阅读说明：本技术 一种六价铬还原菌株、方法及其应用 (Hexavalent chromium reducing strain, method and application thereof ) 是由 刘浩 曹威 李梦新 张小茹 张明怡 于 2021-10-08 设计创作，主要内容包括：本发明公开了一种六价铬还原菌株,所述菌株是通过在出发菌株黑曲霉中依次过表达六价铬还原酶编码基因chrB,铬转运蛋白编码基因sulB和超氧化物酶编码基因sodA获得的。本发明基于黑曲霉具有还原Cr(Ⅵ)的天然特性,通过提高铬还原酶表达增强Cr(Ⅵ)还原水平,引入铬转运元件增强Cr(Ⅵ)摄取能力,提高活性氧分解能力减弱Cr(Ⅵ)还原过程中产生的活性氧对细胞的损伤,从多个维度增强黑曲霉细胞对Cr(Ⅵ)还原水平。(The invention discloses a hexavalent chromium reducing strain, which is obtained by sequentially overexpressing a hexavalent chromium reductase encoding gene chrB, a chromium transport protein encoding gene sulB and a superoxide enzyme encoding gene sodA in an original strain Aspergillus niger. The method is based on the natural characteristic that Aspergillus niger has the capability of reducing Cr (VI), enhances the reduction level of Cr (VI) by improving the expression of chromium reductase, introduces a chromium transport element to enhance the Cr (VI) uptake capability, enhances the decomposition capability of active oxygen to weaken the damage of the active oxygen generated in the reduction process of Cr (VI) to cells, and enhances the reduction level of the Aspergillus niger cells to Cr (VI) from multiple dimensions.)

1. A hexavalent chromium reducing strain, which is characterized in that: the strain is obtained by over-expressing a hexavalent chromium reductase coding gene chrB and/or a chromium transporter coding gene sulB and/or a superoxide enzyme coding gene sodA in an original strain Aspergillus niger.

2. The hexavalent chromium reducing strain of claim 1, wherein: the starting strain Aspergillus niger is Aspergillus niger S469.

3. The hexavalent chromium reducing strain of claim 1, wherein: the promoters for controlling gene transcription are Aspergillus niger 3-glyceraldehyde phosphate dehydrogenase gene promoter PgpdA and pyruvate kinase gene promoter PpkiA.

4. The hexavalent chromium reducing strain of claim 1, wherein: the DNA sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.1 and the DNA sequence with more than 70% of similarity, and the amino acid sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.2 and the amino acid sequence with more than 80% of similarity.

5. The hexavalent chromium reducing strain of claim 1, wherein: the DNA sequence of the chromium transport protein coding gene sulB is SEQ NO.3 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.4 and the amino acid sequence with more than 80% of similarity.

6. The hexavalent chromium reducing strain of any one of claims 1 to 5, wherein: the DNA sequence of the superoxide enzyme coding gene sodA is SEQ NO.5 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.6 and the amino acid sequence with more than 80% of similarity.

7. Use of the hexavalent chromium reducing strain of any one of claims 1 to 6 for reducing hexavalent chromium.

8. The method for obtaining a hexavalent chromium reducing strain according to any one of claims 1 to 6, wherein: the method comprises the following steps:

in Aspergillus niger, the expression level of one or more than two of a hexavalent chromium reductase coding gene chrB, a chromium transport protein coding gene sulB and a superoxide enzyme coding gene sodA is up-regulated.

9. A biological process for reducing Cr (VI) by means of the hexavalent chromium reducing strain according to any of claims 1 to 6, wherein: the method comprises the following steps:

culturing the hexavalent chromium reducing strain, collecting thalli, and adding the thalli into a water body containing Cr (VI) for incubation.

10. The method for reducing Cr (VI) by a biological method according to claim 9, wherein: the concentration of Cr (VI) in the water body is 0-100mg/L, the incubation temperature is 10-40 ℃, the stirring speed is 0-300rpm, and the processing time is 10-96 h.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a hexavalent chromium reducing strain, a method and application thereof.

