The invention provides a PEG / CaCl2 mediated
Penicillium citrinum genetic transformation method, and belongs to the technical field of
microbial genetic transformation, and the method comprises the following steps: adding a
target gene segment into
Penicillium citrinum
protoplast suspension, and carrying out an ice bath reaction; the
target gene segment is obtained by taking pJET-hph-02944
plasmid as a template and carrying out PCR (
Polymerase Chain Reaction) amplification by adopting a
hygromycin B resistance
gene primer pair with
gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; adding a PTC (
Positive Temperature Coefficient)
buffer solution into the mixed
system, uniformly mixing and standing; continuously adding a TB3
liquid culture medium containing
ampicillin, and carrying out
recovery culture; uniformly mixing the resuscitated
protoplast with a molten TB3
solid culture medium, pouring a plate, and carrying out dark culture for the first time; then covering a TB3
solid culture medium containing
hygromycin B and
ampicillin, carrying out secondary dark culture to obtain a transformant, and transferring the transformant to a PDA plate containing
hygromycin B for screening. According to the method, CaCl2-PEG is adopted for mediated transformation of
Penicillium citrinum, the
transformation efficiency is higher, and the transformation speed is higher.