为了克隆桃果实中PpPFK（Prunus persica phosphofructokinase，磷酸果糖激酶）基因，并对其序列和不同低温胁迫下的转录水平进行分析。本研究采用了电子克隆和RT-PCR (reverse transcription PCR)相结合的方法分离桃果实PpPFK基因的cDNA全长序列，采用RT-qPCR (reverse transcription real-time quantitative PCR)技术分析桃果实在0 ℃、5 ℃冷藏过程中目的基因转录水平的变化。试验成功获得了全长1405 bp的PpPFK基因序列，该序列具有完整的开放阅读框（ORF，open reading frame），编码232个氨基酸。对ORF进行PCR的验证结果和电子克隆的结果一致。序列分析显示该基因的氨基酸序列同大豆和葡萄具有很高的相似度。RT-qPCR分析显示，5 ℃贮藏下的桃果实PpPFK基因转录水平在前14 d高于0 ℃果实，这可能导致5 ℃贮藏的桃果实发生更明显的冷害症状。本研究结果表明，桃果实PpPFK转录水平在不同低温贮藏过程中存在差异，这可能在桃果实低温胁迫响应过程中发挥重要作用。

In order to clone PpPFK(Prunus persica phosphofructokinase) gene and analyze its transcript levels of peach fruit stored at different low temperatures, PpPFK gene complete cDNA length was isolated with in silico cloning method and RT-PCR (reverse transcription PCR), and its transcript levels of peach fruit stored at 0 ℃ and 5 ℃ were examined through reverse transcription real-time quantitative PCR (RT-qPCR). The PpPFK gene with 1405 bp was gained, which codes 232 amino acids with an ORF (open reading frame). The ORF check test was conducted and the result suggested that the sequence of PpPFK gene through PCR was the same as that in silico cloning. The amino acid sequence of PpPFK gene in peach fruit was very similar to that of Glycine max and Vitis vinifera. The RT-qPCR analysis of PpPFK gene indicated that its transcript levels of peach fruit stored at 5 ℃were higher than that at 0 ℃ during first 14 days, resulting in an obvious chilling injury of peach fruit stored at 5 ℃. These results revealed that PpPFK was cold response gene, and differential responses existed in peach fruit stored at low temperatures, indicating its important role in low temperature stress response.

国家自然科学基金(31000825)；浙江省自然科学基金(Y3090537)；宁波市农业攻关项目（2010C10021）