油菜AP2/ERF-B4类转录因子克隆及表达载体的构建  

Cloning and Expression Vector Construction of AP2/ERF-B4 Transcription Factor Gene from Brassica napus L.cv Huyou15

作　　者：庄静[1] 熊爱生[2] 乔玉山[1] 章镇[1] 姚泉洪[2] 

机构地区：[1]南京农业大学园艺学院,南京210095 [2]上海市农业科学院生物技术研究所,上海201106

出　　处：《西北植物学报》2008年第8期1521-1532,共12页Acta Botanica Boreali-Occidentalia Sinica

基　　金：863项目(2006AA10Z117;2008AA10Z401);上海市青年科技启明星跟踪计划(08QH14021)

摘　　要：利用油菜UniGene数据库,以拟南芥转录因子保守序列为探针,通过电子克隆方法分离得到一个UniGene库Bna.17538,进一步序列拼接得到一个油菜AP2/ERF-B4亚族的转录因子BnaERFB4-1,长度为672 bp,并进行了相关的生物信息学分析.结果显示BnaERFB4-1是亲水性蛋白,蛋白质三级结构与拟南芥RAP2.6L非常相似,蛋白质无序化程度大于拟南芥RAP2.6L.设计引物通过PCR和RT-PCR方法分别从甘蓝型油菜沪油15幼苗的DNA和cDNA中分离了BnaERFB4-1基因,命名为BnaERFB4-1-Hy15.序列测定和分析显示,来源于沪油15的BnaERFB4-1-Hy15基因与电子克隆的基因序列差异很小,有3个氨基酸位点不同,存在一个内含子.将BnaERFB4-1-Hy15基因通过BamHⅠ和SacⅠ酶切后分别插入酵母表达载体YK3302和植物双元表达载体pYF1404的相应位置,构建了BnaERFB4-1-Hy15基因的酵母体内结合和植物转化载体,为深入研究该基因在油菜抗逆调控中的作用奠定了基础.One AP2/ERF family transcriptional regulators （Named BnaERFB4-1） was isolated from B. hapus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana ERF-B4 subfamily as probe. Then, eDNA and deduced amino acid sequence, composition, hydrophobicty and hydro philicity,physical and chemical characterization, phylogenetic tree, conserved domain sequences, function domain,molecular modeling,and folding state were predicted and analyzed. BnaERFB4-1 was a hydrophilic protein. The protein and RAP2.6L of A. thaliana have similar three-dimension structure. The disordered residues of protein BnaERFB4-1 was higher than that of RAP2.6L. We isolated the BnaERFB4-1-Hy15 gene from winter and spring type B. napus L. cv ‘Huyou 15＇ by RT-PCR and PCR using cDNA and DNA as template. DNA sequencing and analyzing indicated that there only three amino acid residues difference between BnaERFB4-1-Hy15 and BnaERFB4-1. Moreover,the gene was ligated into vector of pYK3302 and pYF1404, respectively. It was successful to construct the recombinant plasmids of plant expression vec tor and yeast expression vector. It established a base for transformation of oilseed and studies of this gene function in abiotic stresses of B. napus.

关 键 词：转录因子 AP2/ERF-B4亚族 RT-PCR 生物信息学 甘蓝型油菜 沪油15 

分 类 号：Q789[生物学—分子生物学]