| 来欢颜,高海,杜荣添,等.耐酸真菌OUCMDZ-4754遗传体系的构建[J].中国海洋药物,2025,(5):-. |
| 耐酸真菌OUCMDZ-4754遗传体系的构建 |
| Construction of The Genetic System of Acid-tolerant Fungus OUCMDZ-4754 |
| 投稿时间：2024-05-15 修订日期：2024-07-14 |
| DOI： |
| 中文关键词: 耐酸真菌 遗传体系 PEG-介导原生质体转化 |
| English Keywords:Acid-tolerant fungi Genetic system PEG-mediated protoplast transformation |
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| 中文摘要: |
| 目的 构建耐酸真菌OUCMDZ-4754的遗传体系,为进一步挖掘其生物资源提供分子生物学的研究基础。方法 制备孢子悬液摸索OUCMDZ-4754的生孢条件以及酶解制备原生质体的条件；通过计算原生质体的再生率以及观察再生状态确定再生培养基的种类；设置不同抗生素的浓度梯度以确定OUCMDZ-4754敏感的抗生素类型及浓度；根据实验结果,利用PEG-CaCl2介导原生质体转化的方式导入对应的抗性片段构建其抗性筛选标记。结果 确定了OUCMDZ-4754的生孢条件为真菌0号培养基,28℃,280 rpm培养9-12 h,用Yatalase酶解2 h后将萌发的孢子酶解制成原生质体,将潮霉素B抗性片段hph导入原生质体后,4754能在潮霉素浓度为100 μg/mL的高渗PDA培养基上生长,成功在4754中构建了潮霉素抗性筛选标记。结论 通过确认生孢条件、酶解条件、再生培养基以及敏感抗生素的种类和浓度,最终构建了OUCMDZ-4754的遗传体系,这有助于进一步对其生物资源进行开发和利用。 |
| English Summary: |
| Objective The genetic transformation system of acid-resistant fungus OUCMDZ-4754 was constructed to provide molecular biology research basis for further exploring its biological resources. Method The sporulation conditions of OUCMDZ-4754 were studied by spore suspension and the conditions of protoplast preparation by enzymatic hydrolysis. The regeneration medium type was determined by calculating the regeneration rate of protoplasts and observing the regeneration state. The concentration gradient of different antibiotics was set to determine the type and concentration of antibiotics to which OUCMDZ-4754 was sensitive. According to the experimental results, PEG-mediated protoplast transformation was used to import corresponding resistant fragments to construct resistance screening markers. Result It was determined that the sporulation condition of OUCMDZ-4754 was cultured in fungus medium No. 0 at 28℃ and 280 rpm for 9-12 h. After enzymolysis with Yatalase for 2 h, the spore germinated was enzymolized into protoplast. After introducing hygromycin resistant fragment hph into protoplasts, 4754 could grow on hypertonic PDA medium with hygromycin concentration of 100 μg/mL, and the hygromycin resistance screening marker was successfully constructed in 4754. Conclusion The genetic system of OUCMDZ-4754 was established by confirming the conditions of sporulation, enzymatic hydrolysis, regeneration medium and the types and concentrations of sensitive antibiotics, which is helpful for further development and utilization of its biological resources. |
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