Xiao Luyao, Shi Tingting, Wang Suying, Zhao Qingyao, Li Wei
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Mar 25;39(3):1217-1231. doi: 10.13345/j.cjb.220663.
The construction of efficient and stable expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from ZY-1 and subjected to functional analysis. The shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene from pNZ5319 and the replicon from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter P of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into , and the transformation efficiency of pLPZ4N (5.23×10-8.93×10 CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into . S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E- constructed with P as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of strains.
构建高效稳定的表达载体对于菌株改良和定制菌株的开发至关重要。在本研究中，从ZY-1中分离出四个内源质粒并进行功能分析。穿梭载体pLPZ3N和pLPZ4N是通过将来自pLPZ3或pLPZ4的复制子、来自pNZ5319的氯霉素乙酰转移酶基因和来自pUC19的复制子组合而构建的。此外，还获得了带有乳酸脱氢酶启动子P和mCherry红色荧光蛋白作为报告基因的表达载体pLPZ3E和pLPZ4E。pLPZ3和pLPZ4的大小分别为6289 bp和5087 bp，其GC含量分别为40.94%和39.51%，相似。两种穿梭载体均成功转化入……，pLPZ4N的转化效率（5.23×10 - 8.93×10 CFU/μg）略高于pLPZ3N。此外，将表达质粒pLPZ3E和pLPZ4E转化入……后，mCherry荧光蛋白成功表达。以P为启动子构建的质粒pLPZ4E-获得的重组菌株的β-半乳糖苷酶活性高于野生型菌株。穿梭载体和表达载体的构建为……菌株的基因工程提供了新的分子工具。
