Tn5 transposase-assisted high-efficiency transformation of filamentous fungus Phoma herbarum YS4108.
Conventionally, filamentous fungi are transformed by using conidia or protoplasts as recipients. However, induction of sporulation is difficult in some fungi, and protoplasting is an awing, frequently frustrating, and batch-dependent work. In this study, we established a simple and convenient method to prepare single cells from mycelia without enzymatic protoplasting. As a case study on the pathogenic fungus Phoma herbarum YS4108, the single cells could be directly and highly efficiently transformed with the aid of Tn5 transposase. The optimal electric pulse delivery parameters were 25 microF in capacitance, 0.75 kV (0.2-cm cuvette) in voltage, and 400 Omega in resistance, under which the efficiency of transposase-assisted transformation (TNAT) was enhanced to two to threefold compared to that of non-TNAT method, resulting in >230 transformants/cuvette (10(6) recipients). Further cell wall weakening of the single cells by lytic enzymes and linearization of the plasmid were found to have no effects on transformation efficiency, but vector linearization apparently lowered the background growth. The present study for the first time explained that Tn5 transposase could be used to increase transformation efficiency in filamentous fungi, and the method presented here may be of wide applicability in different studies and may be the first choice when transformation efficiency and convenience are priorities and mycelia have to be used as transformation recipients.
Feng
Mingqian
M
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Medical School, Nanjing University, Nanjing, People's Republic of China.
Zhou
Tian
T
Wei
Wenjie
W
Song
Yongchun
Y
Tan
Renxiang
R
D001203:Ascomycota / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018274:Electroporation / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D013172:Spores, Fungal / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C473503:Tn5 transposase; D019895:Transposases