State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, People's Republic of China.
Plant Mol Biol. 2011 Sep;77(1-2):117-27. doi: 10.1007/s11103-011-9798-5. Epub 2011 Jun 4.
We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6×Cys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg²⁺ ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass.
我们开发了一种改良的粒子轰击法用于植物转基因。将内含肽标签和 6×Cys 标签依次融合到超活性 Tn5 转座酶的 N 端。通过 6×Cys 标签巯基与金表面的共价结合，将修饰后的转座酶固定在裸露的金微粒子上。在没有 Mg²⁺离子的情况下，连接的转座酶可以在体外结合转座子 DNA 形成转座体。将携带转座体的金颗粒轰击入植物细胞后，由于内含肽标签的激活自切割功能，转座体将从载体中释放出来。我们的数据表明，与传统的粒子轰击法相比，该程序将外源 DNA 整合到植物基因组中，转化频率增加。通过减少与植物细胞生物量相关的组装转座子 DNA 量，也可以获得单拷贝插入。
