WO2017152426A1 - 一种用于培育抗氧化微生物的基因 - Google Patents

一种用于培育抗氧化微生物的基因 Download PDF

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WO2017152426A1
WO2017152426A1 PCT/CN2016/076272 CN2016076272W WO2017152426A1 WO 2017152426 A1 WO2017152426 A1 WO 2017152426A1 CN 2016076272 W CN2016076272 W CN 2016076272W WO 2017152426 A1 WO2017152426 A1 WO 2017152426A1
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dwh
gene
strain
radiodurans
antioxidant
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王劲
林敏�
平淑珍
左开井
江世杰
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中国农业科学院生物技术研究所
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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  • the present invention relates to a gene for cultivating an antioxidant microorganism.
  • Deinococcus radiodurans R1 (DR) is resistant to ionizing radiation, UV radiation, DNA damage agents, etc. It is an ideal strain for studying the adaptive mechanism of microbial abiotic stress. Some people have studied a large number of functionally unknown genes (such as IrrE, Csp, etc.) in Radiation-resistant Aeruginosa, for example, after expressing four genes related to plant drought resistance in D. radiodurans R1 in prokaryotic host cells and plants, Enhance salt tolerance and the like of the host.
  • the object of the present invention is to find a novel gene for the cultivation of antioxidant microorganisms from D. radiodurans R1.
  • the Radiation-resistant Aeruginosa R1 contains a gene encoding a protein similar to Why ( W ater stress and Hy persensitive response), which is named dwh, and its nucleotide sequence is shown in SEQ ID NO. 1, and the protein encoded by the gene (Dwh).
  • the protein has an anti-oxidation function and can be used for cultivating an antioxidant microorganism, thereby providing a reference for cultivating a plant having an antioxidant trait, and the amino acid sequence of the protein is shown in SEQ ID NO.
  • Hydrophobicity analysis of Dwh protein by Kyte & Doolittle method also showed its high hydrophobicity (Fig. 1). Therefore, it is speculated that this protein is a typical class of hydrophobins.
  • secondary structure predictions (Fig. 2) showed that the Dwh protein consisted of six beta sheets and one alpha helix near the C-terminus.
  • the dwh gene upstream and downstream gene fragments (dwh-U and dwh-D) were obtained by PCR, and the kanamycin resistance was obtained.
  • the Km gene fragment (K) of the sex protein (Fig. 3) was fused with the dwh-U, K, and dwh-D fragments by fusion PCR to obtain a dwh-UKD recombinant fragment.
  • the recombinant fragment was then transformed into D.radiodurans R1 competent cells by homologous recombination technique, and subjected to pressure screening on TGY plates containing Km resistance to obtain the dwh gene deletion mutant ⁇ dwh, which was verified by PCR and sequencing (Fig. 4). (See Example 1).
  • the dwh gene was amplified from the genome of D. radiodurans R1 (CGMCC 1.633) by PCR, and the nucleotide sequence thereof is shown in SEQ ID NO. The nucleotide length was 297 bp (Fig. 8), and the gene encodes 99 amino acids, which was cloned into the vector pJET1.2/Blunt to construct a recombinant plasmid pJET-dwh containing the complete dwh gene (Fig. 9);
  • the experiment confirmed that the recombinant pET28a-dwh/BL21 strain containing D.radiodurans R1 dwh gene grew well after treatment with 30 mM H 2 O 2 for 10 min, and the colony viability was much higher than that of the control pET28a/BL21 strain containing only empty plasmid (see Example 4 and Figs. 10 and 11).
  • the results show that the engineered strain has the ability to tolerate high concentrations of H 2 O 2 stress.
  • Figure 1 is a pro-hydrophobicity analysis map of D.radiodurans R1 Dwh protein (where the abscissa indicates the amino acid sequence and the ordinate indicates the hydrophobicity score. The larger the hydrophobic score indicates the stronger the hydrophobicity of the amino acid; Hydrophilic - pro Water, Hydrophobic - hydrophobic);
  • Figure 2 is a secondary structure analysis map of D.radiodurans R1 Dwh protein
  • Figure 3 is a PCR product electrophoresis pattern of dwh-U (1), downstream dwh-D (2) and resistance gene K (3) fragments upstream of the dwh gene;
  • Figure 4 is a PCR verification map of the mutant strain ⁇ dwh (lanes 1-3: primer YZdwh-F/R amplifies the dwh fragment with the mutant strain ⁇ dwh, wild-type DR, sterile water as a template, and verifies that the dwh deletion is complete; lane 4 6: Primer YZdwh-UF/DR amplifies the recombinant fragment with the mutant strain ⁇ dwh, wild-type DR and sterile water as templates, and verified the dwh-UKD insertion position);
  • Figure 5 is a photograph of colonies of wild-type DR and mutant ⁇ dwh viability before H 2 O 2 shock test, wherein:
  • a is a wild type D.radiodurans R1 strain (DR);
  • b is a D.radiodurans R1 mutant strain ( ⁇ dwh) lacking the dwh gene;
  • Figure 6 is a photograph of colonies grown in TGY solid medium after treatment with wild-type DR and mutant ⁇ dwh for 30 min after treatment with 80 mM H 2 O 2 , wherein:
  • a is a wild type D.radiodurans R1 strain (DR);
  • b is a D.radiodurans R1 mutant strain ( ⁇ dwh) lacking the dwh gene;
  • Figure 7 shows changes in the activity of antioxidant enzymes in cells after treatment with wild-type DR and mutant ⁇ dwh for 30 min with 50 mM H 2 O 2 , wherein:
  • a is the change in the activity of catalase (CAT) in the cells of the mutant strain ⁇ dwh compared to the wild-type DR before and after H 2 O 2 treatment;
  • b is the change in intracellular peroxidase (POD) activity of the mutant strain ⁇ dwh compared to wild-type DR before and after H 2 O 2 treatment;
  • c is the change in the activity of superoxide dismutase (SOD) in the cells of the mutant strain ⁇ dwh compared to the wild-type DR before and after H 2 O 2 treatment;
  • Figure 8 is a verified electrophoresis pattern of a PCR product containing the D.radiodurans R1 dwh gene;
  • Figure 9 is a schematic verification electrophoresis map of a prokaryotic recombinant expression plasmid containing the D.radiodurans R1 dwh gene;
  • Figure 10 is a photograph of colonies of E. coli containing an empty plasmid and a prokaryotic expression plasmid containing the D. radiodurans R1 dwh gene before the H 2 O 2 shock test, wherein:
  • a is an E. coli BL21 strain containing an empty expression plasmid
  • b is a recombinant Escherichia coli strain containing a D. radiodurans R1 dwh gene expression plasmid;
  • Figure 11 is a photograph of colonies in E. coli containing the prokaryotic expression plasmid of D. radiodurans R1 dwh gene and empty plasmid in E. coli treated with 30 mM H 2 O 2 for 10 min, in which:
  • a is a control E. coli strain (pET28a/BL21) containing an empty expression plasmid;
  • b is an Escherichia coli recombinant strain (pET28a-dwh/BL21) containing the D. radiodurans R1 dwh gene expression plasmid.
