一种快速提取三孢布拉霉基因组的方法

Rapid Method of Genome Extraction of Blakeslea trispora

摘要:

三孢布拉霉负菌是一种重要的β-胡萝卜素和番茄红素工业生产菌株，由于其基因组提取耗时费力，转化子筛选困难，基因编辑工作严重受阻。作者比较了煮沸法、添加NaOH煮沸法、复合酶液（2 g/dL溶壁酶+3 g/dL纤维素酶+3 g/dL蜗牛酶）酶解法、复合酶液酶解后煮沸法等4种处理方法的效果，发现NaOH为影响三孢布拉霉基因组释放的关键因素；对NaOH浓度、煮沸时间及基因组源的选择（上清液或处理后的菌苔）进行了优化，当NaOH浓度不低于20 mmol/L时，所处理样品的上清液均可扩增出目的基因，煮沸时间对基因组的释放无影响；不同浓度NaOH处理条件下基因组的稳定性实验表明，在所测时间（最长至108 h）内，基因组质量浓度（150~350 ng/μL）与纯度（OD₂₆₀/OD₂₈₀ 1.8~2.0）均较高，且随时间的延长无明显下降趋势；以快速提取法与常规提取法获得的基因组为模板进行PCR，两种方法扩增结果无明显差异，后续对PCR产物的测序与酶切实验证明了NaOH处理不会对后续实验产生影响；最后，从扩增同一菌株不同基因和不同丝状真菌ITS两个方面确定了基因组快速提取方法具有一定的普适性。

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.