Effect of trpl+ Harboring Vectors on Transformation Efficiencies in Coprinopsis cinerea
Genetic transformation of the basidiomycete Coprinopsis cinerea has first been described by Binninger et al.in 1987.For the transfer of genetic material,chromosomal integrative vectors are used,which contain a selectable marker gene and/or a gene of interest.During transformation the genetic material integrates at random sites into the host chromosomes.Binninger et al.created the vector pCcl001,by cloning a 6.5 kb PstI genomic fragment,with the tryptophan synthetase gene (trpl+) of C.cinerea,into the ColE1 vector pUC9.The inserted gene allows the complementation of trpl auxotrophies and can be used as a selection marker.Several transformations experiments using this vector revealed a surprising phenomenon:Single-transformation with solely pCc 1001 gives only low numbers of transformants,whereas co-transformation with an additional plasmid yields about 2x more transformants.To explore this phenomenon,first the existing pUC9 replicon was replaced by a modified ColE 1 replicon.All single-transformations resulted in the same observations,indicating that the E.coii vector part is likely not a reason for the effect.An alternative selection marker of C.cinerea (pabl+ for p-aminobenzoic acid production) subcloned in vector pPABl-2 was tested in co-transformation experiments with pCcl001.Different from the trpl+ vector pCcl001,vector pPAB1-2 with the pabl+ gene gave comparable amounts of transformants,regardless of whether used in single-or in co-transformation.The obtained results lead to the conclusion that tryptophan feedback inhibition might be responsible for the reduced transformation efficiencies in single-transformations of trpl+vectors.
Coprinopsis cinerea transformation tryptophan selection gene
B.D(ǒ)RNTE U.K(ü)ES
Büsgen-Institute, Division of Molecular Wood Biotechnology and Technical Mycology, University of Grttingen,Btsgenweg 2, 37077 G(ǒ)ttingen, Germany
国际会议
The 7th International Medicinal Mushroom Congerence(第七届国际药用菌大会(IMMC7)
北京
英文
328-333
2013-08-26（万方平台首次上网日期，不代表论文的发表时间）