  • the plasmids, strains and reagents given in the examples are as follows:
  • the blunt-end cloning vector pJET1.2/Blunt (Amp) was purchased from Thermo Scientific, and the plasmid pKatAPH3 (carrying the Km resistance gene) was preserved by this experiment.
  • the expression vector pET28a was preserved by the laboratory, pJET-dwh (dwh gene fragment and cloning vector pJET1) .2/Blunt linkage), pET28a-dwh (the dwh gene fragment was ligated with the cloning vector pET28a by restriction enzyme digestion)
  • the recombinant plasmid was constructed from this study.
  • Deinococcus radiodurans Rl wild-type strain (CGMCC 1.633) was provided by China General Microbial Culture Collection; mutant ⁇ dwh (D.radiodurans Rl strain lacking dwh gene) was constructed by this study, D.radiodurans Rl and its mutant strain Both were grown in TGY medium, and the optimum culture temperature was 30 ° C;
  • Escherichia coli trans 109 and BL21 strains were purchased from Beijing Quanjin Biotechnology Co., Ltd.; pET28a/BL21 (recombinant strain obtained by transforming empty vector pET28a into Escherichia coli BL21 strain), pET28a-dwh/BL21 (recombinant plasmid pET28a-dwh transformed large intestine)
  • the recombinant strain obtained from the Bacillus strain BL21 was constructed by this study, and both E. coli and its recombinant strain were grown in LB medium at a culture temperature of 37 °C.
  • Biochemical reagents Restriction enzymes were purchased from NEB; dNTPs, high-purity Primestar HS DNA polymerase, T4 DNA ligase, etc. were purchased from Dalian Bao Biotech Co., Ltd. (TaKaRa); agarose gel DNA recovery kit, common The DNA product purification kit and the common plasmid miniprep kit were purchased from Tiangen Biochemical Technology Co., Ltd. (TIANGEN); 30% H 2 O 2 , IPTG, antibiotics, etc. were purchased from Aladdin; primer synthesis and sequencing used in the experiment All were completed by Huada Gene Company.
  • LB medium tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH adjusted to 7.0, and agar 15 g/L was added to the solid medium.
  • Autoclave (121 ° C, 1.034 ⁇ 10 5 Pa) was sterilized for 30 min.
  • TGY medium tryptone 10 g / L, yeast extract 5 g / L, glucose 1 g / L, agar 15g / L was added to the solid medium. Autoclave (112 ° C, 1.034 ⁇ 10 5 Pa) was sterilized for 30 min.
  • Ampicillin (Amp) (50mg/mL): Weigh 0.5g ampicillin powder, dissolve in 10mL double distilled water, filter and remove impurities, store and store in -20 ° C refrigerator;
  • Kanamycin (Km) 50mg/mL: Weigh 0.5g kanamycin powder, dissolve in 10mL double distilled water, filter and remove impurities, store and store in -20 ° C refrigerator;
  • IPTG 100mmol/L: Weigh 0.24g IPTG, dissolve in 10mL double distilled water, filter and sterilize, and store in -20 °C refrigerator;
  • CaCl 2 (0.3 mol / L): Weigh 4.5g of CaCl 2 ⁇ 2H 2 O powder, dissolved in 80mL of double distilled water, dilute to 100mL, high pressure steam (121 ° C) sterilization;
  • 750 ⁇ mol/L nitroblue tetrazolium NBT solution weigh 0.06133g NBT with phosphate buffer to a volume of 100mL, protected from light;
  • EDTA-Na 2 solution weigh 0.03721g EDTA-Na 2 and dilute to 1000mL with phosphate buffer;
  • 0.05 mol / L guaiacol solution weighed 6.2065g (124.13g / mol ⁇ 0.05mol), and then dissolved in 1L of 95% ethanol, which is 1L of 0.05mol / L guaiacol solution;
  • the PBS solution used was equipped according to the molecular cloning manual.
  • Example 1 Deletion mutant D ⁇ radiudurans R1 dwh gene was obtained ⁇ dwh
  • the D.radiodurans R1 whole genome sequence and the plasmid pKatAPH3 sequence were obtained from the NCBI GeneBank, and primers were designed based on the dwh gene sequence and the Kana resistance gene sequence (see Table 1).
  • the mutant strain ⁇ dwh was constructed by fusion PCR. Using D.radiodurans R1 genomic DNA as a template, the upstream fragment of dwh gene was amplified by primer UF/UR synthesized in Table 1. The length of dwh-U was 749 bp. The downstream fragment of dwh gene was amplified by DF/DR primer. The length of the amplified product dwh-D was 595 bp. The KK/KR primer was used to amplify the Kana resistance gene fragment using the pKatAPH3 plasmid as a template, and the length of the amplified product K was 1007 bp. The amplified products were separately purified and recovered, and the bands were single (Fig.
  • PCR reaction conditions were: 94 ° C 10 min; 94 ° C 1 min; 60 ° C 1 min; 72 ° C 5 min; 17 cycles; 94 ° C 1 min; 60 ° C 1 min; 72 ° C 3 min; 30 cycles; 72 ° C 10 min.
  • the fusion product dwh-UKD (2268 bp) was sequenced and verified, which was completely consistent with the sequence designed by the experiment. Wild type D. radiodurans R1 was transformed by homologous recombination. The deletion mutant ⁇ dwh was screened on Km plates containing 10 ⁇ g/mL. ⁇ dwh was identified by PCR.
