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由于真菌的遗传转化体系中一般以原生质体作为受体细胞,因而对蛹虫草原生质体制备的各种因素进行比较研究。结果表明,蛹虫草原生质体制备的最佳体系为:液体培养4天的蛹虫草菌球,以0.8 mol/L甘露醇作为稳渗剂,加入含1.5%蜗牛酶+1.5%溶壁酶复合酶,在34℃酶解蛹虫草菌球4 h时,原生质体得率达到1.0×10~7个/ml。潮霉素筛选压实验表明,蛹虫草原生质体在PDA固体培养基上的潮霉素最低筛选浓度为650 mg/L。采用正交试验的方法,设计PEG介导蛹虫草原生质体转化,将质粒p SB130-GFP转化蛹虫草原生质体,在荧光倒置显微镜下观察比较,得到最佳的转化体系为:PEG浓度为25%,冰浴时间为10 min,室温时间为20 min,质粒质量为30μg,原生质体个数为10~7个/ml。最终得到PEG法介导蛹虫草遗传转化的转化频率约为100~200个/μg(抗性转化子/质粒+10~7个原生质体)。转化子在含潮霉素的培养基上经4代以上的继代培养后仍可以表达潮霉素抗性并稳定遗传。为通过基因工程手段定向、快速改良蛹虫草药用品质,利用蛹虫草发酵方法生产一些具有重大经济价值的外源蛋白等奠定基础,并且有助于进一步了解蛹虫草这一大型真菌中基因的表达调控机制。
As the fungal genetic transformation system generally takes protoplasts as acceptor cells, and thus the preparation of protoplasts of Cordyceps militaris compared various factors. The results showed that the optimum conditions for the preparation of protoplasts of Cordyceps militaris were as follows: Liquid culture of Cordyceps militaris ball for 4 days, 0.8 mol / L mannitol as stabilizing agent, adding 1.5% snail enzyme + 1.5% lywallzyme complex enzyme The protoplast yield reached 1.0 × 10 ~ 7 / ml at 34 ℃ for 4 h. Hygromycin Screening pressure test showed that the minimum selection concentration of hygromycin on Cordyceps militaris protoplasts was 650 mg / L on PDA solid medium. The orthogonal design was used to design the PEG-mediated protoplast transformation of Cordyceps militaris. The plasmid p SB130-GFP was transformed into protoplasts of Cordyceps militaris, and the best transformation system was obtained under the fluorescence inverted microscope. The optimum conditions were as follows: the concentration of PEG was 25% , The ice bath time was 10 min, the room temperature time was 20 min, the plasmid mass was 30 μg, and the number of protoplasts was 10-7 / ml. Finally, the transformation frequency of PEG-mediated genetic transformation of Cordyceps militaris was about 100-200 μg / μg (resistant transformant / plasmid + 10-7 protoplasts). Transformants can express hygromycin resistance and inherit stably after more than 4 generations of subculture on hygromycin-containing medium. In order to further improve the quality of Cordyceps militaris by genetic engineering, lay a foundation for producing some exogenous proteins of great economic value by using Cordyceps militaris fermentation method, and help to further understand the gene expression in Cordyceps militaris, a large fungus Regulatory mechanism.