PEG法介导蛹虫草遗传转化体系的建立

Establishment of Genetic Transformation System of Cordyceps militaris using PEG Mediated Method

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 protoplast is usually used as the recipient cell in the genetic transformation system of fungi, various factors in the preparation of Cordyceps militaris protoplast were compared. The 4 day old mycelia treated with the mixture of 1.5% snail enzyme and 1.5% lysozyme in 0. 8 mol/L mannitol at 34℃ for 4h could efficiently produce protoplasts with concentration up to 1.0 ×107/ml. Drug resistance test of H. erinaceus against hygromycin B showed that the lowest selection concentration was 650 mg/L. The plasmid pSB130-GFP was transformed into the protoplast of Cordyceps militaris by the method of orthogonal test. The best transformation system was obtained under the fluorescence inverted microscope at PEG was 25% with freezing time of 10 min and temperature for 20 min. The mass of plasmid was 30 p,g, while the number of protoplasts was 107 cells/ml. The transformation frequency of PEG-mediated transformation of Cordyceps militaris was about 100 × 200/μg （resistant transformant/plasmid ＋ 107 protoplasts）. Transformants on medium with hygromycin after 4 or more passages expressed hygromycin resistance with stable inheritance. The study was directed by genetic engineering, rapid improvent of quality and the use of medicinal Cordyceps fermentation methods for producing of some exogenous protein is of great economic value. Such a foundation, also contribute to learn more about this major Cordyceps fungus gene expression regulation mechanism.