甘蓝型油菜BnFAD2基因的克隆、表达及功能分析

高油酸油具备较高的营养价值，在甘蓝型油菜中，脂肪酸去饱和酶基因(FAD2)是控制油酸含量的关键基因。本研究克隆了甘蓝型油菜A5、C5、A1连锁群上3个BnFAD2基因的全长cDNA序列，分别命名为BnFAD2-A5、BnFAD2-C5和BnFAD2-A1，各自编码384、384、136个氨基酸。分别使用TMHMM、Clust X软件分析FAD2基因的跨膜结构域和酶活中心表明，BnFAD2-A1不具备脱氢酶活性。采用酵母功能互补实验对4个基因(含已发表的BnFAD2-C1)进行功能验证，发现BnFAD2-A5和BnFAD2-C5基因去饱和能力接近，均大于BnFAD2-C1基因。采用qRT-PCR分析4个基因在甘蓝型油菜不同组织中的表达规律及血凝素标签法分析BnFAD2-C1、BnFAD2-A5和BnFAD2-C5的蛋白稳定性，表明BnFAD2-A5和BnFAD2-C5是影响油菜种子油酸积累的主效基因。

Cloning and Analyses of Expression and for BnFAD2 Genes in Brassica napus

The oil containing high oleic acid is high nutritional. In Brassica napus, the fatty acid desaturase gene (FAD2) is the key gene controlling oleic acid content. In this study, the full-length cDNA sequences of three genes located on chromosome A5, C5 and A1 in Brassica napus were cloned and named BnFAD2-A5, BnFAD2-C5 and BnFAD2-A1. The three genes encode proteins with 384, 384 and 136 amino acid residues, respectively. TMHMM was used to predict transmembrane domain and Clust X software was used to analyze the activity center of FAD2 genes. Both of the results showed that BnFAD2-A1 did not have the function of dehydrogenase. The yeast complementary experiment on four genes (including published BnFAD2-C1 gene) showed that the desaturation capability of BnFAD2-A5 gene was next to that of BnFAD2-C5 gene, and both of them were greater than that of BnFAD2-C1 gene. The expression patterns of the four genes were analyzed by using qRT-PCR technique in different tissues and the protein stability of BnFAD2-C1, BnFAD2-A5 and BnFAD2-C5 was analyzed by using Hemagglutinin labeling method. Both of the results revealed that BnFAD2-A5 and BnFAD2-C5 aremajor genes affecting the accumulation of oleic acid in rape seed.