苎麻CCoAOMT基因干扰表达载体构建及对烟草的转化

Construction of the RNAi vector of ramie CCoAOMT gene and its transformation in tobacco

根据已克隆的苎麻CCoAOMT(咖啡酰辅酶-A-氧甲基转移酶)基因cDNA序列,以其编码纤维素合成酶底物结合和催化合成结构域的cDNA序列为目标,采用PCR扩增方法引入克隆的酶切位点,分别将其正、反方向克隆到植物RNA干扰表达质粒PFGC5941T-DNA上CHSA内含子两侧,构建了植物表达苎麻CCoAOMT基因的干扰重组Ti载体.将该载体转入根癌农杆菌LBA4404后,采用农杆菌介导法对木质素研究的模式烟草WS38进行了遗传转化,抗性筛选和分子检测表明,成功获得了转基因植株.转基因植株生长延缓,表明可能存在基因表达干扰现象.

A conservative target sequence was amplified from the cloned ramie CCoAOMT（Caffeoyl-CoA-3-O- methyltransferase）, a key gene for ramie lignin synthesis, which is correspondent with substrate binding and catalytic domain in the enzyme.The sequence was inserted into a RNAi vector PFGC5941 T-DNA boarding the CHSA intron in reverse direction enable transcription of an RNAi molecule of CCoAOMT. The recombinant was transformed into Nicotiana tabacum WS38, a model plant for lignin reesearch, via Agrobacteria tumefaciens mediated transformation. Resistance screening and molecular analysis showed that the ramie CCoAOMT inference gene transformed into plant successfully and several transgenic plants were obtained. The transgenic plants showed a slower growth perhaps due to CCoAOMT interference phenomena.