Background

Chromium occurs in nature primarily in two stable valence states, trivalent chromium Cr (iii) and hexavalent chromium Cr (vi). Cr (III) is generally present in nature, participates in the metabolic processes of lipid, glucose and protein in the body, has poor ability of crossing cell membranes and low toxicity. Cr (VI) is mainly generated in the industrial production process and mainly generatedAndthe product has strong oxidizing property, and can penetrate cell membrane to react with biological macromolecule (protein, DNA … …) due to its high water solubility, thereby generating carcinogenic and teratogenic effects. Since chromium is similar in structure to sulfate, it can enter the cell via the sulfate transporter pathway. Chromium is one of the most commonly used heavy metals in metallurgical, leather, steel, plastics, printing and dyeing, painting, paper and pulp processing, and manufacturing industries due to its corrosion resistance, odourlessness, high melting point, etc. Therefore, the chromium-containing wastewater is effectively treated before being discharged, and otherwise, the chromium-containing wastewater causes pollution to soil, underground water, sediment and the like. For example, the discharge standard of electroplating pollutants established in 2008 of China stipulates that the total chromium of a wastewater discharge port of an workshop or a production facility is less than or equal to 0.5mg/L, and the Cr (VI) is less than or equal to 0.1 mg/L. Therefore, the content of chromium, especially Cr (VI), in industrial wastewater is strictly controlled to be discharged into the environment, which is an important problem to be solved in the environmental protection field at present.

At present, the method for converting Cr (VI) into Cr (III) mainly comprises three methods, namely a chemical method, a physical method and a biological method. The chemical method mainly utilizes a chemical precipitation method or an electrolytic method to treat high-concentration chromium-containing wastewater; the physical method mainly utilizes the means of adsorption, ion exchange, membrane filtration and the like to recover and treat the chromium in the wastewater. The two methods are mainly applied to treating high-concentration chromium-containing wastewater, the efficiency is lower when the low-concentration chromium-containing wastewater is treated, the operation cost is higher, and the treated low-concentration chromium-containing wastewater still can not meet the national standard, so the application of the methods is limited. The biological method utilizes the flocculation, absorption, accumulation, enrichment and other effects of microorganisms, and has incomparable advantages of a chemical method and a physical method when treating low-concentration chromium-containing wastewater (1-100mg/L), such as wide sources of biological materials, mild reaction conditions, good selectivity, low cost, no secondary pollution and the like. Is considered to be a novel effective method for removing and recovering heavy metals from wastewater (Wang J.et al, Biotechnology Advances,2006,24(5):427- & 451).

Aspergillus niger is a GRAS strain, has simple requirements for growth nutrition and strong acid resistance, is easy to perform high-density cell culture in liquid submerged culture, has developed hyphae which can be intertwined to form a bacterial ball, has high settling speed and is easy for solid-liquid separation, and is widely applied to the fermentation industry and sewage treatment (Vendruscolo F.et al. International Biodetermination & Biofractionation, 2017,119: 87-95).

A significant progress has been made around Aspergillus niger as a functional strain for the biological treatment of Cr (VI) in industrial wastewater. For example: 1) digging a novel Aspergillus niger strain capable of reducing Cr (VI) (Wangbankun et al. microbiology report, 1998,38: 108-); vala A.K. et. Marine Pollution bulletin.2004,48: 983-; BennettR, et al, chemistry and ecology.2013,29: 320-328; ghosh S.et al.African Journal of Microbiology research.2015,9: 220-; 2) the reaction kinetics of Aspergillus niger inactivated mycelium to reduce Cr (VI) is Cr (III) (Park D.equivalent. Process biochemistry.2005,40: 2559-; park D.et al.Water research.2005,39: 533-; khambery y.et.world Journal of Microbiology and biotechnology.2009,25: 1413; khamhaze Y.et.chemical Engineering journal.2009,145: 489-495;archives of environmental protection.2013,39: 45-56); 3) aspergillus niger Cr (VI) adsorption and reduction molecular machineProduct of general knowledge (Narvekar S.et al. journal of environmental Science)&Engineering,2009,51:233–238；Shugaba A.et al.Journal of Petroleum&Enviromental Biotechnology.2012,03(119): 2; gu Y et al, environmental Science and Pollution research.2015,22: 6271-6279 Xu H.et al, geomicrobiology journal.2021,38: 1-9.); 4) the process exploration for treating chromium-containing wastewater by Aspergillus niger (Shugaba A.et al.Bioremedition journal.2010,14: 142-149; zhang L.et al.advanced Materials research.2011, 236-238: 155-; ) (ii) a 5) Development of a nano material-aspergillus niger composite novel Cr (vi) removal system (Daneshvar m.et al. environmental Science and Pollution research.2018,25: 28654-; chatterjee S.et.chemical Engineering journal.2020,385: 123790.).

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a hexavalent chromium reducing strain, a method and application thereof.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

a hexavalent chromium reducing strain is obtained by over-expressing a hexavalent chromium reductase encoding gene chrB and/or a chromium transporter encoding gene sulB and/or a superoxide enzyme encoding gene sodA in an original strain Aspergillus niger.

Further, the starting strain aspergillus niger is aspergillus niger S469.

The starting strain S469 is a strain used in an issued patent (Chinese patent, patent number: ZL201810985901.9) obtained by the inventor at the previous stage.

