Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai 625021, Tamil Nadu, India.
Plant Sci. 2011 Jun;180(6):766-74. doi: 10.1016/j.plantsci.2011.02.010. Epub 2011 Mar 1.
A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19ΔnptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19ΔnptII and the resultant plasmid pBin19ΔnptII-ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif(r) harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19ΔnptII-ap24) yielded 14 independent hyg+/GUS+ transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separation of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T₁ generation.
构建了一种高拷贝数、可选择标记基因（SMG）-free 的农杆菌双元载体 pBin19ΔnptII，该载体通过删除 pBin19 中的 nptII 基因。该双元载体中 RK2 和 pVS 复制起点分别以 12 个和 3 个拷贝存在于农杆菌中。烟草渗出蛋白基因（ap24）被克隆到 pBin19ΔnptII 中，所得质粒 pBin19ΔnptII-ap24 被转移到含有单拷贝整合载体 pGV2260::pSSJ1 的根癌农杆菌菌株 C58C1 Rif(r)中。整合载体的 T-DNA 携带 hph（SMG）和 gus 基因。将携带根癌农杆菌（pGV2260::pSSJ1、pBin19ΔnptII-ap24）的农杆菌转化为 Oryza sativa L. var. Pusa Basmati1，共获得 14 个独立的 hyg+/GUS+转基因植株。用 hph 和 ap24 探针进行 Southern blot 分析显示，14 个转基因植株中有 12 个发生了共转化，并携带 hph、gus 和 ap24 基因。新的多拷贝二元载体的共转化效率为 86%。通过遗传分离整合 T-DNA 上的 hph/gus 基因和二元载体 T-DNA 上的 ap24 基因，从 10 个初级共转化体中完成了 4 个的基因分离，这些共转化体被进一步传代到 T₁ 代。
