| The lactic acid bacteria（LAB）are closely associated with human as the major probiotic bacteria in human and animal intestine. The long-term, application of LAB has proved their safety in food production, industrial fermentation, micro ecological preparation, hence Lactococcus and Lactobacillu are considered to be GRAS（generally regarded as safe）organisms. LAB exert a positive function on immunity in human and animal mucosa of the gastrointestinal tract, therefor LAB are the most suitabcandidate carrier strains in immune adjuvant and antibody production. With the wide application of LAB in food industry, health care products and pharmaceutical industry, the studies of LAB molecular biology have become deeply. The use of modern molecular biology techniques, construction and application of LAB food grade expression vector system have very important significance for the development of LAB in applied potential and dairy industry, and become a forefront research hotspot in the field of LAB. The wildly distributed Lactococcus lactis are generally regarded as safe Gram-positive bacteria and have an important physiological function in human and animal digestive tract, as exogenous gene safety good expression host bacteria have shown great potential for development.Integration plasmid is often used in constructing chromosomal mutations, and it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate to the host genome without introducing any selectabmarkers or residual bases, and the recombination often happens in non-coding region. Temperature sensitive plasmids can replicate normally within a certain temperature range, while decreased or stopped replication when the temperature is higher than a threshold. Counterselectabmarkers are selectabgenes that can lead to death of strains containing such genes while survival of strains without such genes, thus to achieve the purpose of selection without introducing resistance genes. The upp gene encodes a uracil phosphoribosyl transferace, convert 5-fluorouracil to 5-fluoro-d UMP, lead to cell death. So with this feature upp gene could be used as a counterselectabmarker.This study used the temperature-sensitive p WV01 replicon to construct chloromycetinresistant plasmids p GBWVHC32-upp containing the upp cassette as a counterselective marker for L. lactis MG1363. It suggest that the p GBWVHC32-upp had a shut-off temperature of 40 °C through determination of vector temperature sensitiveness. Therefor 39 °C was the screening of temperature. Based on the p GBWVHC-32 upp as integration vector, to construct 4 gene targeting integration plasmids: p GBWVHC32upp-1L1 R, p GBWVHC32upp-1L2 R, p GBWVHC32upp-2L1 R and p GBWVHC32upp-2L2 R for L. lactis MG1363.According to the screening method of temperature sensitivity test for LAB gene targeting integration system, the integration vectors（4 in total）were respectively electroporated into L. lactis MG1363, and then cultured overnight. Single colonies were picked and cultured in GM17 medium inoculation（1%）by the screening method, then were cultured on a 5-FU containing SDM plate（32 °C, 1–2 d）after 1: 1000–10000 dilution. Single colonies on the 5-FU plate were picked and cultured, and their DNA was isolated for PCR identification, thus obtained upp gene deletion strains Δupp L. lactis MG1363 mutant strain.The results show that Δupp L. lactis MG1363 mutant strain was obtained by gene targeting integration system. Genomic DNA was isolated for PCR identification and sequencing, the target site gene was deleted. The experimental results show that The Δupp mutant and wild type strains showed similar genetic stability after several passages, and the growth curves of the wild type L. lactis MG1363 strain and the Δupp L. lactis MG1363 strain were similar. Analysis of carbon source utilization by testing metabolism of different types of carbon sources, and the results were compared with a database and analysed by software, except for the 5-FU resistance, no significant differences in physiological metabolism were detected between the upp deletion mutant and the wild type strains. Scanning electron microscopy showed that no significant morphological differences were detected between L. lactis MG1363 and △upp L. lactis MG1363 strains in early log phase, late log phase and stationary phase, indicating that upp deletion would not result in morphological changes in L. lactis.In order to prove Δupp L. lactis MG1363 have no altered activity of biochemical pathways and potential harm to the health in food, RNA samples of Δupp L. lactis MG1363 and wild strain were sequenced by RNA-Seq technology. The strains total RNA was extracted, the transcriptomes was sequenced using Hiseq2000, and differential expression gene analysis show there were 6 upregulated genes, and 10 downregulated genes in the contrasts of Δupp L. lactis MG1363 versus wild strain. In the Δupp L. lactis MG1363, upp expression gene as a downregulated genes conform to Δupp strain phenotype. In addition, 11mg0201 was only metabolism related protease gene. Results indicated that, no nottablely changes in biochemical pathways were detected in the upp deletion mutant, so prompted that deletion of upp did not cause cell metabolism toxicity, Δupp L. lactis MG1363 have relatively high food safety.The recombinant Lactococcus lactis were unable to grow in 5-FU containing culture medium, when the upp gene expressed vector was electroporated into Δupp L. lactis MG1363, so the upp gene can be used as a counterselectabscreening marker, construction of upp-defective complementary plasmid vector system. Secreted plasmid p AMJ399 induced by L. lactis MG1363 p H was used as expression vector. Firstly, to construct the recombinant plasmid p AMJ399 uppCa IFN-γ, and then p AMJ399upp-Ca IFN-γ was electroporated into Δupp L. lactis MG1363. Secondly, recombinant Lactococcus lactis p AMJ399upp-Ca IFN-γ/Δupp L. lactis MG1363 was screening by counterselectabmarker upp, which induced by the acid production. Western blot results show that there is about 20 ku protein in cells, which have specific reaction with Ca IFN-γ antiserum from rabbit. So Ca IFN-γ protein is expressed in recombinant Lactococcus lactis.Finally, the gene targeting integration system could be used in deletion of genes at any non essential location of the chromosome of L. lactis MG1363, so the gene integration system can be applyed in L. lactis MG1363, and the gene deletion would not introduce any selectabmarkers or residual bases, which facilitated the investigation of gene function. In addition, using upp as a counterselectabselection marker to construction of upp-defective complementary plasmid vector system expressing heterologous protein. The study above laid the foundation of establishing the biosafety genetic engineering recombinant Lactococcus lactis. |
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