  • the dwh gene fragment was amplified by the YZdwh-F/YZdwh-R primer, and the 346 bp band was not obtained, and the 346 bp band was amplified in the negative control D.radiodurans R1 genomic DNA;
  • the YZdwh-UF/YZdwh-DR primer was used to amplify the YZdwh-UKD fragment to obtain only the 2591 bp band, and the D. radiodurans R1 genomic DNA was used as a template to obtain a 1985 bp band, and sterile water was used as a blank control.
  • Wild-type DR and mutant ⁇ dwh single colonies were picked from streaked TGY plates and inoculated into 2 mL TGY liquid medium (10 ⁇ g/mL Km antibiotic in mutant medium), and cultured at 30 ° C until mid-log phase. 1% was transferred to fresh 50mL TGY liquid medium (10 ⁇ g/mL Km antibiotic in the mutant medium), and cultured to the initial stage of logarithm (OD 600 is about 0.6), each 1 mL of bacterial solution was added and added separately.
  • Wild-type DR and mutant ⁇ dwh single colonies were picked from streaked TGY plates and inoculated into 20 mL TGY liquid medium (10 ⁇ g/mL Km antibiotic in mutant medium), and cultured at 30 ° C until mid-log phase.
  • the cells were washed twice with sterile water and finally resuspended in 5 mL of 50 mM pH 7.0 PBS buffer; the cells were sonicated on ice (power not exceeding 200 W, working for 2 s, interval 3 s, treatment time 45-60 min, change)
  • the rods were ⁇ 3), centrifuged at 12000 rpm for 20 min at 4 ° C, and the total protein concentration of each sample (Bradford method) was determined after dispensing on ice, and the total protein was stored at -20 °C.
  • Reagent Control tube CK Sample tube 0.2M pH 7.8 PBS ( ⁇ L) 100 90 ⁇ 95 0.1M H 2 O 2 ( ⁇ L) 100 100 Crude enzyme solution ( ⁇ L) 0 5 ⁇ 10 Total volume ( ⁇ L) 200 200
  • the enzyme activity is calculated according to the following formula.
  • the amount of enzyme that reduced 0.1 by A 240 in 1 min was 1 unit of enzyme.
  • ⁇ A 240 is the absolute value of the absorbance of the sample tube
  • V 1 is the total volume of the reaction solution ( ⁇ L)
  • V 2 is the volume of the crude enzyme extract for measurement ( ⁇ L)
  • C is the total protein concentration of the crude enzyme extract ( ⁇ g • ⁇ L -1 )
  • 0.1 is 0.1 for each decrease in A 240 enzyme units (U)
  • t is hydrogen peroxide to the last reading time (min).
  • Reagent Control tube CK Sample tube 0.05M pH5.5 PBS ( ⁇ L) 300 290 ⁇ 295 2% H 2 O 2 ( ⁇ L) 100 100 0.05 mol/L guaiacol 100 100 Crude enzyme solution ( ⁇ L) 0 5 ⁇ 10 Total volume ( ⁇ L) 500 500
  • the components are first added according to the sample tube of Table 1-2, and after the enzyme solution is added, it is immediately incubated at 34 ° C for 3 min. Then immediately add 500 ⁇ L of 0.05 M pH 5.5 PBS, rapidly dilute 1 time, measure at 470 nm wavelength, record absorbance once every 1 min, and measure the absorbance values of 1, 2, 3, 4, 5, 6, 7, 8 min, respectively;
  • the enzyme activity is calculated according to the following formula.
  • the change of A 470 per minute is 0.01 for one enzyme activity unit (Unit).
  • POD activity ⁇ A 470 ⁇ V 1 /(0.01 ⁇ V 2 ⁇ t ⁇ C)
  • ⁇ A 470 is the change of absorbance in the reaction time, that is, the measured A 470 reading;
  • V 1 is the total volume of the reaction liquid ( ⁇ L);
  • V 2 is the volume of the crude enzyme extract for measurement ( ⁇ L);
  • C is the crude enzyme extract Total protein concentration ( ⁇ g ⁇ L -1 );
  • 0.01 is A 470 per change of 0.01 for 1 enzyme unit (U);
  • t is reaction time (min).
  • Reagent Control tube CK Sample tube 0.05 mol/L pH 7.8 PBS (mL) 1.5 1.45 ⁇ 1.47 130mmol/L methionine Met solution (mL) 0.3 0.3 750 ⁇ mol/L nitroblue tetrazolium NBT solution (mL) 0.3 0.3 100 ⁇ mol/L EDTA-Na 2 solution (mL) 0.3 0.3 20 ⁇ mol/L riboflavin solution (mL) 0.3 0.3 Distilled water (mL) 0.25 0.25 Enzyme solution (mL) 0 0.05 ⁇ 0.03 Total volume (mL) 3 3 3 3 3
  • the SOD activity unit is expressed by inhibiting 50% of NBT photoreduction as an enzyme activity unit.
  • a CK is the absorbance of the light control tube
  • a E is the absorbance of the sample tube
  • V 1 is the total volume of the reaction solution ( ⁇ L)
  • V 2 is the volume of the crude enzyme extract for measurement ( ⁇ L)
  • C is the crude enzyme extract.
  • Oxidative stress has a strong killing effect on the strain.
  • the effect of high concentration H 2 O 2 on the growth of wild-type DR and mutant ⁇ dwh was analyzed by using hydrogen peroxide solution for oxidative shock treatment.
  • Fig. 5 and Fig. 6 are photographs of colonies of the growth state of D. radiodurans R1 strain before and after the H 2 O 2 impact test, wherein: a is a wild type DR strain; b is a D. radiodurans R1 mutant strain ⁇ dwh lacking the dwh gene; It can be clearly seen in the figure:
  • the viability of the wild-type DR strain and the dwh gene-deficient mutant ⁇ dwh decreased, while the mutant ⁇ dwh was more sensitive to oxidative stress; the mutant ⁇ dwh was more colony than the wild-type DR strain. At least an order of magnitude difference, their viability is much lower than that of wild-type DR strains (see Figure 6).
