In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: pic...
In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue ($1-10{\mu}g$) to $20{\mu}l$ 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at $34^{\circ}C$ for 15 min. Finally, $2{\mu}l$ of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.
In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue ($1-10{\mu}g$) to $20{\mu}l$ 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at $34^{\circ}C$ for 15 min. Finally, $2{\mu}l$ of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.
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제안 방법
fuciformis yeast-like cells (YLCs). Several important parameters such as concentration of enzyme, incubation time, quantity of template, and inactivation of enzyme were tested. The results showed lower concentration Lywallzyme (>0.
The optimal procedure was established: picking up a suitable amount of fungal tissue (approximately 1-10 ig) to 20 il 0.25% Lywallzyme solution, vortexing for 10 s, and then incubation at 34 ℃ for 15 min, and 2 il suspension was used as templates to perform PCR. It was used to screen transformants of T.
대상 데이터
25% Lywallzyme solution, vortexing for 10 s, and then incubation at 34 ℃ for 15 min, and 2 il suspension was used as templates to perform PCR. It was used to screen transformants of T. fuciformis, P. ostreatus, and P. tuberregium. Amplification of the lac3, hph, egfp, or egfphph was achieved successfully in transformants of these mushrooms after enzymolysis treatment (Figure 2).
성능/효과
3125%) failed to obtain the expected products (Figure 1(A)). A reasonable explanation is that PCR amplification was inhibited by higher concentration of Lywallzyme, while lower concentration of Lywallzyme has less inhibitory effect to PCR amplification and was enough to wound the cell wall of YLCs. We also explored whether the incubation time had influence on colony PCR in YLCs of T.
In this study, a rapid and simple colony PCR was developed to screen transformants in several common mushrooms including Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. These strains were preserved in the Laboratory of Food Microbiology, Huazhong Agricultural University.
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