Agrobacterium tumefaciens-mediated transformation (ATMT) for the screening for genes involved in laccase glucose repression in the pathogenic yeast Cryptococcus neoformans

Abstract:

Abstract:［Objective］To identify genes in glucose repression of laccase in the human pathogen Cryptococcus neoformans．［Methods］We created a random insertional mutagenesis library containing over 200000 transformants by Agrobacterium tumefaciens-mediated transformation (ATMT)．We screened the glucose derepression mutants under high-glucose condition and obtained the genes for glucose repression of laccase via inverse polymerase chains reaction ( PCR ) ．[Results］Totally，we isolted 30 glucose derepression mutants from the library． We found that that 83% of the mutants contain a single T-DNA via Southern blot． We preliminarily identified 10 genes，which fall into a broad range of biological processes including: carbohydrate metabolism，sterol biosynthesis，chitin biosynthesis and glycosylphatidylinositol (GPI) anchor biosynthesis． Additionally，we found that three glucose derepression mutants have a single T-DNA insertion in the promoter region of LAC1，which encodes cryptococcal laccase．［Conclusion］As an effective way，ATMT can be utilized for identifying genes in glucose repression of laccase，which sheds lights on the roles of laccase in virulence and provides information for laccase production in industry．