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本文探讨了构巢曲霉(Aspergillus nidulans)、米曲霉(Aspergillus oryzae)和赤霉菌(Gibberella fujikuroi)的原生质体制备条件以及耐药情况,首先以构巢曲霉为受体菌进行了以苯菌灵抗性为选择标记的DNA转化.为在以后的DNA转化中有效地利用抗药标记,分析了构巢曲霉和米曲霉对于抗真菌抗生素潮霉素(Hygromycin B)的天然耐受性.二者尤其是米曲霉,对潮霉素的敏感性非常低,甚至在潮霉素浓度为400μg/mL时,米曲霉仍能较为旺盛地生长;潮霉素浓度为300μg/mL时构巢曲霉也不能完全抑制.假若完全抑制其生长,用药量将很大,鉴于潮霉素价格较为昂贵,故潮霉素抗性作为这两个菌株转化后的筛选标记不太合适.测定了构巢曲霉、米曲霉和赤霉菌对于杀真菌剂苯菌灵的天然耐受性,3个菌种对苯菌灵均十分敏感,最低抑菌浓度(MIC)大约在2.0～3.0μg/mL的范围.因此用苯菌灵抗性作为这3个菌株DNA转化后筛选标记是可取的.由于苯菌灵浓度在2.0～3.0μg/mL范围时上述菌种仍然有十分缓慢的残余生长,筛选转化子时苯菌灵在选择性培养基中的浓度可适当提高.质粒pBT6含有苯菌灵抗性基因[该基因系从脉孢菌(Neurospora crassa)抗性变株中克隆得到的β-tubulin基因],首先用其转化构巢曲霉原生质体.实验中观察到直接把转化后的原生质体涂布到含药?
In this paper, the protoplast preparation conditions and drug resistance of Aspergillus nidulans, Aspergillus oryzae and Gibberella fujikuroi were discussed. Firstly, Aspergillus nidulans Sex-Selectable DNA Transformation For the efficient use of drug-resistant markers in subsequent DNA transformation, the natural tolerance of Aspergillus nidulans and Aspergillus oryzae to the antifungal antibiotic hygromycin B was analyzed both Is Aspergillus oryzae and has a very low sensitivity to hygromycin, and Aspergillus oryzae still grows vigorously even at a hygromycin concentration of 400 μg / mL; Aspergillus nidulans can not grow completely at a hygromycin concentration of 300 μg / mL Inhibition.If the growth is completely inhibited, the dosage will be very large, in view of hygromycin is more expensive, so hygromycin resistance as a screening marker after transformation of these two strains is not appropriate.Aspergillus nidulans, Aspergillus oryzae And Fusarium for the natural tolerance of the fungicide benomyl, the three strains are very sensitive to benomyl, the minimum inhibitory concentration (MIC) is about 2.0 ~ 3.0μg / mL range.Therefore, Ling resistance as DNA transformation of these three strains Screening marker is preferable.Because of the very slow residual growth of benomyl in the range of 2.0 ~ 3.0μg / mL, the concentration of benomyl in the selective medium can be appropriately increased when selecting transformants. Plasmid pBT6 contains the benomyl resistance gene [which is a β-tubulin gene cloned from a Neurospora crassa resistant strain] and was first used to transform A. nidulans protoplasts. Direct experiments were observed Will the transformed protoplast be coated with drug?