Construction And Analysis Of BAC Libraries Of Streptomyces Avermitilis And Paralichthys Olivaceus Genomes

Compared with other large DNA-insert artificial chromosomes, BAC (bacterial artificial chromosome) has more advantages, such as larger insert size, lower recombinant rate and higher transformation efficiency. Thus it is widely used in genome-sequencing, map-based cloning and comparative genomics analysis. In this study, two BAC libraries of Streptomyces avermitilis and Paralichthys olivaceus are constructed, using bacterial protoplast and fish blood.As a low toxic and high performance antibiotic, avermectin, which is a secondary metabolite of Streptomyces avermitilis, plays an important role in agriculture and medicine. To further clarify the biosynthesis and regulation pathways of avermectin and to increase its production, a bacterial artificial chromosome library has been constructed for Streptomyces avermitilis. The Streptomyces avermitilis BAC library consisted of 2,304 clones with an average insert size of 101 Kb, covering 25.9-fold of the Streptomyces avermitilis’s genome. The phosphorothioation feature of the Streptomyces avermitilis’s genome made the DNA very easy to be degraded during electrophoresis. The degradation of DNA can be relieved by adding thiourea into electrophoresis buffer. The successful construction of this BAC library provides a reference for the construction of BAC libraries of other microorganisms, especially those with DNA phosphorothioation.Paralichthys olivaceus is an economically important marine fish that has been widely cultured in China. However, the cultivation of it has suffered from disease outbreaks and germplasm degradation in recent years. A BAC library has been constructed for the genome of Paralichthys olivaceus, whose whole genome sequencing is under the way. The Paralichthys olivaceus BAC library consisted of 94,464 clones and was stored in 246 blocks of 384 well-plates, with an average insert size of 134 Kb and empty clone rate lower than 1%, covering 18-fold of the Paralichthys olivaceus genome. The BAC library will be used as a platform for whole-genome sequencing, gene cloning and genetic breeding of Paralichthys olivaceus.