长喙田菁植物螯合肽合成酶基因在烟草中的表达

Expression of Sesbania rostrata Phytochelatin Synthase 1 Gene in Transgenic Nicotiana tabacum

以pHANNIBAL及pART27为基础,构建了CaMV35S启动子驱动的长喙田菁(Sesbania rostrata)植物螯合肽合成酶SrPCS1基因植物超量表达载体pAM22,采用电击转化方法将pAM22导入根癌农杆菌EHA105,并用该菌株对烟草进行了转化,通过抗生素筛选、PCR及Northern-blotting检测了目的基因的表达水平,并用ClustalW对SrPCS1进行了进化分析。结果表明:SrPCS1编码233个氨基酸与豆科植物PCSs的同源性较高;得到27株抗性植株,23株PCR阳性植株,Northern-blotting证明得到了超量表达该基因的烟草14株,但基因的表达水平不同。

The CaMV35S promoter driving plant expression vector pAM22 with Sesbania rostrata phytochelatin synthase 1（SrPCS1） was constructed using PCR and ligation based on the pHANNIBAL and pART27.By electroporation transformation,pAM22 was transferred into Agrobacterium tumefacien EHA105 and the new engineering bacterium was transferred into Nicotiana tabacum.The expression level of SrPCS1 was indentified by antibiotics screening,PCR and northern-blot analysis.At the same time,phylogenetic analysis of SrPCS1 with other PCSs was conducted by ClustalW.The results indicated that SrPCS1 encoding 233 amino-acids exhibited high homologous with PCS from legumes,27 transgenic tobacco tolerant to antibiotics have been acquired in which 23 are positive plants identified by PCR,and 14 transgenic tobacco expressed SrPCS1 plaqnts were identified with northern-blotting analysis,but their expression levels are different.