Sperling P, Libisch B, Zähringer U, Napier J A, Heinz E
Institut für Allgemeine Botanik, Universität Hamburg, Germany.
Arch Biochem Biophys. 2001 Apr 15;388(2):293-8. doi: 10.1006/abbi.2001.2308.
The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.
利用δ12 -和δ5 -脂肪酸酰基去饱和酶序列之间的相似性构建简并引物，用于对从发育中的琉璃苣种子mRNA转录的cDNA进行PCR实验。用扩增的DNA片段筛选琉璃苣种子cDNA文库，得到了一个对应于推导的446个氨基酸开放阅读框的全长cDNA。该蛋白与植物δ8 -鞘脂去饱和酶以及琉璃苣的δ6 -脂肪酸酰基去饱和酶具有高度相似性。该序列的特征是存在一个N端细胞色素b5结构域。通过对鞘氨醇碱基的二硝基苯基衍生物进行HPLC分析表明，该开放阅读框在酿酒酵母中的表达导致野生型细胞中不存在的δ8 -反式/顺式植物鞘氨醇的形成。对甲基化的二 - O -三甲基硅烷基醚衍生物的GLC - MS分析证实了C18 -和C20 -植物鞘氨醇的δ8 -立体异构体的存在。此外，Northern印迹显示，编码立体非选择性δ8 -鞘脂去饱和酶的基因主要在琉璃苣幼叶中表达。
