基于电击策略的三孢布拉霉遗传转化体系的优化及应用

Optimization and application of the genetic transformation system based on electroshock strategy for Blakeslea trispora

电击转化是一种快速有效的转化方法,可广泛应用于微生物遗传操作。为了建立高效的适用于三孢布拉霉的遗传转化技术,该研究基于电转化策略对其原生质体转化进行了优化。首先对制备原生质体的酶解时间、电压大小与电脉冲时间、缓冲液种类、核酸浓度及再生培养时间等进行了单因素优化。然后通过正交试验分析,结果表明在电压为0.4 kV,电脉冲时间为2 ms条件下进行电击,之后再生培养3 h的转化效率最高,达到35.1 CFU/μg。随后以电转化重组了基因btwc-1c的表达载体为例,其转化效率约为30 CFU/μg。荧光定量PCR分析和表型分析结果表明转化菌中btwc-1 c的表达水平和β-胡萝卜素的合成水平相对野生菌分别提升了2.5倍和2.1倍,该结果表明该研究建立的电转化三孢布拉霉原生质的方法能够高效实现受体菌对外源载体的摄取,为研究三孢布拉霉中关键调控基因的功能奠定了良好的基础。

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 ofβ-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.