摘要
脂肪酸延长酶1(FAE1)是广泛存在于植物中并定位于内质网上的一种能催化脂肪酸碳链延长的酮脂酰CoA合成酶.根据Genbank上已知的植物FAE1基因设计引物,以从甘蓝型油菜叶片中提取的总DNA为模板,进行PCR扩增,获得了1380bp的片段.回收该片段,并连接到pMD18-T载体测序.序列比对结果说明了该片段与植物中已知的FAE1序列有极高的相似性,并且不存在内含子序列.将该片段通过高保真酶扩增,EcoRⅠ酶切消化后定向克隆到pGEX-2T表达载体中,在IPTG诱导下于28℃表达出Mr76×103的蛋白质条带.用兔抗GST多克隆抗体做第一抗体进行Western-blot检测,并获得阳性检测结果.这为甘蓝型油菜脂肪酸链延长酶基因FAE1功能的进一步研究奠定了基础.
Fatty acid elangase 1 （ FAE1 ）, existing widely in plants, is a β-ketoacyl-CoA synthase that can prolong the carbon chain of fatty acid on endoplasmic reticulum. Primers were designed according to the known sequence of plant FAE1 gene from GenBank. From leaves of Brassica napus L. , genomic DNA was extracted as the template for PCR. The fragment was amplified and then cloned into pMD18-T vector. The sequencing result indicated that the fragment maintained significant similarity to the known FAE1 genes in plant and contained no intron. The sequence was amplified by Pfu polymerase, digested by EcoR I and inserted into the prokaryotic expression pGEX-2T vector. Induced by IPTG, protein was expressed at 28 ℃ with an approximately relative molecular weight of 76 x 103 and could be recognized by GST antibody. This study provides basis for further investigation of the function of fatty acid elongasel in B. napus. Fig 4, Tab 1, Ref 14
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2007年第2期152-155,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金资助项目(No.30471095)
四川省"十五"农作物育种攻关启动经费资助(qt2003-013)~~
关键词
甘蓝型油菜
脂肪酸链延长酶1
融合蛋白
原核表达
Brassica napus L.
fatty acid elongase 1
fusion protein
prokaryotic expression
