甘蓝型油菜BnFAD2基因的克隆、表达及功能分析

摘要

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,并用血凝素标签法分析 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。%The oil containing high oleic acid is high nutritional. InBrassica 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 inBrassica napus were cloned and namedBnFAD2-A5,BnFAD2-C5 andBnFAD2-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 soft-ware was used to analyze the activity center ofFAD2 genes. Both of the results showed thatBnFAD2-A1 did not have the function of dehydrogenase. The yeast complementary experiment on four genes (including publishedBnFAD2-C1gene) showed that the desaturation capability of BnFAD2-A5 gene was next to that ofBnFAD2-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 andBnFAD2-C5aremajor genes affecting the accumulation of oleic acid inB. napus.