Sahoo Dipak Kumar, Dey Nrisingha, Maiti Indu Bhushan
KTRDC, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, United States of America.
Department of Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, Odisha, India.
PLoS One. 2014 Jun 4;9(6):e98988. doi: 10.1371/journal.pone.0098988. eCollection 2014.
We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3'rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.
我们构建了一种小型高效的二元Ti载体pSiM24，用于植物转化，以实现最大效率。在pSiM24载体中，早期二元载体pKYLXM24（GenBank登录号HM036220；pKYLX71的衍生物）的骨架大小从12.8 kb减少到7.1 kb。二元载体pSiM24由以下遗传元件组成：左右T-DNA边界、具有重复增强子结构域的改良的全长转录启动子（M24）、三个多克隆位点、一个3'rbcsE9终止子、大肠杆菌（ColE1）和根癌农杆菌（pRK2-OriV）的复制功能以及复制酶trfA基因、卡那霉素抗性（nptII）和氨苄青霉素抗性（bla）的选择标记基因。pSiM24质粒提供了广泛的克隆位点选择、在大肠杆菌中的高拷贝数以及易于操作不同遗传元件的高克隆能力。它已在瞬时（农杆菌浸润、原生质体电穿孔和基因枪）和植物系统（拟南芥和烟草）中稳定地转移绿色荧光蛋白（GFP）和β-葡萄糖醛酸酶（GUS）等转基因方面进行了充分测试，使用了农杆菌介导的转化和基因枪方法。不仅报告基因，使用pSiM24表达载体还能有效表达其他几个导入的基因。因此，pSiM24载体将有助于各种植物生物技术应用。此外，pSiM-24质粒可作为其他应用的平台，如基因表达研究和不同启动子表达分析。
