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[NIES-90 microcystin producing algae strain genome library construction and one isolated gene analysis]
Objective:
To construct a plasmid microcystis genome library for microcystin (MC) pertaining gene family screening or microcystis strain specific gene screening.
Methods:
Extracting genome DNA from MC producing strain NIES-90, then digesting DNA by HindIII and ligating to CIPase treated pUC18/HindIII fragment which later transformed to JM109. After checking the library by Xgal/Antibiotics plate, clones isolation, a few clones were sequenced and specific primers synthesized; finally, PCR was used to testify clone's NIES-90 strain specificity.
Results:
Successfully constructed a NIES-90 genome plasmid library with following functions: a) cloning efficiency more than 95% by comparing the white/blue clones ratio in Xgal Plate; b) 200-700 bp average insert length with 3 x 10(5) independent clones by independent clones analysis; c) 10 times more coverage of original NIES-90 genome; d) strong strain specificity by cross PCR analysis of some clone's sequence in two different microcystis strains compared with FACHB-469.
Conclusion:
We successfully constructed a NIES-90 MC producing microcystis strain's genome plasmid library which could be used for screening MC pertaining gene family because of high strain specificity of some clones.