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目的优化原生质体介导的马尔尼菲青霉转化体系,为其基因功能研究提供良好的平台。方法通过原生质体介导将构巢曲霉(Aspergillus nidulans)pyrG基因插入马尔尼菲青霉尿嘧啶缺陷株ligD(pyrG-,ligD-)中,在不含尿嘧啶的培养基中筛选阳性转化子,运用PCR验证重组子,通过改变影响转化效率的酶解时间、培养基浓度、质粒浓度、不同配方的原生质体洗涤液STC和不同配方的原生质体助融剂PTC 5个条件对体系进行优化。结果 PCR验证A.nidulans pyrG基因成功的插入ligD中,转化子可稳定传代。最适合ligD原生质体转化效率的条件为:酶解时间6h,PTC(60%PEG-4000,100mmol/L Tris-HCl pH8.0,100mmol/L CaCl2),每100μL原生质体(106~107/mL)加入2.5μg质粒,0.6 mol/L蔗糖SD/SDU筛选/再生培养基,每1μg质粒转化子可达68个左右。结论成功优化了原生质体介导马尔尼菲青霉转化体系的条件,优化后该方法转化效率高,为基因功能研究提供良好平台。
Objective To optimize protoplast-mediated Penicillium marneffei transformation system and provide a good platform for the study of its gene function. Methods The pyrG gene of Aspergillus nidulans was inserted into the liga (superscript -) ligD- of uracil-deficiency strain Penicillium marneffei through protoplast-mediated transformation. The positive transformants were screened in uracil- PCR was used to validate the recombinants and the system was optimized by changing the enzymolysis time, medium concentration, plasmid concentration, protoplast washing solution STC with different formulations and PTC protoplasts with different formulations. Results PCR verified that the A.nidulans pyrG gene was successfully inserted into ligD and the transformants could be stably passaged. The most suitable conditions for the transformation efficiency of ligD protoplasts were as follows: enzymatic hydrolysis time 6h, PTC (60% PEG-4000, 100mmol / L Tris-HCl pH8.0, 100mmol / L CaCl2) per 100μL protoplasts (106 ~ 107 / mL) Add 2.5μg plasmid, 0.6mol / L sucrose SD / SDU screening / regeneration medium, per 1μg plasmid transformants up to about 68. Conclusion The conditions of protoplast-mediated transformation of Penicillium marneffei were successfully optimized. The optimized transformation efficiency of this method provided a good platform for the study of gene function.