  • Antioxidase activity in R1 cells decreased; wild type (DR-H 2 O 2 ) and mutant strain ( ⁇ dwh-H 2 O 2 ) treated with 50 mM H 2 O 2 compared to untreated controls, antioxidant enzymes The activity decreased (except for SOD activity) (Fig. 7c), and the antioxidant enzyme activity of the mutant ⁇ dwh decreased more significantly, especially in the H 2 O 2 treated mutant ⁇ dwh in CAT (Fig. 7a) and POD (Fig. 7b). The enzyme activity decreased to 6%-10% of wild-type DR, indicating that the ability to scavenge oxygen free radicals of the dwh gene-deficient mutant ⁇ dwh was decreased under oxidative stress conditions.
  • the wild-type DR strain was more resistant to oxidative stress than the mutant dw gene ⁇ dwh, and the deletion of the dwh gene attenuated the ability of D. radiodurans R1 to scavenge oxygen free radicals (ROS).
  • ROS oxygen free radicals
  • a pair of PCR-specific primers were designed based on the published dwh gene sequence in the D.radiodurans R1 genome to amplify the complete nucleotide sequence from D.radiodurans R1 genomic DNA:
  • the target gene fragment was amplified from the genome of D. radiodurans R1 by PCR.
  • the reaction conditions were: 95 ° C for 10 min, [95 ° C for 30 sec, 62 ° C for 30 sec, 72 ° C for 30 sec] for 35 cycles, 72 ° C for 10 min, and the PCR product was gelatinized. After recovery, it was cloned into the blunt-end vector pJET1.2/Blunt and named as pJET-dwh, and verified by sequencing. Then, the pET28a expression vector containing the sticky end and the dwh gene fragment were obtained by double digestion with BamH I/Hind III. The fragment was ligated to the pET28a vector, and the E.
  • coli recombinant expression plasmid pET28a-dwh was constructed.
  • the expression plasmid was transformed into E. coli BL21, and the inserted sequence was confirmed by PCR, plasmid digestion and sequencing (see Fig. 8, 9). Named pET28a-dwh/BL21.
  • E. coli BL21 containing the pET28a empty plasmid was named pET28a/BL21.
  • the pET28a-dwh/BL21 strain containing the D.radiodurans R1 dwh gene obtained in Example 3 and the pET28a/BL21 strain containing the empty plasmid were inoculated into 20 mL LB liquid medium (containing Km antibiotic), and shaken overnight. After culturing (37 ° C), transfer to 100 mL of LB liquid medium, and keep the inoculum amount consistent. After incubation for 30 min, add IPTG to a final concentration of 0.1 mM, and continue to culture until OD 600 ⁇ 0.5.
  • Fig. 10 and Fig. 11 are photographs of colonies of E. coli growth before and after the H 2 O 2 impact test, wherein: a is an Escherichia coli BL21 strain containing an empty expression plasmid; b is a large intestine containing a D. radiodurans R1 dwh gene expression plasmid. Recombinant strain of Bacillus; as can be clearly seen from the figure:
  • the pET28a-dwh/BL21 strain containing the D.radiodurans R1 dwh gene was almost identical to the pET28a/BL21 strain containing the empty plasmid (see Figure 10);
