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首页> 外文期刊>Russian Journal of Plant Physiology >Transformation of CW-15 mutant cells of Chlamydomonas reinhardtii Dang. with pCTVHyg plasmid
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Transformation of CW-15 mutant cells of Chlamydomonas reinhardtii Dang. with pCTVHyg plasmid

机译：莱茵衣藻CW-15突变细胞的转化。 pCTVHyg质粒

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摘要

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon TO under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10(6) cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10(3) hygromycin-resistant (Hyg(R)) clones per 10(6) Hyg(R) recipient cells. Exogenous DNA integrated in the nuclear genome of C reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using the CW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.
机译：在单细胞绿藻莱茵衣藻中构建了pCTVHyg质粒。通过使用潮霉素磷酸转移酶基因（hpt）作为选择标记，并在早期SV40病毒基因启动子下使用大肠杆菌转座子TO。通过在1kV / cm的电场和2ms的脉冲持续时间内的电穿孔转化无细胞壁的CW-15突变细胞。 10（6）细胞/ ml的悬浮液密度和中等对数生长期是转化的最佳条件，每10（6）Hyg（R）最多产生10（3）潮霉素抗性（Hyg（R））克隆）受体细胞。整合入莱茵衣藻核基因组中的外源DNA在至少8个月的时间内稳定地传给了后代。但是，Hyg R性状表现不稳定。对hpt和赖氏梭菌核基因密码子使用频率的比较分析显着排除了密码子使用偏差是影响外源基因表达的主要因素的可能性。在赖氏梭菌的异源转化的背景下讨论了使用CW-15突变体和所述选择系统的优点。

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