木醋杆菌群体感应基因luxR的克隆及其功能分析

发布时间：2018-04-28 22:32

本文选题：木葡糖酸醋杆菌 + 细菌纤维素　；参考：《天津科技大学》2017年硕士论文

【摘要】：近年来,由于细菌纤维素所具有的高纯度,高亲水性、高生物相容性以及良好的机械性能被广泛应用于生物医药行业,造纸行业以及食品等行业。木葡糖酸醋杆菌Gluconacetobacter xyinus作为细菌纤维素的模式生产菌株,其生产细菌纤维素的确切机制尚不明确。本实验室之前已证实在G.xylinus CGMCC 2955中存在类似费氏弧菌的LuxI/LuxR型群体感应系统。大量文献表明群体感应与生物膜的合成有密切的关系,此本文针对群体感应与细菌纤维素合成之间的相关性进行了探究。首先利用分子生物学手段构建群体感应关键基因luxR过表达载体,将其电转化到G.xylinus,通过静置培养和振荡培养两种培养方式来比较原始菌株与过表达菌株发酵性能的差异,并比较细菌纤维素的结构性能差异。结果发现,在振荡培养条件下,与G.x相比,G.x-pMV24-luxR的菌体生长速度明显减缓,到发酵结束,菌体量降低15.51%,葡萄糖酸的产量提高了 12.11%,对葡萄糖酸的利用率提高了 20.18%,但二者总酸产量无明显差异。在静置条件下,与G.x相比,G.x-pMV24-luxR的纤维素产量提高了 36.45%,且在16d葡萄糖酸含量降低了23.68%。对比各菌株所产的细菌纤维素的性能,傅里叶红外光谱(Fourier Transform infrared spectroscopy, FT-IR)分析由于差异均存在于非结晶区,表明细菌纤维素内官能团结构没有明显差异;X射线衍射(X-ray diffraction.,XRD)表明G.x-pMV24-luxR产的纤维素结晶度明显降低;TG/DTG表明G.x-pMV24-luxR产纤维素的热稳定性降低。利用代谢组学方法,对G.x以及G.x-pMV24-luxR胞内小分子代谢物的的差异进行分析,探究群体感应对胞内代谢的影响。结果表明luxR过表达对中心代谢、氨基酸代谢、脂代谢均有影响。G.x-pMV24-luxR大量合成葡萄糖酸,并且对嘌呤嘧啶的从头合成也具有促进作用,但是却抑制了氨基酸代谢和脂代谢,海藻糖作为一种细胞保护性物质,在葡萄糖酸大量合成的同时其含量也明显增加。
[Abstract]:In recent years, bacterial cellulose has been widely used in biomedical industry, paper industry and food industry because of its high purity, high hydrophilicity, high biocompatibility and good mechanical properties. As a model strain of bacterial cellulose production, the exact mechanism of the production of bacterial cellulose by Gluconacetobacter xyinus is unclear. It has been previously confirmed in our laboratory that there is a LuxI/LuxR type population sensing system in G.xylinus CGMCC 2955 similar to Vibrio flexneri. A large number of literatures have shown that there is a close relationship between population induction and biosynthesis of biofilm. The correlation between group induction and bacterial cellulose synthesis is explored in this paper. Firstly, the overexpression vector of luxR gene was constructed by molecular biology, and transformed into G. xylinus. The fermentation performance of the original strain was compared with that of the overexpression strain by static culture and oscillatory culture. The structure and properties of bacterial cellulose were compared. The results showed that the growth rate of G. x-pMV24-luxR was significantly slower than that of G.x under oscillatory culture conditions, and the growth rate of G. x-pMV24-luxR was significantly slower than that of G.x, until the end of fermentation. The yield of gluconic acid and the utilization rate of gluconic acid were increased by 15.51 and 12.11 respectively, but there was no significant difference between them. Under static conditions, the cellulose yield of G.x-pMV24-luxR increased by 36.45, and the gluconic acid content decreased by 23.68% at 16 days compared with G. x. Comparing the properties of bacterial cellulose produced by each strain, Fourier Transform infrared spectroscopy (FT-IRI) analysis showed that the differences existed in the amorphous region. The results showed that there was no obvious difference in the structure of functional groups in bacterial cellulose. X-ray diffraction (XRD) showed that the crystallinity of cellulose produced by G.x-pMV24-luxR was obviously decreased. The results showed that the thermal stability of cellulose produced by G.x-pMV24-luxR was lower than that of TG / DTG. In order to explore the effect of population induction on intracellular metabolism, the differences between G. x and G.x-pMV24-luxR small molecule metabolites were analyzed by means of metabonomics. The results showed that the overexpression of luxR had an effect on the metabolism of central, amino acid and lipid. G.x-pMV24-luxR also promoted the ab initio synthesis of purine pyrimidine, but inhibited the metabolism of amino acid and lipid. Trehalose, as a cytoprotective substance, increases the content of gluconic acid in a large amount at the same time.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;O636.11

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