Optimization and application of the genetic transformation system based on electroshock strategy for Blakeslea trispora
SHEN Siqiao1, YANG Peilong2, QU Yinbo3, YU Xiaobin1, LUO Wei1*
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China) 2(Key Laboratory of Feed Biotechnology, the Ministry of Agriculture of People's Republic of China, Feed Research Institute Chinese Academy of Agricultural Sciences, Beijing 100081, China) 3(State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China)
Abstract: Electroshock is a rapid and effective transformation method that can be widely used in microbial genetic manipulation. In order to establish an efficient and applicable genetic transformation technique for Blakeslea trispora, this study optimized its protoplast transformation based on the electroshock strategy. The enzymatic digestion time, voltage and electric pulse time, buffer type, nucleic acid concentration and regeneration incubation time for the preparation of protoplasts were first optimized as single factors. Then orthogonal design tests showed that the highest transformation efficiency of 35.1 CFU/μg was achieved at a voltage of 0.4 kV and an electrical pulse time of 2 ms, followed by regeneration incubation for 3 h. Subsequently, taking the expression vector of recombinant gene btwc-1c as an example, it was electrotransformed with a transformation efficiency of approximately 30 CFU/μg. When compared with the wild-type bacteria, the expression level of btwc-1c and the synthesis level of β-carotene in the transformed bacteria by fluorescence PCR and phenotypic analyses increased by 2.5-fold and 2.1-fold, respectively. This indicated that the electrotransformation method established in this study could achieve efficient uptake of exogenous vector by the recipient bacteria and lay a good foundation for the study of the functions of key regulatory genes in B. trispora.
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2022, Vol. 48 
