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WRKY33转录因子基因沉默载体的构建

DOI: 10.7685/j.issn.1000-2030.2008.03.008, PP. 43-46

Keywords: 大白菜,WRKY33基因,基因沉默,载体构建

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Abstract:

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。

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