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[通过同源重组建立裂殖壶菌属的遗传转化系统]

[Establishment of genetic transformation system of Schizochytrium sp. by homologous recombination].

作者信息

Zhuang Xiaoyan, Chen Shenglan, Ji Xiaojun, Xu Xian, Ren Lujing

出版信息

Wei Sheng Wu Xue Bao. 2015 Apr 4;55(4):510-7.

Abstract

OBJECTIVE

Schizochytrium sp. is a marine fungus that can produce DHA efficiently. Genetic engineering has been successfully used in industrial strain improvement and metabolic studies. In order to use genetic engineering to modified Schizochytrium sp., we established an genetic transformation system of Schizochytrium sp.

METHODS

A genetic transformation system of Schizochytrium sp. was established by 18S rDNA-targeted homologous recombination. The targeting vector contained a part of 18S rDNA from Schizochytrium sp. and the ble gene. This targeting vector was transformed into Schizochytrium sp. by electroporation and then selected by Zeocin-containing plates. The incorporation of exogenous ble gene into the genome of Schizochytrium was inspected by PCR amplification.

RESULTS

Fermentation results show that the transformants had similar cell dry weight, lipid yield, DHA content, and composition of other fatty acids to the wild type strain.

CONCLUSION

Our results show that the introduction of resistance gene did not affect the cell growth and lipid metabolism. This system could be used to introduce new functional genes into Schizochytrium sp.

摘要

目的

裂殖壶菌是一种能够高效产生二十二碳六烯酸(DHA)的海洋真菌。基因工程已成功应用于工业菌株改良和代谢研究。为了利用基因工程改造裂殖壶菌,我们建立了裂殖壶菌的遗传转化系统。

方法

通过靶向18S核糖体DNA(rDNA)的同源重组建立裂殖壶菌的遗传转化系统。靶向载体包含来自裂殖壶菌的部分18S rDNA和博来霉素抗性基因(ble基因)。通过电穿孔将该靶向载体转化到裂殖壶菌中,然后用含博来霉素的平板进行筛选。通过聚合酶链反应(PCR)扩增检测外源ble基因是否整合到裂殖壶菌的基因组中。

结果

发酵结果表明,转化子的细胞干重、脂质产量、DHA含量以及其他脂肪酸组成与野生型菌株相似。

结论

我们的结果表明,抗性基因的导入并未影响细胞生长和脂质代谢。该系统可用于将新的功能基因导入裂殖壶菌。

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