Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.030

Abstract

Abstract: The purpose of this work is to optimize the conditions of formatting and transforming the protoplast of Trichoderma atroviride HP35-3,aiming at genetically manipulating the strain for increasing the yield of cellulase. The individual condition for formatting the protoplast of Trichoderma atroviride,i.e.,mycelium age,hydrolysis time of enzyme,constituents and ratios of enzymes,as well as transformation conditions were optimized respectively. Results showed that the combination of 3 mg/mL snailase,3 mg/mL lysing enzyme,and 3 mg/mL lysozyme enzyme was effective in releasing protoplasts from T. atroviride HP35-3 mycelium（at the age of 10 h）after digestion for 2 h,the concentration was over 3.5×10⁷ protoplasts/mL,and the regeneration rate was 61%. The protoplasts were mediated and transformed by PEG,and the transformation rate was 35 transformants/μg DNA when 1×10⁸ protoplasts were employed with 5 μg DNA. In conclusion,establishing efficient formation and transformation of protoplasts may be applied in strain improvement and gene transformation of T. atroviride in biotech industries and laboratories.

Key words: Trichoderma atroviride, filamentous fungi, formation of protoplast, transformation of protoplast

LIN Yan-mei, LI Rui-jie, ZHANG Hui-jie, QIN Xiu-lin, FENG Jia-xun. Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase[J]. Biotechnology Bulletin, 2016, 32(9): 225-231.

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