  • the recombinant strain containing the D.radiodurans R1 dwh gene is much more resistant to oxidative stress than the strain containing only the empty plasmid, and the expression of this gene enhances the antioxidant capacity of Escherichia coli.

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Abstract

提供了一种从耐辐射异常球菌（Deinococcus radiodurans）R1中分离获得的dwh基因，其核苷酸序列如SEQ ID NO.1所示，其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。进一步提供了所述dwh基因用于培育抗氧化微生物的用途。该dwh基因的表达能够提高大肠杆菌的抗氧化能力。

Description

一种用于培育抗氧化微生物的基因 技术领域
本发明涉及一种用于培育抗氧化微生物的基因。
背景技术
耐辐射异常球菌(Deinococcus radiodurans R1,DR)具有对电离辐射、UV辐射、DNA损伤试剂等抗性，是研究微生物非生物胁迫适应机制的理想菌株。曾有人对耐辐射异常球菌中大量功能未知基因(如IrrE、Csp等蛋白)进行研究，比如，将D.radiodurans R1中4个与植物抗旱相关的基因在原核宿主细胞和植物中表达后，能够增强宿主的耐盐抗性等。
然而，对本研究发现的来源于D.radiodurans R1新基因dwh，并未见其在提高细胞抗氧化能力方面的研究报道。
发明内容
本发明的目的是从D.radiodurans R1中发现一种用于培育抗氧化微生物的新基因。
本发明通过如下研究发现：
1、耐辐射异常球菌R1中含有编码Why(Water stress and Hypersensitive response)类似蛋白的基因，命名为dwh，其核苷酸序列如SEQ ID NO.1所示，该基因编码的蛋白(Dwh蛋白)具有抗氧化的功能，可用于培育抗氧化微生物，进而为培育具有抗氧化性状的植物提供参考依据，其蛋白的氨基酸序列如SEQ ID NO.2所示。
2、利用http://web.expasy.org/protparam/对Dwh蛋白氨基酸序列进行在线预测，该蛋白由99个氨基酸组成，分子式C466H772N134O138，其分子量为10.46kDa，理论等电点为9.94，不稳定指数29.04，属于稳定蛋白质；对Dwh蛋白氨基酸序列分析发现，该蛋白富含Leu(15.15％)、Val(12.12％)、Ala(10.1％)、Thr(10.1％)，带负电氨基酸残基总数(Asp+Glu)为7，带正电氨基酸残基总数(Arg+Lys)为9，其中疏水氨基酸残基含量为50.5％，总平均疏水指数(GRAVY)为0.165，同时，采用Kyte & Doolittle方法对Dwh蛋白进行疏水性分析也显示出其高度的疏水特性(图1)。因此，推测该蛋白是一类典型的疏水蛋白。此外，二级结构预测显示(图2)，Dwh蛋白由6个β折叠和靠近C端的1个α螺旋组成。
3、获得D.radiodurans R1 dwh基因的缺失突变株Δdwh
通过PCR获得dwh基因上下游基因片段(dwh-U和dwh-D)，同时获得编码卡那霉素抗 性蛋白的Km基因片段(K)(图3)，采用融合PCR技术将dwh-U、K、dwh-D三片段融合，获得dwh-UKD重组片段。然后将重组片段通过同源重组技术转化D.radiodurans R1感受态细胞，在含有Km抗性的TGY平板上进行压力筛选，获得dwh基因的缺失突变株Δdwh，并进行PCR及测序验证(图4)(见实施例1)。
4、D.radiodurans R1 dwh基因缺失突变株Δdwh的抗氧化实验
1)对野生型DR及突变株Δdwh分别进行氧化胁迫处理(80mM H2O2处理30min)，观察不同菌株在氧化胁迫条件下的存活能力。结果显示，氧化胁迫条件下试验菌株存活能力均有下降，缺失dwh基因的突变株Δdwh与野生型DR菌株相比更加敏感，菌株存活能力下降1个数量级(见实施例2和图5、6)。结果表明dwh基因编码的蛋白对D.radiodurans R1耐受氧化胁迫具有重要贡献。
2)对野生型DR及突变株Δdwh分别进行50mM H2O2处理30min，收集并洗涤菌体，用适量的磷酸缓冲液重悬菌体，超声破碎细胞，离心获得上清液总蛋白，并测定各样品总蛋白浓度。测定野生型DR及突变株Δdwh细胞内的抗氧化酶活性(CAT、POD、SOD)。结果显示，氧化胁迫条件下，突变株Δdwh的抗氧化酶活性与野生型DR菌株相比，均有所下降，CAT和POD下降更加明显(见实施例2和图7)。
以上结果表明，dwh基因在D.radiodurans R1中表达能够增强细胞的抗氧化能力，可能通过保护细胞内抗氧化酶避免氧自由基的损伤，从而发挥保护功能。
5、获得含有D.radiodurans R1 dwh基因的重组工程菌株
1)通过PCR从D.radiodurans R1(CGMCC 1.633)菌株基因组扩增dwh基因，其核苷酸序列如SEQ ID NO.1所示。核苷酸长度为297bp(图8)，该基因编码99个氨基酸，将其克隆于载体pJET1.2/Blunt上，构建了含有完整dwh基因的重组质粒pJET-dwh(图9)；
2)将dwh基因连接于pET28a表达载体上，构建含有完整dwh基因的重组质粒pET28a-dwh；
3)将导入dwh基因的重组质粒pET28a-dwh转入原核宿主细胞受体大肠杆菌BL21中，获得重组工程菌株pET28a-dwh/BL21(见实施例3)；
6、含有D.radiodurans R1 dwh基因工程菌株的抗氧化实验
实验证实，经30mM H2O2处理10min后，含有D.radiodurans R1 dwh基因的pET28a-dwh/BL21重组菌株生长状况良好，菌落生存能力远高于只含空质粒的对照pET28a/BL21菌株(见实施例4及图10、11)。结果显示该工程菌株具有耐受高浓度H2O2胁迫的能力。
此实验表明：D.radiodurans R1 dwh基因具有提高大肠杆菌抗氧化的能力。
附图说明：
图1是D.radiodurans R1 Dwh蛋白的亲/疏水性分析图谱(其中横坐标表示氨基酸序列，纵坐标表示疏水性分值，疏水性分值越大表明该氨基酸疏水性越强；Hydrophilic——亲水，Hydrophobic——疏水)；
图2是D.radiodurans R1 Dwh蛋白的二级结构分析图谱；
图3是dwh基因上游dwh-U(1)、下游dwh-D(2)及抗性基因K(3)片段的PCR产物电泳图谱；
