Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan, Republic of China.
Appl Microbiol Biotechnol. 2013 Oct;97(19):8637-47. doi: 10.1007/s00253-013-5034-3. Epub 2013 Jun 30.
Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.
发根是一种经过转化的根组织，其中的转移 DNA（T-DNA）通过发根农杆菌插入基因组中。为了在发根中建立一个多基因共转化的系统，我们评估了发根农杆菌的四种不同策略。gusA 和 mgfp5 这两个基因位于两个不同的质粒中，它们被转化到两批不同的发根农杆菌（策略 2AR）或一批（策略 2BV）中。这两个报告基因也被插入到一个 T-DNA（策略 1TD）或两个不同的 T-DNA（策略 2TD）中，在一个二元载体中。在所有四组中，超过 90%的感染烟草叶盘形成了发根，与野生型发根农杆菌的感染效率没有显著差异。策略 2AR、2BV、1TD 和 2TD 的共转化发根的比例分别为 65.4%、40.0%、78.6%和 82.1%，这表明所有策略都适用于多个基因的共转化。生长速度和异源蛋白表达的高度变化表明，需要进一步筛选以确定具有最高生产力的克隆。我们的结果表明，策略 1TD 和 2TD 实现了最高的共转化效率。与策略 2AR 或 2BV 相结合为共转化多个转基因提供了额外的选择。
