关键词: 苹果, L-半乳糖-1-磷酸磷酸酶, 原核表达, 多克隆抗体, 抗坏血酸

Abstract:

【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.

Key words: Malus domestica, L-galactose-1-phosphate phosphatase, prokaryotic expression, polyclonal antibody, ascorbate