Construction And Analysis Of A Suppression Subtractive Hybridization Library From Mortierella Isabellina High-yield Mutant Strains

Mortierella isabellina of the Mucorales family, is a filamentous fungus of important economicvalue. When grown in a medium containing carbohydrates as a carbon source, cells accumulate fat, andthe oil is rich in polyunsaturated fatty acid.Suppression subtractive hybridization (SSH) is a simpleand rapid method can identify differentially expressed genes. The study was used to construct thecDNA library of producing mutagenesis strains in Mortierella isabellina in order to identify genesinvolved in the high yield mechanism.For subtractive hybridization,cDNA from the producing mutagenesis strains was used asTester,and cDNA from original strains was used as Driver,After two times of subtractive hybridizationand two times of nested PCR,the products of last PCR amplification were inserted into pMD18-Tvectors and be transformed into the E.coli,The positive clones were preliminarily screened byblue-white colony,and precisely identified by PCR.The inserted fragment sizes focused around250and750bp,91%of the colones were white,96clones were verified by PCR,in which78clones were positiveclones,82%of them showed single band.Further identified by reverse dot-blot hybridization.Fifty-six clones that were identified positivefragments were subjected to sequencing,46ESTs (expressed sequence tags) wereidentified.Bioinformatic analysis of these ESTs showed that42have known functions related toprotein transport, signal transduction, energy metabolism, secondary metabolism, and transcriptionalregulation. The cDNA library of producing mutagenesis strains in Mortierella isabellina weresuccessfully constructed by using suppression subtractive hybridization technique,which lay a solidfoundation for screening and cloning new specific differentially expressed genes in them,and will aid inelucidating the high-yield mechanism of Mortierella isabellina.