Abstract:

Gene cloning is a routine experiment to elucidate its functions, however, many genes are difficult to be cloned, such as those from AT-rich (>60%) fragments. Exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA (ExoCET) cloning combines in vitro homologous recombination mediated by exonucleases and intracellular homologous recombination mediated by E. coli RecET recombinases. ExoCET is a powerful gene cloning technique because it can not only capture fragments larger than 100 kb from genomes but also assemble more than 13 pieces of DNA fragments. So far, there has been no report on the use of ExoCET to clone large fragments from genomes with AT content more than 63%. In this study, the genome of the marine cyanobacteria Prochlorococcus MIT 9301 strain with 69% AT content was used to optimize the ExoCET conditions for cloning of large AT-rich DNA fragments. The results indicate that: (1) Compared to T4 polymerase method, higher cloning efficiency was obtained when Gibson assembly method was used for the in vitro homologous recombination, since the covalently stitched DNA molecule by Gibson assembly is much more stable than the DNA molecule with gap stitched by T4 DNA polymerase; (2) The single-copy bacterial artificial chromosome (BAC) vector should be used, and the multi-copy plasmid vector cannot clone those fragments, because the single-copy BAC can minimize the expression of cloned genes to avoid their toxic effect on the host; (3) The ExoCET-BAC strategy can not only capture fragments larger than 80 kb from the Prochlorococcus genome, but also assemble 11 pieces of DNA fragments with 100% accuracy; (4) ExoCET-BAC can capture 4 pieces of genomic fragments of 7-20 kb simultaneously in one step. Genome sequencing revealed that AT-rich organisms account for more than 30%, and thus the ExoCET-BAC strategy established in this study provides efficient enabling technology for the genome functional research of AT-rich organisms.

Key words: difficult cloning DNA, high AT content, genome, homologous recombination, direct cloning, DNA assembly, ExoCET

摘要：

基因克隆是解析基因功能的重要手段，但仍有很多基因难以克隆，比如高AT含量（>60%）基因组来源的DNA。ExoCET克隆技术通过联合核酸外切酶介导的体外同源重组和大肠杆菌RecET重组酶介导的细胞内同源重组，不仅能从微生物基因组中靶向抓取>100 kb的大片段，而且能高效组装>13个DNA片段，是基因克隆的有力工具，迄今未有利用ExoCET技术从AT含量>63%的基因组克隆大片段的报道。本研究以AT含量为69%的海洋单细胞光合蓝细菌原绿球藻MIT 9301菌株的基因组为研究对象，探究了利用ExoCET技术进行高AT含量基因组大片段克隆的最佳条件。结果显示：①在核酸外切酶介导的体外同源重组时使用Gibson体系较T4聚合酶体系能获得更高的克隆效率；②载体应选择单拷贝的细菌人工染色体（BAC），多拷贝质粒载体会导致克隆失败；③ExoCET可以从原绿球藻基因组上抓取>80 kb的大片段，并且能以100%的正确率组装11个3 kb的DNA片段；④可以一步同时抓取4个7～20 kb的基因组大片段。大规模基因组测序显示高AT含量生物占比超过30%，该研究建立的ExoCET-BAC策略将为高AT含量生物的基因组功能研究提供高效使能技术。

关键词: 难克隆DNA, 高AT含量, 基因组, 同源重组, 直接克隆, DNA组装, ExoCET