甘蓝型油菜BnFAD2基因的克隆、表达及功能分析  

Cloning and Expression Analyses for BnFAD2 Genes in Brassica napus

作　　者：刘睿洋[1] 刘芳[1] 官春云[1] 

机构地区：[1]湖南农业大学农学院/国家油料改良中心湖南分中心,湖南长沙410128

出　　处：《作物学报》2016年第7期1000-1008,共9页Acta Agronomica Sinica

基　　金：湖南省科技创新项目(CX2013A012);国家重点基础研究发展计划(973计划)项目(2015CB150200)资助;supported by Innovation Foundation of Hunan Province(CX2013A012);the National Basic Research Program of China(2015CB150200)

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c DNA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. In Brassica napus, the fatty acid desaturase gene（FAD2） is the key gene controlling oleic acid content. In this study, the full-length c DNA 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 q RT-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 are major genes affecting the accumulation of oleic acid in B. napus.

关 键 词：甘蓝型油菜 BnFAD2基因 生物信息学 蛋白活力 蛋白稳定性 

分 类 号：S565.4[农业科学—作物学]