图4是突变株Δdwh的PCR验证图谱(泳道1-3:引物YZdwh-F/R分别以突变株Δdwh、野生型DR、无菌水为模板扩增dwh片段，验证dwh缺失完全；泳道4-6:引物YZdwh-U-F/D-R分别以突变株Δdwh、野生型DR、无菌水为模板扩增重组片段，验证dwh-UKD插入位置)；
图5是H2O2冲击试验前的野生型DR和突变株Δdwh生存能力的菌落照片，其中：
a是野生型的D.radiodurans R1菌株(DR)；
b是缺失dwh基因的D.radiodurans R1突变体菌株(Δdwh)；
图6是野生型DR和突变株Δdwh经80mM H2O2处理30min后，在TGY固体培养基中生长情况的菌落照片，其中：
a是野生型的D.radiodurans R1菌株(DR)；
b是缺失dwh基因的D.radiodurans R1突变体菌株(Δdwh)；
图7是野生型DR和突变株Δdwh经50mM H2O2处理30min后，细胞内的抗氧化酶活性的变化，其中：
a是突变株Δdwh在H2O2处理前后与野生型DR相比，细胞内的过氧化氢酶(CAT)活性的变化；
b是突变株Δdwh在H2O2处理前后与野生型DR相比，细胞内的过氧化物酶(POD)活性的变化；
c是突变株Δdwh在H2O2处理前后与野生型DR相比，细胞内的超氧化物歧化酶(SOD)活性的变化；
图8含有D.radiodurans R1 dwh基因的PCR产物的验证电泳图谱；
图9是含有D.radiodurans R1 dwh基因的原核重组表达质粒的构建验证电泳图谱；
图10是在H2O2冲击试验前的含有空质粒和含有D.radiodurans R1 dwh基因的原核表达质粒的大肠杆菌的生长状况的菌落照片，其中：
a是含有空表达质粒的大肠杆菌BL21菌株；
b是含有D.radiodurans R1 dwh基因表达质粒的大肠杆菌重组菌株；
图11是含有D.radiodurans R1 dwh基因的原核表达质粒及空质粒的大肠杆菌(E.coli)经受30mM H2O2处理10min后，在LB固体培养基中的生长情况的菌落照片，其中：
a是含有空表达质粒的对照大肠杆菌菌株(pET28a/BL21)；
b是含有D.radiodurans R1 dwh基因表达质粒的大肠杆菌重组菌株(pET28a-dwh/BL21)。
具体实施方式
以下实施例中所举的质粒、菌株只是用于对本发明作进一步详细说明，并不对本发明的实质内容加以限制。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例中所举的质粒、菌株及试剂如下：
(1)质粒：
平末端克隆载体pJET1.2/Blunt(Amp)购自Thermo Scientific公司，pKatAPH3(携带Km抗性基因)质粒由本实验保存，表达载体pET28a由本实验室保存，pJET-dwh(dwh基因片段与克隆载体pJET1.2/Blunt连接)、pET28a-dwh(dwh基因片段与克隆载体pET28a通过酶切连接)重组质粒由本研究构建。
(2)实验菌株：
耐辐射异常球菌Deinococcus radiodurans Rl野生型菌株(CGMCC 1.633)由中国普通微生物菌种保藏中心提供；突变株Δdwh(缺失dwh基因的D.radiodurans Rl菌株)由本研究构建，D.radiodurans Rl及其突变菌株均在TGY培养基中生长，最适培养温度为30℃；
大肠杆菌Escherichia coli trans 109及BL21菌株购自北京全式金生物技术公司；pET28a/BL21(空载体pET28a转化大肠杆菌BL21菌株获得的重组菌株)、pET28a-dwh/BL21(重组质粒pET28a-dwh转化大肠杆菌BL21菌株获得的重组菌株)由本研究构建，E.coli及其重组菌株均在LB培养基中生长，培养温度为37℃。
(3)生化试剂：限制性内切酶购自NEB公司；dNTPs、高保真Primestar HS DNA polymerase、T4 DNA连接酶等购自大连宝生物公司(TaKaRa)；琼脂糖凝胶DNA回收试剂盒、普通DNA产物纯化试剂盒、普通质粒小提试剂盒均购自天根生化科技公司(TIANGEN)；30％H2O2、IPTG、抗生素等购自阿拉丁(aladdin)；实验中所用引物合成及测序均由华大基因公司完成。
(4)培养基：
LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH调至7.0，固体培养基中加入agar 15g/L。高压蒸汽(121℃,1.034×105Pa)灭菌30min。
TGY培养基：胰蛋白胨10g/L，酵母提取物5g/L，葡萄糖1g/L，固体培养基中加入agar 15g/L。高压蒸汽(112℃,1.034×105Pa)灭菌30min。
(5)抗生素及主要溶液配制：
氨苄青霉素(Amp)(50mg/mL)：称取0.5g氨苄青霉素粉末，溶于10mL双蒸水中，滤膜过滤除杂后分装，保存于-20℃冰箱；
卡那霉素(Km)(50mg/mL)：称取0.5g卡那霉素粉末，溶于10mL双蒸水中，滤膜过滤除杂后分装，保存于-20℃冰箱；
IPTG(100mmol/L)：称取0.24g IPTG，溶于10mL双蒸水中，滤膜过滤除菌后分装，保存于-20℃冰箱；
CaCl2(0.3mol/L)：称取4.5g CaCl2·2H2O粉末，溶解于80mL双蒸水中，定容至100mL，高压蒸汽(121℃)灭菌；
0.1mol/L H2O2：30％的H2O2溶液5.68mL稀释至1000mL；
130mmol/L甲硫氨酸Met溶液：称1.9399g Met用磷酸缓冲液定容至100mL；
750μmol/L氮蓝四唑NBT溶液：称0.06133g NBT用磷酸缓冲液定容至100mL，避光保存；
100μmol/L EDTA-Na2溶液：称0.03721g EDTA-Na2，用磷酸缓冲液定容至1000mL；
20μmol/L核黄素溶液：称0.0753g核黄素用蒸馏水定容至1000mL，避光保存；
0.05mol/L愈创木酚溶液：称取6.2065g(124.13g/mol×0.05mol)，然后溶于1L 95％乙醇中，即为1L的0.05mol/L愈创木酚溶液；
2％H2O2：吸取20mL 30％H2O2溶于280mL蒸馏水中，即为300mL的2％H2O2。
所用PBS溶液按照分子克隆手册配备。
实施例1 获得D.radiodurans R1 dwh基因的缺失突变株Δdwh
从NCBI的GeneBank中获得D.radiodurans R1全基因组序列和质粒pKatAPH3序列，根据dwh基因序列和卡那抗性基因序列设计引物(见表1)。
表1 基因片段扩增所用的引物
Figure PCTCN2016076272-appb-000001
Figure PCTCN2016076272-appb-000002
