Construction Of Homologous Recombination Gene Knockout Vector Of Lai And Transformation By Electroporation Of Lactobacillus Plantarum P-8

CLA is currently considered a healthy nutrition, which has a variety of physiological functions such as anti-oxidation, anti-cancer, lipid-lowering diet, regulating immunity etc. It has been widely used in many fields of feed, food, medicine and people on the CLA’s research has become a hot spot. Many microorganisms contain linoleic acid isomerase, which can catalyze the LA generated CLA. Lactobacillus plantarum and Lactobacillus Lactobacillus delbrueckii have the highest conversion efficiency. Lactobacillus P-8is good fermentation bacteria which derived from the Inner Mongolia Urad Middle Banner, the study showed that the bacteria with lowering blood pressure and improving gastrointestinal microbial community structure and other characteristics, and can efficiently produce CLA. Therefore, the study and transformation of LAI gene structure of the Lactobacillus P-8has important theoretical and practical value.Gene knockout technology is one way to study gene function, which can pinpoint modification and transformation the target gene, and genetically modified fragment can be copied and stable genetic with the genome, has been widely used to study various types of organisms.This paper use the principle of homologous recombination gene knockout technology to construct Lactobacillus P-8lai gene knockout vector, and use the exogenous plasmid to study electroporation conditions of Lactobacillus P-8, laid the foundation for lai knockout. According lai sequence registered in GenBank, multiple pairs of primers were designed and synthesized, after several PCR amplified lai-up and lai-down of two homologous arm fragments and cloned into pMD19-T vector. Kan resistance gene was also amplified and built into the pMD19-T vector. The lai-up, lai-down and kan fragments were cut from the pMD19-T vector by use of restriction endonucleases and successfully built into the pUC18vector, finally obtained the homologous recombination knockout vector were designated pUC18△lai. The study showed that the best electroporation conditions of Lactobacillus P-8with plasmid pMG36e is:cells after treatment with3%glycine culture4h, after2kV voltage electric shock, and the last seeded in the recovery medium containing0.4mol/L sucrose.