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| Establishment of Genetic Transformation System of Cordyceps militaris using PEG Mediated Method |
| SUN Dan1,2, ZHANG Min1,2, XIE Chang-rui1,2, GUO Xiao-wei1,2, XU He-han1,2, GAO Hong-tao1,2, LI Xiao-wei1,2, SUN Tian-xu1,2, LI Hai-yan1,2 |
1 College of Life Science, Jilin Agricultural University, Ministry of Education, Changchun 130118, China;
2 Engineering Research Center of Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun 130118, China |
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Abstract 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 μg, 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.
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Received: 22 November 2016
Published: 25 April 2017
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Cite this article:
SUN Dan, ZHANG Min, XIE Chang-rui, GUO Xiao-wei, XU He-han, GAO Hong-tao, LI Xiao-wei, SUN Tian-xu, LI Hai-yan. Establishment of Genetic Transformation System of Cordyceps militaris using PEG Mediated Method. China Biotechnology, 2017, 37(4): 76-82.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170410 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I4/76
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