Furthermore, the promoters controlling gene transcription are the Aspergillus niger glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the pyruvate kinase gene promoter PpkiA, and other promoters capable of exerting transcription in Aspergillus niger can also achieve the purpose of up-regulating the expression of the genes.

Furthermore, the DNA sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.1 and the DNA sequence with more than 70% of similarity, and the amino acid sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.2 and the amino acid sequence with more than 80% of similarity.

Furthermore, the DNA sequence of the chromium transport protein coding gene sulB is SEQ NO.3 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.4 and the amino acid sequence with more than 80% of similarity.

Furthermore, the DNA sequence of the superoxide enzyme coding gene sodA is SEQ NO.5 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.6 and the amino acid sequence with more than 80% of similarity.

The use of a hexavalent chromium reducing strain as described above for reducing hexavalent chromium.

The strain for efficiently reducing Cr (VI) has the capability of reducing Cr (VI) in water bodies such as Cr (VI) in an aqueous solution containing Cr (VI) and industrial wastewater.

The strain for efficiently reducing and synthesizing the Cr (VI) containing aqueous solution and the Cr (VI) in the industrial wastewater can meet the capability of reducing Cr (VI) in other media through strain transformation or process improvement.

The method for obtaining a hexavalent chromium reducing strain as described above, comprising: the method comprises the following steps:

in Aspergillus niger, the expression level of one or more than two of a hexavalent chromium reductase coding gene chrB, a chromium transport protein coding gene sulB and a superoxide enzyme coding gene sodA is up-regulated.

A biological method for reducing Cr (VI) by utilizing the hexavalent chromium reducing strain comprises the following steps:

culturing the hexavalent chromium reducing strain, collecting thalli, and adding the thalli into a water body containing Cr (VI) for incubation.

Furthermore, the concentration of Cr (VI) in the water body is 0-100mg/L, the incubation temperature is 10-40 ℃, the stirring speed is 0-300rpm, and the treatment time is 10-96 h.

Further, the specific method comprises the following steps: aspergillus niger strains obtained by the above method are harvested for spores at 2X 10⁶one/mL of the strain is inoculated in an Aspergillus niger liquid culture medium, and the strain is obtained after culturing for 36-72h under the conditions of 10-40 ℃ and 100-300 rpm. Filtering to harvest thallus and buffering with PBSFlushing the thalli for 2-4 times, transferring the thalli to a water body containing 0-100mg/L Cr (VI), incubating for 96h under the conditions of 10-40 ℃, 100-300rpm, taking supernatant to determine the residual amount of hexavalent chromium, wherein the removal rate of Cr (VI) is more than 90%.

Further, the Aspergillus niger liquid culture medium in the Cr (VI) reduction technology mainly comprises the following components: 0 to 10 percent of cane sugar and 0 to 0.6 percent of (NH)₄)₂SO₄，0％～0.2％MgSO₄·7H₂O，0％～0.2％KH₂PO₄0-0.1% of yeast extract.

The invention has the beneficial effects that:

1. the method is based on the natural characteristic that Aspergillus niger has the capability of reducing Cr (VI), enhances the reduction level of Cr (VI) by improving the expression of chromium reductase, introduces a chromium transport element to enhance the Cr (VI) uptake capability, enhances the decomposition capability of active oxygen to weaken the damage of the active oxygen generated in the reduction process of Cr (VI) to cells, and enhances the reduction level of the Aspergillus niger cells to Cr (VI) from multiple dimensions.

2. The hexavalent chromium reducing strain can effectively reduce the water body containing 0-100mg/L Cr (VI) by treating for 96 hours at 10-40 ℃ and at 100-300rpm, and the reduction rate reaches more than 90 percent. The invention can be used for treating hexavalent chromium-containing industrial wastewater. .

3. The hexavalent chromium reducing strain can form bacteria balls, has high settling speed, is easy for solid-liquid separation, and can not cause secondary pollution when used for treating chromium-containing wastewater.

4. The hexavalent chromium reducing strain has simple and mild reaction conditions, and the energy consumption cost for treating the chromium-containing wastewater is lower than that of a chemical method and a physical method.

5. The invention establishes a method for obtaining a Cr (VI) bioremediation aspergillus niger strain which can be efficiently and without secondary pollution by enhancing the Cr (VI) reduction level, enhancing the Cr (VI) uptake efficiency of cells and weakening the damage of active oxygen generated in the Cr (VI) reduction process to the cells, and establishes a technical process for treating the water body containing Cr (VI) by using the strain.

