中文摘要：

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。

英文摘要：

Objective This study aims to optimize the protoplast-mediated transformation system of Penicillium marnef- fei ,it would provide a good platform for the gene function research of P.marneffei. Methods We inserted the selectable marker pyrG gene （from A.nidulans） into P.marneffei strain ligD （pyrG-,ligD-） by protoplast method, screened positive transformant by medium without uracil,and used PCR to verify the restructuring.Through changing some important effecting factors including enzymolysis period, concentration of plasmid, protoplast fusion agents PTC and screening culture medium to find out the optimal protocol.Results As a result,The positive transformants growed well in the media without uracil,and PCR analysis showed that pyrG was inserted into the transformants, the appropriate conditions for protoplast-mediated transformation of P.marneffei were as follows ： enzymatic time is 6 h ; PTC ： 60 %PEG-4000,100 mmol/L Tris- HC1 pH8.0, 100 mmol/L CaC12 -0.6 mol/L sucrose screening culture medium,add 2.5 peg plasmid per 100μ L protoplast （10^6 -10^7/mL）. The transformation efficiency was about 68 transformants/txg plasmid DNA under these conditions.Conclusions Protoplasts- mediated transformation in P.rnarneffei was successfully optimized, this method could get high transformation efficiency and provide a good platform for P.marneffei gene function research.