通过构建三段T-DNA载体高频率获得无标记转基因大豆植株的研究

被引：11

作者：

叶兴国

秦华

机构：

[1] 中国农业科学院作物科学研究所/国家基因资源与基因改良重大科学工程/农业部作物遗传育种重点实验室

来源：

生物工程学报 | 2007年 / 01期

关键词：

大豆; 农杆菌; 三段T-DNA载体; 转化; 无标记转基因植株;

D O I：

10.13345/j.cjb.2007.01.026

中图分类号：

Q943.2 [植物基因工程];

学科分类号：

摘要：

高频率获得无选择标记转基因植株有利于转基因植物的环境释放和安全性生产,农杆菌介导的共转化法是获得无标记转基因植株的方法之一。含二段T-DNA载体的共转化法已被人们成功应用,而二段以上T-DNA载体的共转化法还未见报道。基于这一目的,通过几个中间质粒构建了含有三段T-DNA的双元表达载体pNB35SVIP1,其中包含1个拷贝bar基因选择标记基因表达盒和2个拷贝VIP1目的基因表达盒。利用EHA101农杆菌菌系介导法转化大豆子叶节,经过在含3～5mg/Lglufosinate培养基上多次筛选,获得了一定数量抗性再生植株,然后对抗性再生植株进行叶片涂抹除草剂、Southernblot和Northernblot检测,共鉴定出51棵T0代转基因植株,转化频率0·83%3·16%,二个基因的共转化频率为86·4%。在对T1代群体进行叶片涂抹除草剂检测的基础上,不抗除草剂植株进行PCR、Southernblot和Northernblot检测,共鉴定出41棵无选择标记转基因植株,无标记植株获得率为7·6%。检测结果还表明,T1代群体中22·7%的株系发生了基因丢失现象,27·3%的株系发生了bar基因沉默现象,目的基因在37·1%的无标记植株中发生了沉默现象。三段T-DNA的双元表达载体是获得无标记转基因植株的理想途径。

引用

页码：138 / 144

页数：7

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