Tai H, Jaworski J G
Chemistry Department, Miami University, Oxford, Ohio 45056.
Plant Physiol. 1993 Dec;103(4):1361-7. doi: 10.1104/pp.103.4.1361.
A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.
分离出了一个编码菠菜（Spinacia oleracea）3-酮脂酰-酰基载体蛋白合酶III（KAS III）的cDNA克隆，该酶催化脂肪酸生物合成中的初始缩合反应。根据纯化的菠菜KAS III胰蛋白酶消化产物的氨基酸序列，设计了简并聚合酶链反应（PCR）引物，并用于从菠菜叶RNA的第一链cDNA中扩增出一个612 bp的片段。用该PCR片段探测根cDNA文库，分离出一个1920 bp的克隆。其推导的氨基酸序列与从纯化的KAS III获得的胰蛋白酶消化产物的序列匹配。Northern分析证实它在叶和根中均有表达。该克隆包含一个1218 bp的开放阅读框，编码405个氨基酸。通过在大肠杆菌BL 21中作为谷胱甘肽S-转移酶融合蛋白表达来确认该克隆的身份。推导的氨基酸序列与紫菜假定的KAS III和大肠杆菌的KAS III分别有48%和45%的同一性。它与植物查尔酮合酶也有很强的局部同源性，但与来自植物、细菌或动物的其他KAS同工型几乎没有同源性。
