Studies Of Cholesterol-lowering Mechanism Based On Transcriptom Of Lactobacillus Plantarum

Cholesterol has diverse probiotic functions,and it is an important nutritional component of human body.However,excessive intake of cholesterol can cause cardiovascular and cerebrovascular diseases,which is seriously harmful to human health.In clinical,conventional therapeutic drugs have many side effects,and it is hoped to find a more effective and safer way to reduce cholesterol.Studies have shown that Lactobacillus plantarum can lower serum cholesterol and reduce the incidence of cardiovascular and cerebrovascular diseases.At present,many research teams have carried out in vivo and in vitro experiments on the cholesterol lowering characteristics of L.plantarum,aiming to study its mechanism.The mechanism of cholesterol-lowering effects by L.plantarum includes coprecipitation,assimilation,and adsorption of extracellular polysaccharide（EPS）,etc.In this study,two strains of L.plantarum Lp10 and Lp11 with quite different cholesterol-lowering capacity were investigated on their cholesterol-lowering mechanisms.First of all,experimental operations including gene cloning,bioinformatics analysis,heterologous expression,and RT-qPCR were performed on bile salt hydrolase gene（bsh）,which is a key enzyme with cholesterol lowering.Results showed that the lengths of bsh1,bsh2,bsh3 and bsh4genes were 975bp,1017bp,987bp and 954bp respectively.The constructed expression vector pET-32a-bshs was successfully expressed in E.coli BL21（DE3）and the molecular weight of the protein was approximately 37 KDa.However,only BSH3 was soluble expression,and BSH1,BSH2,and BSH4 were all expressed as inactive inclusion bodies.The crude enzyme supernatant of BSH3 was purified and a single band was displayed by electrophoresis suggesting the purification effect was good.The enzyme activity was determined and found to be inactive.Subsequently,under the conditions of adding bile salts,the expression of bshs gene was determined by RT-qPCR.The results indicated that the relative expression levels of bsh1 and bsh2 were upregulated by 7.303 and 4.256 times,and bsh3 and bsh4 were downregulated by 1.805and 3.247 times,respectively.That is,bsh1 plays a major role in the cholesterol-lowering process,followed by bsh2.However,the expression of bsh3 and bsh4 genes were inhibited during this processIn order to avoid the interference of coprecipitation,used transcriptome sequencing technology to explore other cholesterol lowering mechanisms of L.plantarum without adding bile salts.Transcriptome sequencing obtained were3.12×10¹⁰ bp data,the quatily of the orginal sequencing data was qualifity.Compared with the reference genome L.plantarum ST-III,the mapping rate of all samples was as high as 99%.Analysis of differential expression for sequencing data,304 differentially expressed genes（DEGs）were found from the comparison of Lp10_M vs Lp10_CM,of these,79 genes were up-regulated and 225 genes were down-regulated.272 DEGs were found from the comparison of Lp11_M vs Lp11_CM,of these,127 genes were up-regulated and 145 genes were down-regulated.In the two comparison groups,there were 85 of the same significant difference genes.GO and KEGG enrichment analysis about DEGs showed that the the fructose and mannose metabolic pathways were related to the cholesterol-lowering mechanism of EPS adsorption by L.plantarum.Therefore,the L-rhamnulose 1-phosphate aldolase（rhaD）gene in this metabolic pathway is identified as cholesterol-lowering related gene and verified by RT-qPCR.The results showed that the relative expression of rhaD gene was reduced by 1.140 times during the cholesterol lowering process,which was consistent with the transcriptome data.Analysis of the expression of bshs in the transcriptome data revealed that the expression of bsh1,bsh2,bsh3,and bsh4 were not significant,suggesting that BSH must be coprecipitated in the presence of bile salt,and the main cholesterol lowering mechanism of L.plantarum is quite different under diverse conditions.Finally,the pNZ5319-rhaD knockout plasmid was constructed based on homologous recombination technology and transformed into L.plantarum Lp10competent cells by the method of electroporation.Results demonstrated the knockout vector pNZ5319-rhaD was constructed successfully,and the DNA sequencing results indicated that rhaD gene was successfully knocked out.In order to verify the function of rhaD gene,the physical and chemical indexes of original strains and knockout strains were determined,such as growth curve,cholesterol lowering ability,acid resistance,antioxidation,bile salt resistance and hydrophobicity.The results showed that the growth of knockout vector was slower than original strain.When the pH value was 2.5and 3.0,the growth rate of two strains was the same.Under pH 3.5,the acid resistance of the original strain was better.The hydrophobicity of the original strain was 32.2%and the knockout strain was 8.9%,and the hydrophobicity of the original strain was better.Determination of cholesterol lowering ability with the o-phthalaldehyde method,cholesterol degradation rate of the original strain rate is 18.96%/OD600,and knockout strain is 19.22%/OD600.After the down-regulated gene rhaD was knocked out,the cholesterol lowering ability of the knockout strain increased slightly,which was consistent with the analysis of transcriptome data.In this experiment,the key gene of cholesterol lowering（bshs）was analyzed in various aspects,and DEGs in cholesterol lowering were excavated from the transcriptional level.The mechanism of cholesterol lowering in L.plantarum was studied under different treatment conditions.This paper not only provides new ideas for the study of cholesterol lowering mechanism of L.plantarum,but also provides a theoretical basis for the development of functional lactic acid bacteria products.