Abstract:Blakeslea trispora (-) strain is a very important industrial strain producing β-carotene and lycopene. The genome extraction of Blakeslea trispora (-) is time-consuming and laborious, which brings difficulties to the transformants selection and genome edition. Four different lawn pretreatment methods were compared, boiling, boiling with NaOH, enzymolysis with mixed enzymes(2 g/dL lysozyme, 3 g/dL cellulose and 3 g/dL snailase), boiling after enzymolysis with mixed enzymes. The result showed that NaOH was the most prominent factor in the genome releasing process. The optimization was carried out including the NaOH concentration, the boiling time and the genome source (the supernatant or the pretreated lawn).The results showed that if the concentration of NaOH was not lower than 20 mmol/L, the target gene could be well exemplified with the supernatant as the genome source, and the boiling time did not play an important role in the process of genome releasing. The stability test of genome under different NaOH concentration showed that the genome concentration (150~350 ng/μL) and the purity (OD260/OD280 1.8~2.0) were both high during the whole testing period (up to 108 h), and there was no obvious decline with the time. There was no prominent difference between the PCR results with the genome extracted by the rapid method and the traditional method as the template. The subsequent sequencing and restriction enzyme digestion of PCR products showed that NaOH treatment had no effect on the following experiments. Finally, it was confirmed that the rapid genomic extraction method had certain universality from two aspects of amplifying different genes and different filamentous fungi ITS of the same strain.
