PEG介导的广东虫草原生质体遗传转化体系建立及其应用

Establishment and Application of PEG-Mediated Protoplast Genetic Transformation System of Cordyceps guangdongensis

【目的】广东虫草是我国特有珍稀食药用菌,其营养成分丰富、活性功效显著,已获批新资源食品,产业化应用前景广阔。建立广东虫草原生质体制备及遗传转化方法,可为广东虫草新品种选育及基因功能研究提供参考依据。【方法】以广东虫草菌丝体为材料,采用单因素分析方法对原生质体制备过程中酶解组合、酶解时间、酶解温度、复苏培养基及抗性标记进行筛选。同时以潮霉素抗性标记质粒pCAMBIA1300为转化片段,采用单因素分析方法对PEG介导的原生质体转化过程中亚精胺浓度、PEG加入间隔时间、抗生素添加时间进行筛选。并以广东虫草中的锌指半胱氨酸转录因子CgPro1敲除片段为目的转化片段,对转化系统进行验证。【结果】广东虫草原生质体制备的最佳组合条件为:采用含有裂解酶+崩溃酶的混合酶体系(1∶1)对广东虫草的菌丝体进行酶解,且酶解温度28℃、酶解时间为5.5 h时原生质体得率最高;原生质体复苏过程中,TB3复苏培养基对广东虫草原生质体的复苏效果最好、其次为0.6mol/L KCl-PDA;PEG介导原生质体转化过程中,加入5 mmol/L亚精胺、PEG加入间隔时间10 min、原生质体复苏3 d后添加250μg/mL潮霉素抗性标记进行筛选,转化效率最高。此外,通过建立的转化系统获得3个CgPro1敲除突变株及3个杂合子,表型分析结果显示,该基因参与广东虫草子实体发育过程。【结论】建立了成熟的广东虫草原生质体制备及PEG介导的遗传转化体系,可为今后广东虫草基因功能、蛋白定位等遗传操作研究奠定良好基础,同时为广东虫草遗传育种提供重要应用参考。

【Objective】Cordyceps guangdongensis,a rare edible-medicinal fungus in China,has been approved as a new resource food,with a broad prospect of industrial application.The fruitingbodies of this fungus are rich in nutrients and have significant active effect.The protoplast preparation and genetic transformation methods of Cordyceps guangdongensis were established in order to provide references for new variety breeding and gene function research of this fungus.【Method】Protoplasts were separated from the mycelia of C.guangdongensis by various enzymes.During the process of protoplasts preparation,the enzyme combinations,enzyme digestion time and temperature,regeneration medium of protoplasts and resistance marker were selected by using single-factor analyses.Plasmid pCAMBIA1300 was used as transformation fragment to establish a genetic transformation system,and the concentration of spermidine,PEG addition interval and resistance marker addition time during PEG-mediated protoplast transformation were also screened by single-factor analyses.The knockout fragment of Zn2Cys6-type transcription factor CgPRo1 in C.guangdongensis was used as transformation fragment to verify the transformation system.【Result】The optimal condition for protoplast preparation was mycelium treated with lytic enzyme and driselase(1:1)for 5.5 h at 28℃.The highest protoplast regeneration rate of C.guangdongensis was observed on TB3 medium,followed by 0.6 mol/L KCl-PDA.During the transformation process,adding 5 mmol/L spermidine,PEG at an interval of 10 min and screening medium with 250μg/mL hygromycin after protoplast regeneration for 3 days showed the optimal transformation efficiency.In addition,three CgPro1 knockout mutants and three heterozygotes were obtained through the established transformation system.Phenotypic analysis results showed that CgPro1 was involved in the fruit body development of C.guangdongensis.【Conclusion】In the study,the optimized protoplast preparation method and PEG-mediated protoplast transformation system for C.guangdongensis were established.The techniques and procedures described will lay a solid foundation for future researches on gene function and protein localization,and also provide important application value in genetic breeding of in C.guangdongensis.