Angulo-Bejarano Paola Isabel, Sharma Ashutosh, Paredes-López Octavio
Centro de Investigación y de Estudios Avanzados-IPN, Unidad Irapuato, Km 9.6 Libr. Norte Carr. Irapuato-León, Apdo. Postal 629, Irapuato, 36824 Guanajuato, Mexico.
2Tecnologico de Monterrey, School of Engineering and Sciences, Epigmenio González No. 500 Fracc. San Pablo, 76130 Queretaro, Queretaro Mexico.
3 Biotech. 2019 Mar;9(3):98. doi: 10.1007/s13205-019-1627-6. Epub 2019 Feb 20.
In the present study, a novel transformation protocol for was generated by means of particle bombardment. The best conditions obtained were: 900 psi rupture disk pressure, 8 cm microprojectile travel distance, and 4 h of exposition to 0.2 M mannitol. For all experiments, gold particles coated with 1.0 µg/µL of pBI426 plasmid DNA were used. With all these conditions, a 23% of transformation efficiency in terms of regeneration in selection media (100 mg/L kanamycin) was obtained. Interestingly, the presence of both transgenes: and , by means of PCR and RT-PCR assays was detected. The regeneration percentage achieved in terms of stable integration for both genes was 10%. In addition, we also detected adequate amounts of -glucuronidase activity by means of the GUS fluorometric assay. The procedure described in the present investigation reveals the feasibility of using nopal for the introduction, expression, and possible production of heterologous proteins.
在本研究中，通过粒子轰击产生了一种新的 转化方案。获得的最佳条件为：900 psi的破裂盘压力、8 cm的微弹飞行距离以及在0.2 M甘露醇中暴露4小时。对于所有实验，均使用包被有1.0 µg/µL pBI426质粒DNA的金颗粒。在所有这些条件下，在选择培养基（100 mg/L卡那霉素）中再生方面获得了23%的转化效率。有趣的是，通过PCR和RT-PCR分析检测到了两个转基因： 和 的存在。两个基因稳定整合的再生百分比为10%。此外，我们还通过GUS荧光测定法检测到了足够量的β-葡萄糖醛酸酶活性。本研究中描述的方法揭示了使用胭脂树进行异源蛋白导入、表达以及可能生产的可行性。
