cDNA cloning, characterization, and functional expression of 4S-(-)-limonene synthase from Perilla frutescens.
cDNA cloning, characterization, and functional expression of 4S-(-)-limonene synthase from Perilla frutescens.
复制标题
紫苏 4S-(-)-柠檬烯合酶的 cDNA 克隆、表征和功能表达。
DOI：
10.1006/abbi.1996.0343
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Croteau,R
中科院分区：
文献类型：
--
作者：
Yuba,A;Yazaki,K;Tabata,M;Honda,G;Croteau,R
对紫苏中柠檬烯合成酶进行了分子生物学研究，该酶催化香叶基二磷酸环化生成紫苏醛生物合成的中间产物4（S）-柠檬烯。我们分离并鉴定了10个与留兰香柠檬烯合成酶cDNA同源的cDNA，这些cDNA来自于由两对显性基因GandH纯合的紫苏株子叶构建的表达文库，GandH负责4（S）-柠檬烯的形成。这些cDNA克隆中的两个在大肠杆菌中功能性表达，产生在从香叶基二磷酸生成4（S）-柠檬烯中具有催化活性的酶。在代表性cDNA克隆PFLC 1中最长的开放阅读框架由1812个核苷酸组成，对应于603个氨基酸。该基因与留兰香柠檬烯合酶、烟草5-表马兜铃烯合酶和蓖麻子卡宾合酶的ORF的同源性分别为65%、35%和30%。对不同基因型（GGHH、GGhh、ggHH和gghh）的P. frutescens的结果表明，在具有HH基因型的菌株中存在不止一个拷贝的PFLC 1 DNA。相比之下，没有PFLC 1 DNA序列中发现的菌株的基因组与thehh基因型，不能生产环己烷类单萜缺乏柠檬烯合酶活性。北方印迹分析表明，PFLC 1基因在GGHH植株的所有地上部分都有表达，尤其是在叶片中。另一方面，在ggHH植物中，PFLC 1 mRNA仅在茎和花萼中检测到微量。
A molecular biological study on limonene synthase that catalyzes the cyclization of geranyldiphosphate to yield the olefin 4(S)-limonene, an intermediate in the biosynthesis of a monoterpenoid, perillaldehyde, inPerilla frutescensBritton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of aPerillastrain homozygous for two pairs of dominant genes,GandH,which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed inEscherichia coli,yielding enzymes which were catalytically active in generating 4(S)-limonene from geranyldiphosphate. The longest open reading frame in the representative cDNA clone PFLC1 consisted of 1812 nucleotides corresponding to 603 amino acids. Its identity to the ORFs of spearmint limonene synthase, tobacco 5-epi-aristolochene synthase, and castor bean casbene synthase were 65, 35, and 30%, respectively. Genomic Southern blot analyses of various genotypes (GGHH, GGhh, ggHH,andgghh) ofP. frutescenssuggested that more than one copy of the PFLC1 DNA exists in strains having theHHgenotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with thehhgenotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC1 3′-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of theGGHHplants, particularly in the leaves. In theggHHplants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.