Dry I B, Robinson S P
CSIRO Division of Horticulture, Adelaide, Australia.
Plant Mol Biol. 1994 Oct;26(1):495-502. doi: 10.1007/BF00039560.
Polyphenol oxidase (PPO) was purified to homogeneity from Sultana grapes yielding a single protein with an apparent molecular mass of 40 kDa as determined by SDS-PAGE. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified 40 kDa grape PPO protein was used to amplify a 1650 bp cDNA clone (GPO1M) by 3' rapid amplification of cDNA ends (3'-RACE). GPO1M hybridized to a single 2.2 kb transcript from grape berry mRNA indicating the presence of further upstream sequence which was cloned using 5'-RACE PCR. The complete 1990 bp cDNA (GPO1) encodes a 67 kDa protein consisting of a 10.6 kDa chloroplast transit peptide, a 40.5 kDa catalytic unit containing two copper-binding regions and a 16.2 kDa C-terminal extension. Southern analysis suggested the presence of only one PPO gene in grapevine. High levels of gene expression were found in young developing berries, leaves and roots, but there was little expression in mature tissues. Biogenesis of PPO in grapevine tissues, appears to involve synthesis of a 67 kDa precursor protein which is imported into the chloroplast and processed to remove a 10.6 kDa chloroplast transit peptide from the N-terminus and a 16.2 kDa peptide of unknown function from the C-terminus.
从苏丹娜葡萄中纯化出了均一的多酚氧化酶（PPO），通过SDS-PAGE测定，得到一种表观分子量为40 kDa的单一蛋白质。基于这种纯化的40 kDa葡萄PPO蛋白的N端氨基酸序列设计了简并寡核苷酸引物，通过3' cDNA末端快速扩增（3'-RACE）扩增出一个1650 bp的cDNA克隆（GPO1M）。GPO1M与葡萄浆果mRNA的一个2.2 kb转录本杂交，表明存在进一步的上游序列，该序列通过5'-RACE PCR进行克隆。完整的1990 bp cDNA（GPO1）编码一个67 kDa的蛋白质，该蛋白质由一个10.6 kDa的叶绿体转运肽、一个包含两个铜结合区域的40.5 kDa催化单元和一个16.2 kDa的C端延伸组成。Southern分析表明葡萄中仅存在一个PPO基因。在幼嫩的发育中的浆果、叶片和根中发现了高水平的基因表达，但在成熟组织中几乎没有表达。葡萄组织中PPO的生物合成似乎涉及一种67 kDa前体蛋白的合成，该前体蛋白被导入叶绿体并进行加工，从N端去除一个10.6 kDa的叶绿体转运肽，从C端去除一个功能未知的16.2 kDa肽段。
