生物
克隆(编程)
合成生物学
计算生物学
遗传学
基因组DNA
功能基因组学
基因
转化(遗传学)
DNA
基因组
基因组学
计算机科学
程序设计语言
作者
Yongchun Zhao,Jingluan Han,Jiantao Tan,Yugui Yang,Shuangchun Li,Yajun Gou,Yuyu Luo,Tie Li,Wenyu Xiao,Xue Yang,Hao Yu,Xianrong Xie,Yao‐Guang Liu,Qinlong Zhu
摘要
Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.
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