Transformation of CryK13V gene into protoplasts of Trichoderma viride
The lysine in site 13 of cryptogein protein was mutated to valine (K13V) through PCR site-directed mutagenesis. The mutant fragment (CryK13V) was confirmed by enzyme digestion and DNA sequencing. The CryK13V gene was expressed in Trichoderma viride, with constructed vector pCSNTCCm. Transformation w...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2006-05-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/1008-9209.2006.03.0270 |
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| Summary: | The lysine in site 13 of cryptogein protein was mutated to valine (K13V) through PCR site-directed mutagenesis. The mutant fragment (CryK13V) was confirmed by enzyme digestion and DNA sequencing. The CryK13V gene was expressed in Trichoderma viride, with constructed vector pCSNTCCm. Transformation was carried out by restriction enzyme Xho Ⅰ mediated integration and transformants were obtained on the CM media contained 200 μg·mL-1 hygromycin B, and the transformation rate was 1-2 transformants per microgramme vector DNA. The optimum of isolation, regeneration of the protoplasts from T. viride was that: pH=6.98 phosphate buffer, 4 mg·mL<sup>-1</sup> glucanex, hypha cultured 24 hours, digested at 30 ℃for 4 hours, and the yields of the protoplasts was 4.7×10<sup>7</sup> per·mg<sup>-1</sup>. On the CM medium containing 0.3 mol·L<sup>-1</sup> KCl and 0.3 mol·L<sup>-1</sup> Inositol, the regeneration rate was 14.5%. |
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| ISSN: | 1008-9209 2097-5155 |