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

Obtaining Marker-free Transgenic Soybean Plants with Optimal Frequency by Constructing Three T-DNAs Binary Vector

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/L glufosinate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、Southern blot和Northern blot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、Southern blot和Northern blot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

Abstract:

Obtaining marker-free plants by high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2～3 months regeneration and selection on 3～5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83%～3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T₁ progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6%. Among the ₁ populations tested, the loss of the alien genes happened in 22.7% lines, the silence of Tbar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.