采用融合PCR方法构建突变株Δdwh。以D.radiodurans R1基因组DNA为模板，分别用表1中合成的引物U-F/U-R扩增dwh基因上游片段，扩增产物dwh-U长度为749bp；用D-F/D-R引物扩增dwh基因下游片段，扩增产物dwh-D长度为595bp；以pKatAPH3质粒为模板，用K-F/K-R引物扩增卡那抗性基因片段，扩增产物K长度为1007bp。分别对各扩增产物进行纯化回收，条带单一(图3)，在反应体系中加入等摩尔的三片段产物，引物U-F/D-R，采用一步融合法进行融合PCR反应，得到三片段的融合产物dwh-UKD(2268bp)。PCR反应条件为：94℃ 10min；94℃ 1min；60℃ 1min；72℃ 5min；17个循环；94℃ 1min；60℃ 1min；72℃ 3min；30个循环；72℃ 10min。
对融合产物dwh-UKD(2268bp)进行测序验证，完全与实验设计的序列一致。通过同源重组转化野生型D.radiodurans R1。在含有10μg/mL的Km平板上筛选缺失突变株Δdwh。通过PCR方法对Δdwh进行鉴定。以突变株Δdwh基因组DNA为模板，用YZdwh-F/YZdwh-R引物扩增dwh基因片段，未获得346bp条带，而在阴性对照D.radiodurans R1基因组DNA中能够扩增出346bp大小条带；用YZdwh-U-F/YZdwh-D-R引物扩增YZdwh-UKD片段仅获得2591bp条带，以D.radiodurans R1基因组DNA为模板扩增获得1985bp条带，同时以无菌水作空白对照。结果表明卡那抗性基因完全替代了dwh基因，完整的整合到dwh基因在D.radiodurans R1基因组DNA上所在的位置(图4)。
实施例2 D.radiodurans R1 dwh基因缺失突变株Δdwh的抗氧化实验
一、实验方法
(1)菌株的抗氧化表型实验
分别从划线培养的TGY平板上挑取野生型DR与突变株Δdwh单菌落接种到2mL TGY液体培养基中(突变株培养基中含有10μg/mL Km抗生素)，30℃振荡培养至对数中期，1％分别转接到新鲜的50mL TGY液体培养基中(突变株培养基中含有10μg/mL Km抗生素)，培养至对数初期(OD600约为0.6)，各取1mL菌液，分别加入8μL 30％H2O2溶液，至菌液的H2O2终浓度为80mM(10mM H2O2≈1μL 30％H2O2)，并设置对照组(未加入H2O2)，30℃摇床分别温育30min后，立即用无菌去离子水等比稀释(10-1至10-5)。每个稀释度各取10μL点在TGY固体培养基表面，经30℃恒温培养2-3天，观察野生型和突变株在TGY培养基平板上的生长情况。分别进行三次独立重复实验。
(2)细胞内抗氧化酶活测定
分别从划线培养的TGY平板上挑取野生型DR与突变株Δdwh单菌落接种到20mL TGY液体培养基中(突变株培养基中含有10μg/mL Km抗生素)，30℃振荡培养至对数中期，1％分别转接到200mL新鲜TGY液体培养基中(突变株培养基中含有10μg/mL Km抗生素)，30℃ 220rpm培养至对数初期(OD600约为0.6)，分别用50mM H2O2处理野生型DR与突变株Δdwh，并设置对照组(未加入H2O2的野生型DR与突变株Δdwh)，摇床避光处理30min后，立即收菌(4℃ 8000rpm 10min)，用无菌水洗涤菌体两次，最后重悬于5mL 50mM pH 7.0的PBS缓冲液中；于冰上进行超声破碎处理细胞(功率不超过200W，工作2s，间隔3s，处理时间45～60min，变幅杆Φ3)，4℃ 12000rpm 20min离心，于冰上分装后测定各个样品总蛋白浓度(Bradford方法)，并保存总蛋白于-20℃。
A细胞内过氧化氢酶(CAT)活性测定
①室温条件下(25℃)，取1.5mL EP管，其中一个为样品测定管(每个样品1个)，另一个为空白对照管(CK)，按表2顺序加入试剂。
表2 过氧化氢酶(CAT)活性测定各组分配方表
试剂 对照管CK 样品管
0.2M pH7.8 PBS(μL) 100 90～95
0.1M H2O2(μL) 100 100
粗酶液(μL) 0 5～10
总体积(μL) 200 200
用UV-3010分光光度计测定，关掉钨灯WI(340～1200nm)，打开氘灯D2(190～340nm)，先调零，内侧比色皿加入1mL 0.2M pH 7.8PBS，外侧比色皿先加入CK溶液(200μL)，迅速调零；
②样品测定时，按表2顺序加入各成分，并迅速混合，立即开始计时，240nm下测定其吸光度，分别于15s、30s、45s、1min、1.5min、2min、3min读数。
③待所有样品全部测完后，按下列公式计算酶活性。以1min内A240减少0.1的酶量为1个酶活单位(Unit)。
CAT活性(Unit/μg·μL-1·min)＝ΔA240·V1/(0.1·V2·t·C)
式中：ΔA240为样品管吸光值的绝对值；V1为反应液总体积(μL)；V2为测定用粗酶提取液体积(μL)；C为粗酶提取液总蛋白浓度(μg·μL-1)；0.1为A240每下降0.1为1个酶活单位(U)；t为过氧化氢到最后一次读数时间(min)。
B细胞内过氧化物酶(POD)活性测定
①酶活性测定显色反应体系如下：
表3 过氧化物酶(POD)活性测定各组分配方表
试剂 对照管CK 样品管
0.05M pH5.5 PBS(μL) 300 290～295
2％H2O2(μL) 100 100
0.05mol/L愈创木酚 100 100
粗酶液(μL) 0 5～10
总体积(μL) 500 500
②首先调零：按照反应体系设置对照组CK，34℃保温3min后加入500μL 0.05M pH 5.5PBS，分别于内侧石英比色皿加入1mL 0.05M pH 5.5PBS，外侧比色皿加入CK溶液(200μL)，470nm波长下调零；
③同样，样品测定时首先按照表1-2的样品管加入各组分，待加入酶液后，立即于34℃保温3min。然后立即加入500μL 0.05M pH 5.5PBS，迅速稀释1倍，470nm波长下测定，每隔1min记录1次吸光度，分别测定1、2、3、4、5、6、7、8min的吸光值；
④待所有样品全部测完后，按下列公式计算酶活性。以每分钟内A470变化0.01为1个酶活性单位(Unit)。
⑤按下列公式计算出过氧化物酶活性Unit/(μg·μL-1·min)。
POD活性＝ΔA470·V1/(0.01·V2·t·C)
ΔA470为反应时间内吸光度的变化，即所测得的A470读数；V1为反应液总体积(μL)；V2为测定用粗酶提取液体积(μL)；C为粗酶提取液总蛋白浓度(μg·μL-1)；0.01为A470每变化0.01为1个酶活单位(U)；t为反应时间(min)。
C细胞内过氧化物酶(POD)活性测定
①SOD酶活性测定反应体系，在具塞试管(10mL)中按表4依次加入以下溶液：
表4 超氧化物歧化酶(SOD)活性测定各组分配方表
