油菜FAD2基因的克隆与功能分析

油酸去饱和酶FAD2基因催化C18∶1脂肪酸脱氢生成C18∶2脂肪酸,直接决定了油料作物的油脂品质,同时对植物的抗冻能力至关重要。通过RT-PCR从甘蓝型油菜品种＂中双9号＂中克隆了Bn FAD2基因的全长c DNA序列,该基因编码一个长384aa的脂肪酸去饱和酶蛋白,分子量44.2 ku,等电点pI值为8.54,对应的基因组序列无内含子。使用荧光定量RT-PCR发现它在种子发育到25 d时转录水平最高;使用生物信息学手段发现该蛋白具有6个跨膜区,定位在内质网膜上,不含信号肽。将该基因克隆到植物转基因载体pBILP2PTNap中,通过农杆菌LBA4404介导转化了拟南芥fad2基因突变体,使其...

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Published in广东农业科学 Vol. 43; no. 1; pp. 15 - 23
Main Author 胡亚平 郭雁君 吉前华 蒋惠 郭丽英 张小凤
Format Journal Article
LanguageChinese
Published 肇庆学院果树研究所/肇庆学院生命科学学院,广东肇庆,526061 2016
Subjects
基因克隆
油菜
油酸去饱和酶
转基因,功能分析
油酸去饱和酶
转基因,功能分析
油菜
基因克隆
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ISSN1004-874X

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Summary:油酸去饱和酶FAD2基因催化C18∶1脂肪酸脱氢生成C18∶2脂肪酸,直接决定了油料作物的油脂品质,同时对植物的抗冻能力至关重要。通过RT-PCR从甘蓝型油菜品种＂中双9号＂中克隆了Bn FAD2基因的全长c DNA序列,该基因编码一个长384aa的脂肪酸去饱和酶蛋白,分子量44.2 ku,等电点pI值为8.54,对应的基因组序列无内含子。使用荧光定量RT-PCR发现它在种子发育到25 d时转录水平最高;使用生物信息学手段发现该蛋白具有6个跨膜区,定位在内质网膜上,不含信号肽。将该基因克隆到植物转基因载体pBILP2PTNap中,通过农杆菌LBA4404介导转化了拟南芥fad2基因突变体,使其在种子中超量表达,共获得20株转基因阳性植株。使用气相色谱法比较野生型植株、突变体植株、转基因阳性植株种子中脂肪酸的组成成分,结果发现转基因前后,种子中油酸（C18∶1）的含量从53.8%降低到12.4%-34.1%,亚油酸（C18∶2）含量从2.9%提高到11.5%-62.7%,说明这些转入的Bn FAD2基因在不同植株中均能不同程度地互补fad2突变体的表型,证明克隆得到的油菜BnFAD2基因具有完整的催化功能。
Bibliography:44-1267/S
HU Ya-ping, GUO Yan-jun, JI Qian-hua, JIANG Hui, GUO Li-ying, ZHANG Xiao-feng （ Fruit Tree Research Institute of Zhaoqing University/Life College of Zhaoqing University, Zhaoqing 526061, China ）
oleic acid desaturase; Brassica napus; gene clone; transgene; function analysis
The conversion from oleic acid to linoleic acid,which is catalyzed by oleic acid desaturase FAD2,determines oil quality of oil crops and also plays an important role in cold resistance of plants. In this study,the fulllength of Brassica napus FAD2 gene（Bn FAD2） was cloned from＂Zhongshuang 9＂via RT-PCR. The Bn FAD2 encoded a 384 aa oleic acid desaturase with predicted molecular weight of 44.2 ku and isoelectric point（p I） value of 8.54. The properties of this protein were further analyzed by bioinformatics methods. The results demonstrated that it contained six transmembrane regions and located in the endoplasmic reticulum omentum,and there was no evidence that signal peptides existed. The transcription of Bn FAD2 gene reached the hi
ISSN:1004-874X