农杆菌介导的多裂叶荆芥转化体系建立

摘要

The Agrobacterium-mediated transformation system for Nepeta multifida was established in this study.The Agrobacterium tumefacien strain used in this study was the LBA4404 carrying with the pBI121-gfp plasmid.The segment of stem with axils was used as explants.The factors affecting transgenic efficiency,such as pre-culture time,bacterial concentration,infection time,co-culture time were studied.Each factor was set up with four levels.L16 (45)orthogonal experiment was conducted three times independently.The transgenic buds were confirmed by PCR assays and GFP fluorescence detection.The results showed that the gfp gene was successfully integrated in the genome of the transgenic plants.Transgenic expression was observed in buds,leaves,stems,roots.The optimum condition for transformation was pre-culture for 5 days,Agrobacterium OD600 value at 0.7,infection for 15 min,and co-culture for 3 days.The transgenic efficiency was 20％,and the average number of transgenic buds was 5 per explant.The establishment of N.multifida transformation system lays the groundwork for the improvement of the genetic traits via plant genetic engineering.%为了建立根癌农杆菌介导的多裂叶荆芥转化体系,以携带pBI121-gfp质粒的根癌农杆菌LBA4404菌株介导,荆芥试管苗带叶腋茎段为转化受体材料,探究了预培养时间、菌液浓度、侵染时间、共培养时间4个因素对荆芥转化效果的影响.每个因素设置4个水平,以L16(45)正交实验进行,筛选的卡那霉素抗性芽进行GFP荧光检测和PCR检测.结果表明,gfp基因成功导入转化植株,预培养5d、菌液浓度OD600为0.7、侵染15min、共培养3d为多裂叶荆芥的最优转化条件.转化率为20％,每个外植体再生抗性不定芽数平均为5个.荆芥转化体系的建立为利用植物基因工程改良其遗传性状奠定基础.