含双边界序列植物双价表达载体的构建

Construction of a Plant Binary Expression Vector Harboring Two T-DNAs and Two Separate Exogenous Gene Cassettes

转基因植物的安全性是基因工程改良作物的一个重要问题。构建含双边界序列(双T-DNA区)载体,将选择标记基因和目标外源基因分开在不同的T-DNA区,通过转基因植株有性杂交及后代分离,可在转基因后代中获得无选择标记的转基因安全植株。将2个外源基因置于同一载体上,可以提高其共转化的效率。本研究构建了1个中间载体pAHC17-PTA和1个含双边界序列的植物双价表达载体pDB13PS。pAHC17-PTA含有由Ubiquitin启动子引导的具有抗虫效果的半夏凝集素基因(PTA)。pDB13PS含2个独立的T-DNA区,在其中一个T-DNA区,含两个目的基因的完整的表达盒,一个是由Ubiquitin启动子引导的半夏凝集素基因(PTA),另一个是由水稻胚乳特异表达启动子(Glutelin-B1promoter)驱动的马铃薯高赖氨酸蛋白基因的cDNA(SB401)。pDB13PS的成功构建,为提高PTA和SB401的共转化效率和获得无选择标记的转基因后代植株奠定了基础。

Environmental and edible safety issues over transgenic rice plants are important problems for improvement of crops through genetic engineering. To obtain transgenic rice plants without selective marker genes, an efficient approach is to transform rice with plant expression vectors containing two separate T-DNAs in a single binary expression vector, of which a selective marker gene and exogenous target genes are on two separate T-DNAs, by which transgenic plants without selective marker genes could be selected in their progenies by crossing deletion. Construction of a vector containing two exogenous genes will increase efficiency of integration of two genes. This study aims to make construction of a transition plant expression vector pAHC 17-PTA and a plant expression vector containing two T-DNAs, designated as pDB 13PS. The pAHC 17-PTA vector contains Pinellia ternata agglutinin gene （U/A） under the control of Ubiquitin promoter. In pDB13PS with two T-DNAs, the selectable marker gene Hpt conferring resistance to hygromycin phosphotransferase, and two target genes, PTA gene under the control of Ubiquitin promoter and POTA TO LYSINE-RICH gene （SB401） under the control of Glutelin-B 1 promoter, are on two separate T-DNAs. The construction of pDB 13PS provides a basis to improve the co-integration efficiency of PTA and SB401 and to achieve marker-flee transgenic plants in the progenies.