Song Guannan, Wang Wan, Hu Lina, Liu Yu, Li Aifang, Du Jingxia, Wang Jiao, Jia Mengyuan, Feng Shuying
Medical College, Henan University of Science and Technology, No. 263 Kaiyuan Avenue, Luoyang, 471023, Henan, China.
Medical Research Center, Henan University of Science and Technology, Luoyang, 471023, Henan, China.
AMB Express. 2019 Nov 9;9(1):181. doi: 10.1186/s13568-019-0905-3.
As a new expression system, Dunaliella salina (D. salina) has bright prospects and applications in various fields. However, its application is currently restricted because of the low expression and instability of foreign gene in D. salina cells. During genetic operation, transformation is a crucial step for genes expression in D. salina system. Although several transformation methods are existing currently, many inherent deficiencies and limitations still can be found in actual practice. Thus, we attempted to set up a rapid transformation method using the change of salt concentrations for D. salina. Based on osmotic pressure difference, exogenous genes can be spontaneously transferred into D. salina cells. After that, transformed D. salina cells were subjected to histochemical and molecular analysis. The results showed that the reporter gene, beta-glucuronidase genes were successfully expressed in the positive transformants, and detected in all of transformed cells by PCR analysis. Moreover, different transformation parameters, containing the salt gradient, time, dye dosage and Triton X-100 concentration, were optimized to obtain an optimal transformation result. Taken together, we preliminarily established a rapid transformation method with the features of fast, simple, economic, and high-efficient. This method will provide a strong genetic manipulation tool for the future transformation of D. salina system.
作为一种新的表达系统，盐生杜氏藻在各个领域都具有广阔的前景和应用价值。然而，由于外源基因在盐生杜氏藻细胞中的低表达和不稳定性，其应用目前受到限制。在基因操作过程中，转化是盐生杜氏藻系统中基因表达的关键步骤。虽然目前存在几种转化方法，但在实际操作中仍可发现许多固有的缺陷和局限性。因此，我们尝试建立一种利用盐浓度变化对盐生杜氏藻进行快速转化的方法。基于渗透压差异，外源基因可以自发地转入盐生杜氏藻细胞。之后，对转化后的盐生杜氏藻细胞进行组织化学和分子分析。结果表明，报告基因β-葡萄糖醛酸酶基因在阳性转化体中成功表达，并通过PCR分析在所有转化细胞中检测到。此外，对不同的转化参数，包括盐梯度、时间、染料用量和Triton X-100浓度进行了优化，以获得最佳的转化结果。综上所述，我们初步建立了一种具有快速、简便、经济、高效特点的快速转化方法。该方法将为未来盐生杜氏藻系统的转化提供强有力的基因操作工具。
