本文選題：沼澤紅假單胞菌 + 鎳鐵氫酶��； 參考：《成都理工大學(xué)》2015年碩士論文

【摘要】：生物制氫基于生物化學(xué)反應(yīng)原理,在常溫常壓下,利用微生物自身產(chǎn)氫代謝系統(tǒng)或基因改良產(chǎn)氫代謝系統(tǒng),以有機(jī)質(zhì)或自由水為底物產(chǎn)生氫氣。這種方式不僅能夠在常溫常壓下進(jìn)行,而且更加環(huán)保,消耗的能源也較少,是一種可持續(xù)生產(chǎn)氫氣的方式。論文主要研究含鎳鐵氫酶基因的重組大腸桿菌的構(gòu)建及產(chǎn)氫能力。實(shí)驗(yàn)通過RCVBN培養(yǎng)液富集,采用平板篩選法,篩選沼澤紅假單胞菌,并對篩選到的沼澤紅假單胞菌進(jìn)行16S rDNA分子學(xué)鑒定；根據(jù)GenBank上查找的沼澤紅假單胞菌鎳鐵氫酶大小亞基基因序列,設(shè)計PCR引物,分別擴(kuò)增鎳鐵氫酶大小亞基基因,即hupS和hupL,將PCR產(chǎn)物經(jīng)電泳回收后,與克隆質(zhì)粒pBM19-T載體進(jìn)行連接,轉(zhuǎn)化到大腸桿菌DH5α中,獲得含有鎳鐵氫酶大小亞基基因的pBM19-T載體,進(jìn)行測序,測序結(jié)果與GenBank上的同源序列進(jìn)行比對；然后,實(shí)驗(yàn)分別設(shè)計含有限制性內(nèi)切酶位點(diǎn)的鎳鐵氫酶大小亞基基因引物,經(jīng)PCR擴(kuò)增、雙酶切實(shí)驗(yàn)后,與表達(dá)載體pETDuet-1進(jìn)行連接,構(gòu)建成含有鎳鐵氫酶大小亞基基因的雙啟動子表達(dá)載體pETD-SL。實(shí)驗(yàn)選用大腸桿菌BL21(DE3)作為重組對象,將獲得的雙啟動子表達(dá)載體pETD-SL轉(zhuǎn)化到大腸桿菌中。挑取陽性克隆,進(jìn)行PCR、限制性酶切驗(yàn)證,并測序保證序列的一致性。以含pETDuet-1質(zhì)粒的大腸桿菌BL21(DE3)作為對照組,將重組大腸桿菌BH20用1 mM的IPTG誘導(dǎo)產(chǎn)生重組蛋白,并利用SDS-PAGE電泳檢測重組蛋白誘導(dǎo)情況。同時,利用氫氧化鈉排水法收集對照組和重組大腸桿菌的氣體組分,經(jīng)過氣相色譜分析氣體樣本組分,考察大腸桿菌是否產(chǎn)氫。實(shí)驗(yàn)還考察了在不同條件下(即葡萄糖濃度、溫度、pH),重組大腸桿菌產(chǎn)氫情況。通過上述實(shí)驗(yàn),我們得到以下結(jié)果：(1)通過顯微鑒定和16S rDNA鑒定,獲得了一株沼澤紅假單胞菌株。此菌為革蘭氏陰性菌,在顯微鏡下呈桿狀,有鞭毛。菌落在固體培養(yǎng)基上呈棕紅色,呈規(guī)則圓形,直徑約0.4-1.3mm,表面濕潤光滑且邊緣整齊。經(jīng)光譜分析,結(jié)果顯示分離的沼澤紅假單胞菌的特征吸收峰為380、592、806和875 nm。(2)按照已知的鎳鐵氫酶大小亞基基因序列設(shè)計引物,經(jīng)PCR擴(kuò)增得到長度約為1400 bp、2000 bp的兩個片段,經(jīng)連接到測序載體上測序后,測序結(jié)果顯示鎳鐵氫酶大小亞基基因所編碼的蛋白質(zhì)大小分別為65.5 kDa和42.7 kDa。將兩段序列在GenBank上進(jìn)行同源序列分析,分析比對結(jié)果顯示所克隆的基因確為沼澤紅假單胞菌的鎳鐵氫酶的hupS和hupL基因�？寺〉降暮邢拗菩詢�(nèi)切酶位點(diǎn)的大小亞基克隆片段連接到雙啟動子表達(dá)載體pETDuet-1上,最終成功構(gòu)建了沼澤紅假單胞菌鎳鐵氫酶表達(dá)載體pETD-SL質(zhì)粒。(3)將重組質(zhì)粒pETD-SL轉(zhuǎn)化到大腸桿菌BL21(DE3)中,并篩選出一株能夠表達(dá)沼澤紅假單胞菌鎳鐵氫酶并能夠產(chǎn)生氫氣的重組大腸桿菌BH20。產(chǎn)氫實(shí)驗(yàn)現(xiàn)象和氣體組分分析表明,重組大腸桿菌BH20能夠產(chǎn)生氫氣,而實(shí)驗(yàn)對照組不能產(chǎn)生氫氣。(4)檢測大腸桿菌BH20菌株在不同溫度下的產(chǎn)氫效率,結(jié)果表明,當(dāng)溫度為35℃時,產(chǎn)氫量最高,達(dá)到115.8±1.0 mL,葡萄糖的消耗率約為61.9%。大腸桿菌BH20菌株在不同pH值下的產(chǎn)氫效率,結(jié)果表明,當(dāng)pH值為6.5時,產(chǎn)氫量最高,達(dá)到115.8±1.0 mL,葡萄糖的消耗率約為63.0%。大腸桿菌BH20菌株在不同葡萄糖濃度下的產(chǎn)氫效率,結(jié)果表明,當(dāng)葡萄糖濃度為2%時,產(chǎn)氫量最高,達(dá)到氫氣的產(chǎn)量為105.3±2.4 mL,葡萄糖消耗率為63.1%。因此,大腸桿菌BH20的最適產(chǎn)氫條件為溫度35℃,pH為6.5,葡萄糖濃度為2%。在最適條件下產(chǎn)氫量為122.9±2.4 mL,同時葡萄糖的總利用率約為5.3%。論文利用基因工程技術(shù),將獲得沼澤紅假單胞菌鎳鐵氫酶中hupS和hupL基因序列,借助雙啟動質(zhì)粒載體pETDuet-1成功地轉(zhuǎn)化到大腸桿菌BL21(DE3)中,獲得了一株重組大腸桿菌BH20。產(chǎn)氫實(shí)驗(yàn)結(jié)果表明重組大腸桿菌BH20能夠產(chǎn)氫,說明pETD-SL載體能增強(qiáng)以甲酸代謝系統(tǒng)產(chǎn)氫的微生物的產(chǎn)氫能力,這為未來應(yīng)用于提升含有甲酸代謝系統(tǒng)的微生物的產(chǎn)氫能力方面有很好的應(yīng)用價值。
[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.

【學(xué)位授予單位】：成都理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TQ116.2;Q78

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相關(guān)期刊論文 前2條

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本文編號：1885065