摘要
参照拟南芥WRKY33转录因子基因序列设计引物,利用RT-PCR技术从大白菜中克隆了WRKY33基因编码区460bp的序列,利用双链RNA介导的基因沉默技术,构建WRKY33基因沉默植物表达载体,为进一步研究该基因在植物与软腐病菌互作中的功能及其作用机制奠定基础。首先将WRKY基因片段连接至pUCm-T载体上,用PstⅠ/BamHⅠ和PstⅠ/XhoⅠ分别对pUCm-WRKY载体进行酶切,得到2个WRKY基因片段;先后将其连接到pBSSK-in载体上,构建成pBSSK-WRKY-in-WRKY载体,该载体中的2个WRKY片段大小一致,反向重复;并用SacⅠ/KpnⅠ酶切pBSSK-WRKY-in-WRKY载体得到WRKY-intron-WRKY片段,连入表达载体pCAMBIA1301中,构建成该基因的沉默植物表达载体。
PCR primers were designed based on WRKY33 transcription factor of Arabidopsis thaliana. The 460 bp cDNA of WRKY33 transcription factor was cloned from Brassica campestris ssp. pekinensis using RT-PCR. The RNA interference technology was used to construct gene silencing vector harborin WRKY33 gene. This study may provide a basic matarial for further studies on the bio-function of WRKY33 and the mechanism of signal transduction induced by Erwinia in plant. Two 460 bp fragments of WRKY with Pst Ⅰ/BamH Ⅰ and Pst Ⅰ/Xho Ⅰ obtained through digesting pUCm-WRKY, the combination of vector pUCm-T and gene WRKY, were connected into vector pBSSK-in to form pBSSK-WRKY-in-WRKY, in which the two fragments were inverse duplication. The transform unit WRKY-intron-WRKY, got by digesting vector pBSSK-WRKY-in-WRKY with Sac Ⅰ/KpnⅠ , was connected into expression vector pCAMBIA1301. Gene silencing vector harboring WRKY33 gene was constructed, and the combined vector would be employed.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2008年第3期43-46,共4页
Journal of Nanjing Agricultural University
关键词
大白菜
WRKY33基因
基因沉默
载体构建
Brassica campestris ssp. pekinensis
WRKY33 gene
gene silencing
vector construction
