基于iTRAQ技术对三株植物乳杆菌的比较蛋白质组学分析

发布时间：2018-09-06 19:27

【摘要】：目前,越来越多的植物乳杆菌被证实为益生菌。植物乳杆菌具有诸多有利于人体健康的生理功能。除此之外,植物乳杆菌在工业上的应用也引起了人们越来越多的关注。然而在这些应用中,植物乳杆菌会遭遇高盐浓度。植物乳杆菌运用诸多策略来应对不利的渗透压条件。尽管已经有一些关于植物乳杆菌渗透压反应的研究,目前对这方面的的理解还是有限的。本研究以不同来源的植物乳杆菌ATCC14917、植物乳杆菌FS5-5和植物乳杆菌208为研究对象,绘制了其在无NaCl培养基中的生长曲线,然后收集对数期菌体使其分别在NaCl浓度为0%、24%(w/v)培养基中应激3h,运用iTRAQ研究了其在两种盐应激下的蛋白质表达情况,最后运用RT-qPCR技术对所选差异蛋白质的基因表达进行了验证。1.通过测定其OD600nm分别绘制了它们在无NaCl培养基中的生长曲线。由生长曲线可知,植物乳杆菌ATCC14917、植物乳杆菌FS5-5和植物乳杆菌208在无NaCl培养基中,生长至对数生长期中期的时间点分别为5h、5h和6h。2.收集三株植物乳杆菌在无NaCl培养基中生长至对数生长期中期的菌体进行盐应激试验。然后收集菌体,进行iTRAQ试验。试验结果表明:在uniprot_taxonomy_1578库中鉴定到350957个谱图,匹配上16669个谱图,鉴定到1628个肽段种类,匹配上878个蛋白质。3.以差异倍数在1.2倍以上,且P-value0.05为筛选条件对三株植物乳杆菌在NaCl浓度为24%与0%(w/v)相比、0%(w/v)相比和24%(w/v)相比出现的差异蛋白质点进行筛选,共筛选出867个差异蛋白质点。在所有比较中上调蛋白质点和下调蛋白质点均是植物乳杆菌ATCC14917植物乳杆菌FS5-5植物乳杆菌208,并且植物乳杆菌ATCC14917与植物乳杆菌FS5-5的上调蛋白质点和下调蛋白质点个数相差较小,而均与植物乳杆菌208相差较大。4.通过 Gene ontology(GO)数据库和 Kyoto encyclopedia of genes and genomes(KEGG)数据库对差异蛋白质进行功能和代谢通路的注释,然后再对三株植物乳杆菌的注释差异蛋白质进行比较。结果再次说明植物乳杆菌ATCC14917与植物乳杆菌FS5-5的上调蛋白质点和下调蛋白质点个数相差较小,而均与植物乳杆菌208相差较大。这可能说明植物乳杆菌ATCC14917的盐应激能力与植物乳杆菌FS5-5的盐应激能力相当,植物乳杆菌208的盐应激能力较差。因此,不同来源的三株植物乳杆菌的盐应激能力有差异。在盐应激条件下,表达水平明显变化的蛋白质主要为参与糖代谢、氨基酸代谢、核苷酸代谢、蛋白质合成、应激反应和转运的蛋白。5.应用RT-qPCR技术对iTRAQ试验中所选差异蛋白质进行基因表达水平的验证试验。结果表明,基因表达水平与蛋白质表达水平基本一致,这在RNA水平上验证了iTRAQ结果的准确性。
[Abstract]:At present, more and more Lactobacillus plantarum have been confirmed as probiotics. Lactobacillus plantarum has many physiological functions beneficial to human health. In addition, the application of Lactobacillus plantarum in industry has attracted more and more attention. In these applications, however, Lactobacillus plantarum suffers from high salt concentrations. Lactobacillus plantarum uses a number of strategies to cope with adverse osmotic conditions. Although there have been some studies on the osmotic response of Lactobacillus plantarum, the understanding is limited. In this study, the growth curves of Lactobacillus plantarum ATCC14917, and Lactobacillus plantarum FS5-5 and Lactobacillus plantarum 208 from different sources in NaCl free medium were drawn. Then we collected the bacteria in logarithmic phase to stress them in 0 ~ 24% (w / v) NaCl medium for 3 h, studied their protein expression under two kinds of salt stress by iTRAQ. Finally, the gene expression of the selected differential protein was verified by RT-qPCR technique. 1. Their growth curves in NaCl free medium were plotted by measuring their OD600nm. According to the growth curve, the growth time points of Lactobacillus plantarum ATCC14917, Lactobacillus plantarum FS5-5 and Lactobacillus plantarum 208 in NaCl free medium were 5 h / 5 h and 6 h 路2 h respectively. Three strains of Lactobacillus plantarum were collected to grow to the middle stage of logarithmic growth in NaCl free medium for salt stress test. Then the bacteria were collected and iTRAQ test was carried out. The results showed that 350957 spectra were identified in uniprot_taxonomy_1578 library, 16669 spectra were matched, 1628 peptides were identified and 878 proteins were matched. Three strains of Lactobacillus plantarum were screened at 24% of NaCl concentration compared with 0% (w / v) and 24% (w / v) compared with 0% (w / v). A total of 867 differentially expressed protein spots were screened by using P-value0.05 as the screening condition and the multiple of difference was more than 1.2.The results showed that three strains of Lactobacillus plantarum had different protein spots compared with 0% (w / v) and 24% (w / v) respectively. In all the comparisons, the up-regulated and down-regulated protein spots were Lactobacillus plantarum ATCC14917, Lactobacillus plantarum FS5-5, Lactobacillus plantarum FS5-5, and the number of up-regulated protein spots and down-regulated protein spots of Lactobacillus plantarum ATCC14917 and Lactobacillus plantarum FS5-5 were smaller than those of Lactobacillus plantarum. The difference between the two groups was larger than that of Lactobacillus plantarum 208. The functional and metabolic pathways of differential proteins were annotated by Gene ontology (GO) database and Kyoto encyclopedia of genes and genomes (KEGG) database, and then the annotated differential proteins of three strains of Lactobacillus plantarum were compared. The results showed that the number of up-regulated protein spots and down-regulated protein spots of Lactobacillus plantarum ATCC14917 and Lactobacillus plantarum FS5-5 were smaller than those of Lactobacillus plantarum FS5-5. This may indicate that the salt stress ability of Lactobacillus plantarum ATCC14917 is equal to that of Lactobacillus plantarum FS5-5, and the salt stress ability of Lactobacillus plantarum 208 is poor. Therefore, the salt stress ability of three Lactobacillus plantarum strains from different sources is different. Under salt stress, the main proteins involved in glucose metabolism, amino acid metabolism, nucleotide metabolism, protein synthesis, stress response and transport were protein. RT-qPCR technique was used to verify the gene expression level of differential protein in iTRAQ test. The results showed that the gene expression level was basically the same as the protein expression level, which verified the accuracy of the iTRAQ results at the RNA level.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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