Red重组技术研究进展

Advances in Red Recombination Applied in Knockout of Bacteria Chromosomal Gene

主要从Red系统组成元件、作用机理、重组策略以及先进性和发展前景四个方面综述了利用Red重组系统敲除或替换细菌染色体目的基因的方法。首先简要介绍了传统的细菌染色体重组技术，指出了其中的缺陷。然后提出了Red重组技术的定义：利用噬菌体Red系统介导来实现外源线性DNA片断与细菌染色体的靶基因进行同源重组的方法，外源线性DNA通常是PCR产物、寡核苷酸片断等，在它们的两翼各含有与染色体靶基因两翼同源的序列40—60bp。这种Red重组技术省去了体外DNA酶切和连接等步骤，使细菌染色体靶基因的敲除与替换操作相对简单，逐渐成为基因功能探索以及新菌株构建的有力手段。

Traditional recombination technology of bacteria chromosome and its limitation were introduced. The definition of Red recombination technology is put forward： a method of homologous recombination between foreign linear DNA and the target gene in chromosomes mediated by λ phage Red system. The linear DNA referred here is general PCR product or oligonucleotide, which has a 36 - 50bp homologous sequence with the target gene in chromosome at both flanking. Red recombination technology leaves out the in vitro DNA restriction enzyme digestion and link process, which makes the knockout and alternation of target gene in bacteria chromosome relatively easier, and becomes an effective method to exploring genes and constructing new strains gradually. The gene inactivation and alternation method aiming at bacteria chromosome applied to Red recombination system was summarized by the structure element, action mechanism, and strategy of recombination, advantage and developing prospect. The Red system includes three genes： bet （akaβ）, exo and gain （aka γ）. Exo is a 5＇→43＇ exonuclease, which degrades the 5＇ ends of linear DNA molecules. Bet is a single-stranded DNA binding protein that binds to the single stranded 3＇ ends generated by Exo and promotes annealing to complementary DNA. Gam binds to the host RecBCD complex and inhibits its exonuclease activity. Red recombination system may be constructed in such plasmids as pKD20 and pKD46 or in chromosome of bacteria. Most bacteria are not readily transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA. But when bacteria cells are transformed with pKD20 or pKD46 plasmid, or integrated with a detective λ prophage, Red recombination enzymes may be expressed in host cells, which make linear DNA with 36 -50bp extensions that are homologous to both flanking of target genes transform E. coli readily and knock-out or alternate target gene. The Red recombination method is not only useful in chromosomal gene inactivation in E. coli, but also in other bacteria or virus, such as Salmonella, Shigella flexneri and virus HaSNPV. With the proceeding research, Red system will be applied for more and more purposes, and contribute a lot for gene improvement and gene function investigation in the coming Postgenome Era.