放阅读框(open reading frame,ORF)为813 bp,编码270个氨基酸残基,预测分子量为29 kD,该基因与其它植物报道的GPP基因具有较高的相似性,但与肌醇-1-磷酸磷酸酶基因差异较大。构建的pET-32a(+)-GPP载体在大肠杆菌BL21(E.coli BL21)中异源表达后,获得主要以包涵体存在约50 kD的融合蛋白GPP-His(His约21 kD)。以该蛋白制备抗体,与重组蛋白的Western杂交表明该抗体能与GPP发生特异反应。对苹果可溶性蛋白杂交显示,苹果体内GPP蛋白约33 kD。在苹果不同组织中,GPP mRNA与蛋白质的相对表达水平与AsA含量存在明显的一致性。【结论】以单体形式存在的苹果GPP蛋白具有翻译后修饰特性,且该基因的表达在苹果AsA合成调控中可能起重要作用。

【Objective】This study is to understand the characteristics of apple L-galactose-1-phosphate phosphatase（GPP） and the relationship between its expression pattern and ascorbic acid（AsA） in apple.【Method】 GPP cDNA was cloned from young fruit of Malus domestica Borkch cv.Gala,expressed in E.coli,and the specific antibody was prepared,then the relationship between AsA level and the expression level among different tissues of apple were analyzed.【Result】cDNA of GPP from young fruit of apple has an 813 bp open reading frame（ORF） and encodes a protein of 270 amino acid residues with a putative molecular mass of 29 kD.Its accession No.in Genbank is FJ752240.Both of its cDNA and amino acid sequence showed high similarity with GPP genes reported in other plants,but not with myo-inositol-1-phosphate phosphatase.After GPP was heterologously expressed in E.coli,the fusion protein（GPP-His,His was about 21 kD,and GPP was about 29 kD） was about 50 kD,and mainly showed as inclusion body.Then a polyclonal antibody was prepared in rabbit.By western blotting,the antibody could have a specific reaction with recombined protein of apple GPP.However,the size of GPP protein in apple was about 33 KD with protein extracted from apple tissues.Additionally,both of GPP mRNA and protein expression levels showed a good consistency with AsA content among different tissues of apple.【Conclusion】These results suggest that GPP protein in apple,as a monomer protein,has a character of post-translational modification,and its expression might play an important role in regulating AsA synthesis.