Hao G, Liu W, O'Connor M, Roelofs W l
Department of Entomology, New York State Agricultural Experiment Station, Cornell University, 14456, Geneva, NY, USA
Insect Biochem Mol Biol. 2002 Sep;32(9):961-6. doi: 10.1016/s0965-1748(01)00176-x.
Two cDNAs encoding acyl-CoA Z9-desaturase from the fat body and Z10-desaturase from the pheromone gland of the greenhead leafroller moth, Planotortrix octo, were obtained by RACE PCR. The Z9-desaturase (Pocto-Z9) cDNA spans 2291 nt with an ORF encoding a 352 amino-acid protein, which has 65% identity to Trichoplusia ni Delta 9 desaturase (Tni-Z9). The Z10-desaturase (Pocto-Z10) cDNA spans 2777 nt with an ORF encoding a protein with 356 amino acids. Pocto-Z10 shows lower identity to Pocto-Z9 and Tni-Z9 (48 and 46%, respectively) and relatively higher identity to the Delta 11 desaturases of T. ni and Helicoverpa zea (57 and 56%, respectively). The ORFs of these two P. octo cDNAs were constructed into an expression vector, YEpOLEX, that complemented the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient ole1 strain of Saccharomyces cerevisiae. Expression of Pocto-Z9 produced a 5:2 ratio of Z9-16 and Z9-18 acids, with minor amounts (<4%) of Z9-14, Z9-15, and Z9-17 acids. Pocto-Z10 was successfully expressed in the YEpOLEX system when complemented with Z11-18:Me, and the major desaturase product proved to be Z10-16:Acid. The results confirm the regio- and stereo-selectivity of this unusual Delta 10 desaturase.
通过RACE PCR获得了两条编码绿头卷叶蛾（Planotortrix octo）脂肪体中酰基辅酶A Δ9-去饱和酶和信息素腺中Δ10-去饱和酶的cDNA。Δ9-去饱和酶（Pocto-Z9）cDNA全长2291 nt，其开放阅读框编码一个352个氨基酸的蛋白质，该蛋白质与粉纹夜蛾（Trichoplusia ni）的Δ9-去饱和酶（Tni-Z9）有65%的同一性。Δ10-去饱和酶（Pocto-Z10）cDNA全长2777 nt，其开放阅读框编码一个含356个氨基酸的蛋白质。Pocto-Z10与Pocto-Z9和Tni-Z9的同一性较低（分别为48%和46%），而与粉纹夜蛾和棉铃虫（Helicoverpa zea）的Δ11-去饱和酶的同一性相对较高（分别为57%和56%）。将这两个P. octo cDNA的开放阅读框构建到一个表达载体YEpOLEX中，该载体可互补酿酒酵母去饱和酶缺陷型ole1菌株的不饱和脂肪酸（UFA）营养缺陷。Pocto-Z9的表达产生了Z9-16和Z9-18酸的5:2比例，以及少量（<4%）的Z9-14、Z9-15和Z9-17酸。当与Z11-18:Me互补时，Pocto-Z10在YEpOLEX系统中成功表达，主要的去饱和酶产物被证明是Z10-16:酸。结果证实了这种不寻常的Δ10-去饱和酶的区域和立体选择性。