试剂 对照管CK 样品管
0.05mol/L pH 7.8PBS(mL) 1.5 1.45～1.47
130mmol/L甲硫氨酸Met溶液(mL) 0.3 0.3
750μmol/L氮蓝四唑NBT溶液(mL) 0.3 0.3
100μmol/L EDTA-Na2溶液(mL) 0.3 0.3
20μmol/L核黄素溶液(mL) 0.3 0.3
蒸馏水(mL) 0.25 0.25
酶液(mL) 0 0.05～0.03
总体积(mL) 3 3
②混匀后将1支对照管置暗处不照光，其他各管于4000lx日光下反应20min。
③至反应结束后，以不照光的对照管设置空白，分别取样品200μL于酶标板中，测定各管在560nm处的吸光值。
④按下列公式计算出SOD活性U/(μg·μL-1)。SOD活性单位以抑制NBT光化还原的50％为一个酶活性单位表示。
SOD活性＝(ACK-AE)·V1/(0.5·ACK·C·V2)
ACK为照光对照管的吸光值，AE为样品管的吸光值，V1为反应液总体积(μL)，V2为测定用粗酶提取液体积(μL)，C为粗酶提取液总蛋白浓度(μg·μL-1)。
二、实验结果
(1)氧化胁迫对菌株具有强烈的杀伤作用，利用过氧化氢溶液进行氧化冲击处理，分析高浓度H2O2对野生型DR和突变株Δdwh生长的影响。图5和图6是H2O2冲击试验前后的D.radiodurans R1菌株的生长状况的菌落照片，其中：a是野生型DR菌株；b是缺失dwh基 因的D.radiodurans R1突变株Δdwh；从图中可清楚看出：
H2O2处理前：
野生型DR菌株与缺失dwh基因的突变株Δdwh生长能力基本一致(见图5)；
H2O2处理后：
经80mM H2O2处理后，野生型DR菌株与缺失dwh基因的突变株Δdwh的生存能力均有所下降，而突变株Δdwh对氧化胁迫更加敏感；突变株Δdwh较野生型DR菌株的菌落形成相差至少一个数量级，其生存能力远低于野生型DR菌株(见图6)。
(2)分别用50mM H2O2处理野生型DR菌株与缺失dwh基因的突变株Δdwh 30min，测定四个不同菌株样品(DR、DR-H2O2、Δdwh、Δdwh-H2O2)细胞内抗氧化酶(CAT、POD、SOD)活性(图7)，结果显示，未处理的Δdwh细胞内的三种抗氧化酶活性均低于野生型DR菌株，表明dwh基因缺失导致D.radiodurans R1细胞内的抗氧化酶活性下降；经50mM H2O2处理的野生型(DR-H2O2)和突变株(Δdwh-H2O2)与未处理的对照相比，抗氧化酶活性均有所下降(SOD活性除外)(图7c)，突变株Δdwh的抗氧化酶活性下降更加显著，尤其是H2O2处理的突变株Δdwh中CAT(图7a)和POD(图7b)的酶活性下降为野生型DR的6％-10％，表明氧化胁迫条件下，缺失dwh基因的突变株Δdwh清除氧自由基的能力下降。
三、实验结论
野生型DR菌株对氧化胁迫的抗性高于缺失dwh基因的突变株Δdwh，dwh基因的缺失减弱了D.radiodurans R1清除氧自由基(ROS)的能力。
实施例3 D.radiodurans R1 dwh基因在大肠杆菌中的表达
根据已公布的D.radiodurans R1基因组中的dwh基因序列设计1对PCR特异性引物，从D.radiodurans R1基因组DNA中扩增完整的核苷酸序列：
Up    5′CGGGATCCATGCTCACGAGTTTCAGTTT3′；
Down  5′CCCAAGCTTTCACGTCAGAGTTCCGTCCA3′。
通过PCR方法从D.radiodurans R1的基因组中扩增出目的基因片段，反应条件：95℃ 10min，[95℃ 30sec，62℃ 30sec，72℃ 30sec]35个循环，72℃ 10min，PCR产物经胶回收后，克隆于平末端载体pJET1.2/Blunt上，命名为pJET-dwh，并测序验证；然后通过BamH I/Hind III双酶切获得含有粘性末端的pET28a表达载体及dwh基因片段，将dwh片段连接于pET28a载体上，构建大肠杆菌重组表达质粒pET28a-dwh，将该表达质粒转化大肠杆菌BL21，经PCR、质粒酶切、测序验证插入序列正确(见图8,9)，将该重组菌株命名为pET28a-dwh/BL21。
将含有pET28a空质粒的E.coli BL21命名为pET28a/BL21。
实施例4 含D.radiodurans R1 dwh基因重组菌株的抗氧化实验
一、实验方法
1、将实施例3中获得的含有D.radiodurans R1 dwh基因的pET28a-dwh/BL21菌株与含空质粒的pET28a/BL21菌株分别接种于20mL LB液体培养基(含Km抗生素)中，摇瓶过夜(37℃)培养后，再分别转接于100mL的LB液体培养基中，保持接种量的一致，培养30min后加入终浓度为0.1mM的IPTG，继续培养至OD600≈0.5即可。
2、取1mL的菌液，加入30％的H2O2溶液3μL使菌液终浓度为30mM，处理10min后，每个样品立即用无菌去离子水倍比稀释至10-5，取10μL点在LB固体培养基表面，经37℃培养16h，观察菌落形成情况并照相。
二、实验结果
图10和图11是H2O2冲击试验前后的大肠杆菌的生长状况的菌落照片，其中：a是含有空表达质粒的大肠杆菌BL21菌株；b是含有D.radiodurans R1 dwh基因表达质粒的大肠杆菌重组菌株；从图中可清楚看出：
H2O2处理前：
含有D.radiodurans R1 dwh基因的pET28a-dwh/BL21菌株与含空质粒的pET28a/BL21菌株生长能力基本一致(见图10)；
H2O2处理后：
经30mM H2O2处理后，只含空质粒的pET28a/BL21菌株几乎不能正常生长；
含有D.radiodurans R1 dwh基因的pET28a-dwh/BL21重组菌株生长状况良好，其生存能力远高于只含空质粒的pET28a/BL21菌株，对照菌株pET28a/BL21几乎不能正常生长(见图11)。
三、实验结论
含有D.radiodurans R1 dwh基因的重组菌株对氧化胁迫的抗性远高于只含空质粒的菌株，该基因的表达提高了大肠杆菌的抗氧化能力。

Claims (8)

  1. 一种用于培育抗氧化微生物的基因，其核苷酸序列如SEQ ID NO.1所示。
  2. 权利要求1所述基因培育抗氧化微生物的用途。
  3. 含权利要求1所述基因的重组质粒。
  4. 权利要求3所述的重组质粒培育抗氧化微生物的用途。
  5. 用权利要求3所述的重组质粒转化的原核宿主细胞。
  6. 含权利要求3所述的重组质粒的重组工程菌株。
  7. 一种能够提高微生物抗氧化能力的蛋白，其氨基酸序列SEQ ID NO.2所示。
  8. 权利要求1所述蛋白培育抗氧化微生物的用途。
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