Construction Of Lentinan High-yield Strains Of Lentinus Edodes Using Promoter Swapping

The content of lentinan in mycelia and fruit bodies of Lentinus edodes is very low,which results that the industrial production of lentinan is few,the price of lentinan is high,and lentinan does not satisfy the market's needs.Therefore,it is necessary to breed lentinan high-producing strains by genetic breeding technology.In this study,lentinan high-yield engineered strains of Lentinus edodes were constructed using a promoter swapping.The promoter of the glucan synthase gene gls of Lentinus edodes was replaced by the gpd promoter of Pleurotus ostreatus,the yield of lentinan of the transfertants increased.The results were as follows:(1)The hygromycin resistance gene expression cassette D2(3321 bp)was cloned via PCR from the PAN7-1 plasmid.The gls gene promoter upstream sequence(upstream homology arm)D1(870 bp)and the downstream from the start codon of the gls gene in shiitake mushrooms(downstream homology arm)D4(867 bp)were cloned from the genome of shiitake mushrooms L26 by PCR.The gpd promoter D3(950 bp)was cloned from the genome of Pleurotus ostreatus 831 by PCR.The D1 was fused with D2 by fusion PCR and ligated to PMD19-T vector,generating PMDF19-T plasmid.The D3 was fused with D4 by fusion PCR and ligated into the p EASY-BLunt E1 vector,resulting in the PEASY-BLunt-F plasmid.The fusion segment of D1 and D2 was obtained by digesting PMDF19-T plasmid with ASCI and Pac I.The PEASY-BLunt-F plasmid was linearizated by digesting with Pac I.The D1 and D2 fusion segment was ligated with the linearizated PEASY-BLunt-F,thus generating the Lhg-R plasmid.(2)The constructed homologous recombination fragment was transformed into Lentinus edodes L26 using liposome-mediated protoplast transformation.The protoplast concentration was 8.9×107/ml,and the ratio of DNA fragment to liposome was 1:1.5.The concentration of the DNA fragment was 400 ng/?L.The mixture of protoplasts,liposomes and DNA fragments was scribbled on a hygromycin-containing CYM solid medium and cultivated.After cultivation for 8 days,the better growing hyphae were inoculated on RCM solid medium containing hygromycin.Multiple screenings were performed.Finally,the hyphae that were able to grow on RCM medium containing hygromycin were verified by PCR.The hygromycin resistance gene,the fragment harboring the partial sequence of the gpd promoter and the hygromycin resistance gene,and the fragment harboring the partial sequence of the hygromycin resistance gene and the downstream homologous arm were all amplified.(3)The polysaccharide content,gls gene expression and mycelial biomass of the transformants were assayed.Compared with the original strain,the polysaccharide content of the transformant was 1.12 times higher,the expression of gls gene was doubled higher,and the dry weight of the mycelia was increased by 16.7%.The above results indicated that the homologous recombination fragment comprised four segments were obtained by fusion PCR and digestion-ligation.The homologous recombination fragment was transfored into Lentinus edodes L26 by liposome-mediated transformation to construct the gls gene promoter swap transformants.The lentinan content of the transformants was increased significantly compared to the original strain.