Karvouni Z, John I, Taylor J E, Watson C F, Turner A J, Grierson D
Department of Physiology and Environmental Science, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.
Plant Mol Biol. 1995 Mar;27(6):1153-62. doi: 10.1007/BF00020888.
A cDNA clone (MEL5), encoding a protein homologous to phytoene synthase (PSY), has been isolated from a climacteric melon fruit cDNA library, using the tomato cDNA clone TOM5 [34] as a heterologous probe. MEL5 hybridised to a transcript of 1.65 kb which suggested that the 1.36 kb clone, isolated originally, was not full-length. The missing 5' end was isolated by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method. This enabled the full sequence of the protein to be deduced and the cleavage site of the transit peptide for chromoplast import to be predicted. Northern analysis of RNA extracted from fruit samples of different ripening stages as well as from roots, leaves and flower petals was used to examine the expression pattern of the corresponding mRNA. The corresponding mRNA. The transcript corresponding to MEL5 is present at low quantities in unripe (green) fruit, reaches its highest levels when the fruit turns from green to orange and persists at lower levels during later ripening stages. A similar transcript was also detected in flower petals and in trace amounts in leaves and roots. Genomic Southern analysis indicates that the clone is homologous to a low-copy-number gene family. Sequence analysis showed a high degree of conservation among plant PSYs.
利用番茄cDNA克隆TOM5 [34]作为异源探针，从跃变型甜瓜果实cDNA文库中分离出一个编码与八氢番茄红素合酶（PSY）同源蛋白的cDNA克隆（MEL5）。MEL5与一个1.65 kb的转录本杂交，这表明最初分离的1.36 kb克隆不是全长的。通过基于逆转录酶-聚合酶链反应（RT-PCR）的方法分离出缺失的5'端。这使得能够推导该蛋白的完整序列，并预测向质体转运肽的切割位点。对从不同成熟阶段的果实样品以及根、叶和花瓣中提取的RNA进行Northern分析，以检测相应mRNA的表达模式。对应于MEL5的转录本在未成熟（绿色）果实中含量较低，在果实从绿色变为橙色时达到最高水平，并在随后的成熟阶段维持在较低水平。在花瓣中也检测到类似的转录本，在叶和根中检测到微量。基因组Southern分析表明该克隆与一个低拷贝数基因家族同源。序列分析显示植物PSY之间具有高度保守性。
