Pechous Steven W, Whitaker Bruce D
Produce Quality and Safety Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, MD 20705-2350, Beltsville, USA.
Planta. 2004 May;219(1):84-94. doi: 10.1007/s00425-003-1191-4. Epub 2004 Jan 22.
Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple ( Malus domestica Borkh.) fruit. The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood. Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, reverse transcription-polymerase chain reaction with RNA isolated from peel tissue of 'Law Rome' apples yielded an approx. 800-bp gene fragment. This was used to screen a cDNA library generated from the peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1,931 nucleotides long was isolated. The 1,728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa. Sequence analysis of the apple TS showed it to be most similar to several monoterpene synthases. Oddly, the TS includes an RR(X(8))W motif near the N-terminus that is common among monoterpene synthases but it lacks the plastid transit peptide sequence typically associated with genes of that group. Expression of the apple TS gene in Escherichia coli gave myc-epitope-tagged and untagged proteins estimated at approx. 68 and approx. 66 kDa, respectively. In assays of sesquiterpene synthase activity, with farnesyl diphosphate as substrate, the untagged bacterially expressed TS gene product synthesized ( E, E)-alpha-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only ( E)-beta-ocimene, albeit at much reduced levels. Addition of a C-terminal myc tag appeared to completely prevent production of soluble protein under all of the expression conditions tested. This is the first report of an ( E, E)-alpha-farnesene synthase gene ( AFS1; GenBank accession number AY182241) from a flowering plant. RNA gel blots showed that AFS1 transcript increased about 4-fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5 degrees C. In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and fell to nearly undetectable levels by 8 weeks.
苹果（Malus domestica Borkh.）果实成熟时，萜类化合物和许多其他与香气相关的挥发性物质的产量会增加。气态植物激素乙烯在挥发性物质合成的诱导中起关键作用，但其机制尚不清楚。基于几种倍半萜合酶共有的短保守序列设计简并引物，以‘Law Rome’苹果果皮组织分离的RNA进行逆转录-聚合酶链反应，得到一个约800 bp的基因片段。该片段用于筛选由果皮组织mRNA构建的cDNA文库。分离出一个全长1931个核苷酸的萜类合酶（TS）cDNA。1728 bp的开放阅读框编码一个576个氨基酸的蛋白质，分子量为66 kDa。苹果TS的序列分析表明它与几种单萜合酶最为相似。奇怪的是，TS在N端附近包含一个RR(X(8))W基序，这在单萜合酶中很常见，但它缺乏通常与该类基因相关的质体转运肽序列。苹果TS基因在大肠杆菌中的表达产生了带myc表位标签和不带标签的蛋白质，估计分子量分别约为68 kDa和约66 kDa。在以法呢基二磷酸为底物的倍半萜合酶活性测定中，未带标签的细菌表达TS基因产物几乎只合成了（E, E）-α-金合欢烯。在以香叶基二磷酸为底物的单萜合酶测定中，未带标签的苹果TS只产生了（E）-β-罗勒烯，尽管产量大大降低。在所有测试的表达条件下，添加C端myc标签似乎完全阻止了可溶性蛋白质的产生。这是开花植物中（E, E）-α-金合欢烯合酶基因（AFS1；GenBank登录号AY182241）的首次报道。RNA凝胶印迹显示，在0.5℃下储存的前4周，苹果果实果皮组织中AFS1转录本增加了约4倍。相反，果实采收时用乙烯作用阻断剂1-甲基环丙烯处理后，AFS1 mRNA在冷藏的最初4周内急剧下降，到8周时降至几乎检测不到水平。
