一个硫链丝菌素抗性基因标记、用于DNA导入链霉菌的pSET152衍生载体

A pSET152 derivative vector marked with thiostrepton resistance gene for introducing DNA into Streptomyces

摘要:

【目的】在链霉菌PCR-targeting系统中, 安普霉素是使用最普遍的选择标记。然而在基因敲除阶段利用安普霉素选择标记后, 在遗传补偿时就不能使用相同选择标记的许多重要载体, 如pSET152。这常给研究带来不便, 特别是当研究对象基因如一些调控基因, 其生理功能对剂量敏感时更是如此。基于此, 拟以pSET152为基础构建一个不以安普霉素为抗性标记的通用整合型载体。【方法】利用融合PCR和λ Red重组等方法构建载体。【结果】来自pHZ1358上的硫链丝菌素抗性基因tsr和来自pCR2.1的氨苄抗性基因bla以“tsr在前bla在后”的次序融合。融合后的抗性片段替换pSET152上的安普霉素抗性基因aac(3)-IV, 从而获得新载体pGIM6626。利用该载体将删除的榴菌素最小聚酮合酶基因重新导入到Streptomyces vietnamensis突变株中, 该突变株恢复了产榴菌素的能力, 证实了该载体的有效性。【结论】构建了一个新的pSET152衍生载体pGIM6626。该载体包含氨苄和硫链丝菌素抗性基因, 分别在大肠杆菌和链霉菌中作为选择标记。pGIM6626与pSET152用途相似, 但前者由于与PCR-targeting系统不存在选择标记的冲突而与该系统更兼容。

Abstract:

[Objective] In the PCR-targeting system of Streptomyces, apramycin is the most widely used selection marker. However, using apramycin as selection marker at disruption stage precludes, during subsequent genetic complementation, the use of some very useful and widely used vectors with the same marker, such as pSET152. This often caused unwanted inconvenience, especially in the situation that the physiological function of interested gene is sensitive to gene dosage, such as regulatory genes. The current study aims to provide an integrative plasmid with selection marker rather than apramycin. [Methods] Fusion-PCR and λ Red recombination were adapted to construct the new vector. [Results] The bla gene from pCR2.1 and the tsr gene from pHZ1358 were fused in the tsr-bla order. This fused fragment replaced the aac(3)-IV gene on pSET152 to generate pGIM6626. This new vector was validated by successfully restoring granaticin production of a granaticin-deficient S. vietnamensis mutant by re-introducing the deleted minimal polyketide synthase genes. [Conclusion] We constructed a new pSET152 derivative vector, pGIM6626, which contains ampicillin and thiostrepton resistance genes for selection in E. coli and Streptomyces, respectively. pGIM6626 and pSET152 have similar uses, but the former is more compatible with the PCR-targeting system because of no conflict of selection marker.