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A Protoplast Transformation System for Gene Deletion and Complementation in Sclerotinia sclerotiorum
Author(s) -
Ge ChangYan,
Duan YaBing,
Zhou MingGuo,
Chen ChangJun
Publication year - 2013
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/jph.12137
Subject(s) - sclerotinia sclerotiorum , complementation , protoplast , biology , transformation (genetics) , mutant , gene , expression vector , selectable marker , microbiology and biotechnology , hypha , mycelium , heterokaryon , biochemistry , botany , recombinant dna
Protoplast transformation is an important technique for establishing a mutation library and determining gene function for S clerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid‐sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60–85 transformants per microgram of DNA . In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of S hk1 for gene complementation based on the S hk1 deletion mutant △ S hk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain △ S hk1+ S hk1. The techniques and procedures described will improve our ability to study gene function in S . sclerotiorum and are likely applicable to other plant pathogens.