甘蓝型油菜FAE1基因启动子的克隆和瞬时表达载体的构建及功能分析

油菜脂肪酸延长酶基因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基因启动子序列设计引物,以从甘蓝型油菜(Braccica napus.L)叶片中提取的总DNA为模板,进行PCR扩增,获得了1 328 bp的片段.回收该片段,并连接到pMD18-T载体测序.序列在NCBI进行blast比对,同源性为99.78%,说明该片段与植物中已知的FAE1基因的启动子序列有极高的相似性.将该片段替换掉改造后的pBI221上面的35S启动子从而构建了原生质体瞬时表达载体.然后通过PEG介导的方式将含有FAE1启动子驱动的荧光素酶报告基因(LUC)表达载体导入油菜原生质体中,研究报告基因表达量,证明出脱落酸(ABA)影响了报告基因的表达,随着ABA浓度的不断增加,LUC的表达量呈现逐步下降的趋势.

Cloning and Transient Expression Assay of FAE1 Promoter from Brassica napus

Fatty Acid Elongase(FAE1),existing widely in plants,is a ketoacyl-CoA,which can catalyse the prolongation reaction of fatty acid cabon chain on endoplasmic reticulum.Design primers according to the known sequence of Brassica napus FAE1 gene are promoted from GenBank.The total DNA,which is taken as the template for PCR,is extracted from the leaves of Braccica napus.L.The fragment was amplified and then cloned into pMD18-,T vector,and the homology is 99.78 percent after sequence alignment on the NCBI.The sequencing results indicated that the fragment maintained significant similarity to the known FAE1 gene promoter in plants.Then the fragment was inserted into pBI221 vector.The reconstructed plasmid,pBI221-,FP-,LUC,which had the Brassica napus FAE1 promoter and the reporter gene LUC,was transferred into protoplasts of Arabidopsis by PEG.Transient expression showed that the expression of LUC decreased when ABA was added.