构建含镍铁氢酶基因的重组大肠杆菌及产氢研究

发布时间：2018-05-13 22:18

本文选题：沼泽红假单胞菌 + 镍铁氢酶　；参考：《成都理工大学》2015年硕士论文

【摘要】：生物制氢基于生物化学反应原理,在常温常压下,利用微生物自身产氢代谢系统或基因改良产氢代谢系统,以有机质或自由水为底物产生氢气。这种方式不仅能够在常温常压下进行,而且更加环保,消耗的能源也较少,是一种可持续生产氢气的方式。论文主要研究含镍铁氢酶基因的重组大肠杆菌的构建及产氢能力。实验通过RCVBN培养液富集,采用平板筛选法,筛选沼泽红假单胞菌,并对筛选到的沼泽红假单胞菌进行16S rDNA分子学鉴定；根据GenBank上查找的沼泽红假单胞菌镍铁氢酶大小亚基基因序列,设计PCR引物,分别扩增镍铁氢酶大小亚基基因,即hupS和hupL,将PCR产物经电泳回收后,与克隆质粒pBM19-T载体进行连接,转化到大肠杆菌DH5α中,获得含有镍铁氢酶大小亚基基因的pBM19-T载体,进行测序,测序结果与GenBank上的同源序列进行比对；然后,实验分别设计含有限制性内切酶位点的镍铁氢酶大小亚基基因引物,经PCR扩增、双酶切实验后,与表达载体pETDuet-1进行连接,构建成含有镍铁氢酶大小亚基基因的双启动子表达载体pETD-SL。实验选用大肠杆菌BL21(DE3)作为重组对象,将获得的双启动子表达载体pETD-SL转化到大肠杆菌中。挑取阳性克隆,进行PCR、限制性酶切验证,并测序保证序列的一致性。以含pETDuet-1质粒的大肠杆菌BL21(DE3)作为对照组,将重组大肠杆菌BH20用1 mM的IPTG诱导产生重组蛋白,并利用SDS-PAGE电泳检测重组蛋白诱导情况。同时,利用氢氧化钠排水法收集对照组和重组大肠杆菌的气体组分,经过气相色谱分析气体样本组分,考察大肠杆菌是否产氢。实验还考察了在不同条件下(即葡萄糖浓度、温度、pH),重组大肠杆菌产氢情况。通过上述实验,我们得到以下结果：(1)通过显微鉴定和16S rDNA鉴定,获得了一株沼泽红假单胞菌株。此菌为革兰氏阴性菌,在显微镜下呈杆状,有鞭毛。菌落在固体培养基上呈棕红色,呈规则圆形,直径约0.4-1.3mm,表面湿润光滑且边缘整齐。经光谱分析,结果显示分离的沼泽红假单胞菌的特征吸收峰为380、592、806和875 nm。(2)按照已知的镍铁氢酶大小亚基基因序列设计引物,经PCR扩增得到长度约为1400 bp、2000 bp的两个片段,经连接到测序载体上测序后,测序结果显示镍铁氢酶大小亚基基因所编码的蛋白质大小分别为65.5 kDa和42.7 kDa。将两段序列在GenBank上进行同源序列分析,分析比对结果显示所克隆的基因确为沼泽红假单胞菌的镍铁氢酶的hupS和hupL基因。克隆到的含有限制性内切酶位点的大小亚基克隆片段连接到双启动子表达载体pETDuet-1上,最终成功构建了沼泽红假单胞菌镍铁氢酶表达载体pETD-SL质粒。(3)将重组质粒pETD-SL转化到大肠杆菌BL21(DE3)中,并筛选出一株能够表达沼泽红假单胞菌镍铁氢酶并能够产生氢气的重组大肠杆菌BH20。产氢实验现象和气体组分分析表明,重组大肠杆菌BH20能够产生氢气,而实验对照组不能产生氢气。(4)检测大肠杆菌BH20菌株在不同温度下的产氢效率,结果表明,当温度为35℃时,产氢量最高,达到115.8±1.0 mL,葡萄糖的消耗率约为61.9%。大肠杆菌BH20菌株在不同pH值下的产氢效率,结果表明,当pH值为6.5时,产氢量最高,达到115.8±1.0 mL,葡萄糖的消耗率约为63.0%。大肠杆菌BH20菌株在不同葡萄糖浓度下的产氢效率,结果表明,当葡萄糖浓度为2%时,产氢量最高,达到氢气的产量为105.3±2.4 mL,葡萄糖消耗率为63.1%。因此,大肠杆菌BH20的最适产氢条件为温度35℃,pH为6.5,葡萄糖浓度为2%。在最适条件下产氢量为122.9±2.4 mL,同时葡萄糖的总利用率约为5.3%。论文利用基因工程技术,将获得沼泽红假单胞菌镍铁氢酶中hupS和hupL基因序列,借助双启动质粒载体pETDuet-1成功地转化到大肠杆菌BL21(DE3)中,获得了一株重组大肠杆菌BH20。产氢实验结果表明重组大肠杆菌BH20能够产氢,说明pETD-SL载体能增强以甲酸代谢系统产氢的微生物的产氢能力,这为未来应用于提升含有甲酸代谢系统的微生物的产氢能力方面有很好的应用价值。
[Abstract]:Biological hydrogen is based on the principle of biochemical reaction. At normal temperature and atmospheric pressure, hydrogen production is produced by the use of microorganism's own hydrogen production and metabolic system or genetically modified hydrogen production system. Organic matter or free water is used as the substrate to produce hydrogen. This method can not only be carried out under normal temperature and pressure, but also are more annulus and consume less energy. It is a kind of sustainable production. The paper mainly studies the construction and hydrogen production capacity of recombinant Escherichia coli containing nickel iron hydrogenase gene. The experiment was enriched by RCVBN culture and screened by plate screening method to screen Rhodopseudomonas marshes and determine the 16S rDNA classification of Rhodopseudomonas marshes; according to the marsh red Pseudomonas found on GenBank PCR primers were designed to amplify the subunit gene of nickel iron hydrogenase, hupS and hupL, respectively. After the recovery of the PCR products, the PCR products were connected with the plasmid pBM19-T vector and transformed into the Escherichia coli DH5 alpha, and the pBM19-T carrier containing the nickel iron hydrogenase subunit gene was obtained and sequenced and sequenced. The results were compared with the homologous sequences on the GenBank; then, the experiment was designed to design the nickel iron hydrogenase subunit gene primers containing the restriction endonuclease site. After PCR amplification, the double enzyme digestion experiment was used to connect with the expression vector pETDuet-1 to construct a double promoter expression vector containing the nickel iron hydrogenase subunit gene, pETD-SL. experimental selection. Escherichia coli BL21 (DE3) was used as a recombinant object to convert the obtained double promoter expression vector pETD-SL into Escherichia coli. The positive clones were picked up, PCR, restriction enzyme digestion, and sequencing guaranteed consistency. The recombinant Escherichia coli BL21 (DE3) containing pETDuet-1 plasmid was used as the control group, and the recombinant Escherichia coli BH20 was induced by 1 mM IPTG. The recombinant protein