基于同源重组的裂殖壶菌遗传转化体系的构建及验证

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

摘要:

摘要:【目的】裂殖壶菌是一种能高效生产DHA的海洋真菌;基因工程技术已经成功应用在微生物改造和代谢机理研究中，利用基因工程技术对裂殖壶菌进行改造首先需要构建适合裂殖壶菌的遗传转化体系;【方法】本文利用电转化的方法将含有18S rDNA同源重组片段的ble基因导入裂殖壶菌中，通过zeocin抗性平板筛选出阳性菌株，并设计ble基因引物，以裂殖壶菌基因组为模板，进行PCR验证ble基因是否成功结合到裂殖壶菌染色体上。【结果】筛选获得的抗性菌株基因组上确实PCR出ble基因片段，对改造菌株与原始菌株进行发酵培养，发现改造后菌株在生物量、油脂含量、DHA含量及脂肪酸分布等方面和原始菌株基本一致。【结论】抗性基因的插入不会影响菌株的正常代谢，该体系的构建为后续其他外源基因导入奠定基础。

Abstract:

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．．