利迪链霉菌A02细菌人工染色体基因组文库的构建

 利迪链霉菌A02是从京郊森林土壤中分离筛选出的植物真菌病害高效生防菌,其活性产物为安全高效广谱的抗真菌剂纳他霉素。为了克隆纳他霉素生物合成基因簇和调控其表达相关的功能基因,通过基因改良的方式进行A02的定向分子改造,提高纳他霉素的效价和产量,提取了利迪链霉菌A02的基因组DNA,用Hind III和Bam HI部分酶解后,回收了97~194kb和48.5~97kb大小的高分子量DNA,与质粒载体Copycontrol pCC1BAC连接,分别构建了含有800个和1500个克隆的两个BAC文库。从文库中随机挑选20个克隆,酶切检测平均插入片段分别为133kb和65kb,空载率小于1%,假定利迪链霉菌的基因组有8×10⁶kb,计算文库基因组覆盖率分别为12.28倍和11.25倍。因此,从文库筛选到目的片段的概率达99.99%以上。

Construction of Bacterial Artificial Chromosome Library of Streptomyces lydicus A02

Streptomyces lydicus strains A02 is isolated from the soil of suburban vegetable and forest fields in Beijing (China), which is capable of producing natamycin and has proved to be a potential biocontrol agent to several plant fungal diseases. In order to clone the biosynthetic gene clusters and regulate genes of natamycin, the genomic DNA of Streptomyces lydicus A02 is extracted and partially digested with HindIII and BamHI to increase the yield of natamycin by gene modification, the 97-194kb and 48.5-97kb high molecular weight DNA are extracted and ligated to pCC1BAC. The ligation mixtures were transformed into EPI300 competent cells. A total of 800 and 1500 colonies are obtained by white-blue screening. The analysis of enzyme digestion shows that the average size of the insert fragments are about 133kb and 65kb, and the frequency of clones without inserts is less than 1%. The libraries can cover 12.28 times and 11.25 times of genome if the Streptomyces lydicus A02 contains 8Mb chromosome. Therefore, the probability of screening the target fragment from the libraries is more than 99.99%. This BAC library will be an important resource used in gene cloning, the secondary metabolic pathways and new antibiotics.