六个甘蓝型油菜油酸脱氢酶（FAD2）假基因的克隆和分析

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基因拷贝。它们与公布的甘蓝型油菜FAD2基因(AY577313)具有87%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基因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基因拷贝为假基因。

Cloning and Characterization of Six Oleic Acid Desaturase Pseudogenes of Brassica napus

The phenomenon of multi-copy genes is common in plants. Pseudogene is defined as an inactive gene, which can not synthesize functional proteins but share the similar DNA sequences with normal functional genes. In this study, 56 FAD2 DNA clones and 47 FAD2 seed cDNA clones of Brassica napus cv. Xiangyou 15 were investigated, and 6 new copies of FAD2 were detected, designated as FAD2P1-6 respectively. This sequence length of 6 copies ranged from 1 141–1 157 bp and there were no introns in their open reading frames (ORF). These 6 copies share 96.1% identity in nucleotides from one another, and share more than 87% nucleotides identity with AY577313. Deduced amino acid sequences revealed that 1–12 stop codons occurred in the coding region of six copies which will prevent them from coding for a functional protein. These six copies were investigated in vivo in Saccharomyces cerevisiae through being cloned into yeast expression vector pYES2.0, and the 16:2 and 18:2 fatty acids were determined by gas chromatographic analysis. The results revealed that the products of the six copies were not able to synthesize 16:2 and 18:2 fatty acids, suggesting that they are pseudogenes of FAD2. These multiple pseudogenes of FAD2 within the B. napus genome might result from the duplication of large chromosomal segments simultaneously following mutation. Because of the existence of multiple pseudogenes for FAD2 in B. napus genome, we should be careful in genetic research to identify true and false, to avoid wrong conclusions.