was produced and the recombinant protein was induced by SDS-PAGE electrophoresis. At the same time, the gas components of the control group and the recombinant Escherichia coli were collected by the sodium hydroxide drainage method, and the gas samples were analyzed by gas chromatography to investigate whether the Escherichia coli produced hydrogen. The experiment also examined the glucose concentration, temperature, and the temperature, PH) recombinant Escherichia coli hydrogen production. Through the above experiments, we obtained the following results: (1) a strain of Pseudomonas marshes was obtained by microscopic identification and 16S rDNA identification. The bacteria were Gram-negative bacteria, rod-shaped under the microscope and flagellum. The colony was brown red on the solid medium, with a regular round and about 0.4-1.3mm in diameter. The surface was smooth and smooth and the edge was neat. The spectral analysis showed that the characteristic absorption peaks of the isolated Rhodopseudomonas marshes were 380592806 and 875 nm. (2), and the primers were designed according to the known subunit gene sequence of the nickel iron hydrogenase size subunit, and two fragments of the length of 1400 BP and 2000 BP were amplified by PCR amplification, and then sequenced on the sequencing vector. The sequencing results showed that the size of the nickel iron hydrogenase subunit gene was 65.5 kDa and 42.7 kDa., respectively, to carry out the homologous sequence analysis of two segments on GenBank. The results showed that the cloned gene was the hupS and hupL gene of the nickel iron hydrogenase of Rhodopseudomonas marshes. The size subunit cloned fragment of the loci was connected to the double promoter expression vector pETDuet-1, and the pETD-SL plasmid was successfully constructed. (3) the recombinant plasmid pETD-SL was transformed into the Escherichia coli BL21 (DE3), and a nickel iron hydroenzyme could be expressed and hydrogen could be produced to produce hydrogen. The hydrogen production of recombinant Escherichia coli BH20. and gas component analysis showed that recombinant Escherichia coli BH20 could produce hydrogen, while the experimental control group could not produce hydrogen. (4) the hydrogen production efficiency of Escherichia coli BH20 strain at different temperatures was detected. The results showed that when the temperature was 35, the hydrogen production was the highest and reached 115.8 + 1 mL, glucose was 115.8. The consumption rate is about the hydrogen production efficiency of 61.9%. Escherichia coli BH20 under different pH values. The results show that when the pH value is 6.5, the hydrogen production is the highest, reaching 115.8 + 1 mL. The consumption rate of glucose is about the hydrogen production efficiency of 63.0%. Escherichia coli BH20 strain at different glucose concentrations. The results show that when the glucose concentration is 2%, the amount of hydrogen production is the highest. The production of hydrogen is 105.3 + 2.4 mL and the glucose consumption rate is 63.1%., so the optimum hydrogen production condition of Escherichia coli BH20 is 35 C, pH 6.5, and the glucose concentration is 122.9 + 2.4 mL under the optimum condition, and the total utilization rate of glucose is about 5.3%. on the gene engineering technology, and the swamp red Pseudomonas will be obtained. The sequence of hupS and hupL gene in nickel iron hydrogenase was successfully transformed into Escherichia coli BL21 (DE3) with the double starting plasmid carrier pETDuet-1. A recombinant Escherichia coli BH20. hydrogen production experiment results showed that the recombinant Escherichia coli BH20 could produce hydrogen, indicating that the pETD-SL carrier could strengthen the hydrogen production of the microorganism with the formic metabolic system. This will provide a good application value for the future production of hydrogen producing microorganisms containing formic acid metabolic system.

【学位授予单位】：成都理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ116.2;Q78

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