基于漩涡振荡茎尖生长点转化法的Lyz-GFP双元基因在‘陇东苜蓿’中的转化和表达

Transformation and expression of the dual genes of Lyz-GFP genes in alfalfa('Long Dong') based on the method of vortex concussion growing point of stem tip

以‘陇东苜蓿’茎尖生长点为受体材料,利用农杆菌介导法并结合漩涡振荡,将双元基因溶菌酶(Lyz)与绿色荧光蛋白(GFP)导入其中,并对转化过程中的干扰因素和适宜条件进行了研究.结果表明:70mg/L的卡那霉素对陇东苜蓿转化植株的生长具有抑制效应;250mg/L头孢霉素能够有效地抑制农杆菌LBA4404的生长;‘陇东苜蓿’茎尖生长点经携有pBI121-Lyz-GFP的农杆菌LBA4404(D600值0.5～0.6)漩涡振荡侵染30min,培养4～5d后,转化材料生长良好,其转化率为24.47%.试验成功获得了含有该双元基因的‘陇东苜蓿’转基因植株,通过荧光检测和PCR扩增分析,转化植株有较强的荧光表达和GFP基因片段.

The growing point of stem tip of alfalfa（＇Long Dong＇） was receptor materials and the dual genes of Lyz-GFP genes was transfered intio the alfalfa（＇Long Dong＇） by agrobacterium-mediated technique combined with whirlpool oscillation.In the research,interference factors and the optimal conditions of the transformation process was studied.The results showed that 70 mg/L Kanamycin could restrain plants growth of alfalfa,250 mg/L Cef can restrain growth of Agrobacterium LBA4404 effectively.The growing point of stem tip of alfalfa（＇Long Dong＇） was infected for 30 min by Agrobacterium LBA4404（D6000.5~0.6） with the plasmid which contains pBI121-Lyz-GFP genes.Then these receptor materials mentioned above were conducted vortex oscillation,and then cultured 4~5 d.The transformation plants grow well and the transformation rate is 24.47%.Through the fluorescence detection and PCR amplification analysis,the transformation plant has fluorescence expression and GFP gene fragment significantly.