Cloning and Characterization of Six FAD2 Pseudogenes of Brassica napus

FAD2 gene is essential for converting oleic acid to linoleic acid in plant seeds. From Xiangyou 15, a cultivar of Brassica napus, 56 FAD2 DNA clones and 47 FAD2 cDNA clones were obtained including 6 new copies of FAD2, which were designated FAD2P1 to FAD2P6. These new FAD2 copies ranged from 1141 bp to 1157 bp in length and contained no introns in their open reading frames. They had 96.1% homology in nucleotide sequence and shared more than 87.0% of nucleotides identity to the published FAD2 gene (AY577313). The deduced amino acid sequences of the 6 FAD2 copies presented 1–12 stop codons within the coding regions, which prevented from coding functional proteins. These copies were transferred into Saccharomyces cerevisiae through vector pYES2.0, and the expression products showed no functions to synthesize 16:2 and 18:2 fatty acids. These results indicate that they are pseudogenes of FAD2.

摘 要以甘蓝型油菜湘油15为材料, 采用PCR方法克隆并分析了56个FAD2基因克隆和47个FAD2基因cDNA克隆, 从中发现6个新的FAD2拷贝。它们与公布的甘蓝型油菜FAD2基因(AY577313)具有87.0%以上的同源性, 没有内含子, 在开放阅读框中存在1∼12个终止密码子, 其中有2个拷贝具有转录功能。将这6个FAD2拷贝在酿酒酵母中进行体内表达实验, 通过气相色谱检测脂肪酸组成证明其不具备油酸脱氢酶功能, 与对照相比, 也没有改变酵母体内脂肪酸组成。由此推测这6个FAD2拷贝为假基因。