纤维素降解球毛壳菌NK-102的高效遗传转化方法

Two optimized transformation protocols for a cellulose-utilizing fungus Chaetomium globosum NK-102

【目的】在球毛壳菌(Chaetomium globosum)NK-102中,建立菌株特异性转化体系。【方法】构建新的抗性标记pUCATPH-Pgap,转化效率优于pUCATPH和pCM768。建立了PEG-原生质体和根癌农杆菌(Agrobacterium tumefaciens)EHA105介导的两种转化方法。【结果】原生质体转化效率为30-50个转化子/10μg DNA,抗性标记pUCATPH-Pgap效率最高。EHA105介导转化率达到3.2×102转化子/107孢子。Southern blot证明DNA随机整合于基因组中,转化子抗性稳定。【结论】两种转化体系的建立为筛选高效利用纤维素的球毛壳菌突变株奠定了基础。

[Objective] We developed the transformation methods of the strain Chaetomium globosum NK-102.[Methods] We constructed plasmid pUCATPH-Pgap and compared the transformation efficiency with pUCATPH and pCM768.We established the PEG mediated protoplast transformation and Agrobacterium tumefaciens EHA105 mediated transformation methods.[Results] In protoplast approach,approximately 3-5 transformants/μg DNA could be obtained.The highest efficiency of transformation was obtained by employing pUCATPH-Pgap.A.tumefaciens EHA105 successfully mediated T-DNA insertion into the genome of C.globosum NK-102 and the transformation rate was 3.2×102 transformants /107 spores.The transformants retained stable after generations.Southern blot analyses confirmed that the DNA had integrated into the chromosomal DNA of C.globosum NK-102.[Conclusion] The transformation systems were good basis for selection of C.globosum mutant strains that effectively utilizing cellulose.