红曲菌9903A转化体系影响因素的研究

Influencing Factors on the Efficiency of Gene Transformation System of the Monascus purpureus 9903A

摘要:

采用PEG介导的原生质体转化方法，研究了将含有潮霉素抗性基因的质粒pMP-Hygro转入红曲菌9903A的影响因素。实验结果表明，原生质体转化最佳条件为：1.0mol/L的山梨醇为渗透压稳定剂；以含有50mmol/L 的Ca²⁺和最终质量分数为40％的PEG4000为转化介质；最佳原生质体浓度和载体DNA用量分别为1×10⁸个/mL、1μg/100μL原生质体；原生质体再生培养基采用不含无机盐的培养基，再生方式采用原生质体液涂布单层再生培基平板法。得到的平均DNA转化率可达160个/μg DNA。本文所研究的PEG介导的原生质体转化方法可以较好的向红曲菌细胞导入外源DNA，并使外源DNA在红曲菌细胞内大量表达。

Abstract:

The protoplasts from Monascus purpureus 9903A were transformed with hygromycin B resistance plasmid pMP-Hygro.The transformation medium contained 50 mmol/L Ca²⁺ and 40％PEG4000 with 1.0mol/L sorbitol an osmotic stabilizer.The protoplasts concentration is1×10⁸ /mL and the quantity of vectors is 1 μg/100μL protoplasts.In this study，the average transformation ratio is 160 colonies/μg DNA.