Drawings

FIG. 1 is a map of the chrB gene expression plasmid pLH864 obtained in the present invention;

FIG. 2 is a single restriction enzyme digestion verification map (Kpn I, 9994bp/609bp) of chrB gene expression plasmid pLH864 in the present invention, wherein M is DNA Marker, N is negative control, and 1 is a Kpn I single restriction enzyme digestion verification plasmid;

FIG. 3 is a sulB gene expression plasmid pLH969 map obtained in the present invention;

FIG. 4 is a diagram showing the double restriction enzyme digestion verification of the sulB gene expression plasmid pLH969 (EcoR I/Xba I, 9251bp/3111bp) in the present invention, wherein M is a DNA Marker, N is a negative control, and 1 is an EcoR I and Xba I double restriction enzyme digestion verification plasmid;

FIG. 5 is a map of sodA gene expression plasmid pLH1032 obtained in the present invention;

FIG. 6 is a diagram showing the double restriction enzyme digestion verification of the sodA gene expression plasmid pLH1032 (EcoR I/Xba I, 9357bp/1205bp) in the present invention, wherein M is a DNA Marker, N is a negative control, and 1 is a double restriction enzyme digestion verification plasmid of EcoR I and Xba I;

FIG. 7 is a fluorescent micrograph of the active oxygen species of Aspergillus niger before and after Cr (VI) treatment of the S469 and (chrB, sulfoB, sodA)/OE strains of the present invention;

FIG. 8 shows the active oxygen intensities in Aspergillus niger before and after Cr (VI) treatment for S469 and (chrB, sulB, sodA)/OE strains according to the present invention;

FIG. 9 is a graph showing the change in the concentration of Cr (VI) in a water body treated with the S469 and (chrB, sulB, sodA)/OE strains according to the present invention;

FIG. 10 is a graph showing the change with time of the Cr (VI) concentration in wastewater from a plating plant treated with the (chrB, sulfoB, sodA)/OE strain of the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A hexavalent chromium reducing strain is obtained by over-expressing a hexavalent chromium reductase encoding gene chrB and/or a chromium transporter encoding gene sulB and/or a superoxide enzyme encoding gene sodA in an original strain Aspergillus niger.

Preferably, the starting strain aspergillus niger is aspergillus niger S469.

The starting strain S469 is a strain used in an issued patent (Chinese patent, patent number: ZL201810985901.9) obtained by the inventor at the previous stage.

Preferably, the promoters controlling gene transcription are the Aspergillus niger glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the pyruvate kinase gene promoter PpkiA, and other promoters capable of performing transcription in Aspergillus niger can also achieve the purpose of up-regulating the expression of the genes.

Preferably, the DNA sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.1 and the DNA sequence with more than 70% of similarity, and the amino acid sequence of the hexavalent chromium reductase coding gene chrB is SEQ NO.2 and the amino acid sequence with more than 80% of similarity.

Preferably, the DNA sequence of the chromium transport protein coding gene sulB is SEQ NO.3 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.4 and the amino acid sequence with more than 80% of similarity.

Preferably, the DNA sequence of the superoxide enzyme coding gene sodA is SEQ NO.5 and the DNA sequence with more than 70% of similarity, and the amino acid sequence thereof is SEQ NO.6 and the amino acid sequence with more than 80% of similarity.

The use of a hexavalent chromium reducing strain as described above for reducing hexavalent chromium.

The strain for efficiently reducing Cr (VI) has the capability of reducing Cr (VI) in water bodies such as Cr (VI) in an aqueous solution containing Cr (VI) and industrial wastewater.

The strain for efficiently reducing and synthesizing the Cr (VI) containing aqueous solution and the Cr (VI) in the industrial wastewater can meet the capability of reducing Cr (VI) in other media through strain transformation or process improvement.

The method for obtaining the hexavalent chromium reducing strain comprises the following steps:

in Aspergillus niger, the expression level of one or more than two of a hexavalent chromium reductase coding gene chrB, a chromium transport protein coding gene sulB and a superoxide enzyme coding gene sodA is up-regulated.

A biological method for reducing Cr (VI) by utilizing the hexavalent chromium reducing strain comprises the following steps:

culturing the hexavalent chromium reducing strain, collecting thalli, and adding the thalli into a water body containing Cr (VI) for incubation.

Preferably, the concentration of Cr (VI) in the water body is 0-100mg/L, the incubation temperature is 10-40 ℃, the stirring speed is 0-300rpm, and the treatment time is 10-96 h.

Preferably, the specific method comprises the following steps: aspergillus niger strains obtained by the above method are harvested for spores at 2X 10⁶one/mL of the strain is inoculated in an Aspergillus niger liquid culture medium, and the strain is obtained after culturing for 36-72h under the conditions of 10-40 ℃ and 100-300 rpm. Filtering the filter cloth to harvest thallus, washing the thallus for 2-4 times by PBS buffer solution, transferring the thallus to a water body containing 0-100mg/LCr (VI), incubating for 96h under the conditions of 10-40 ℃ and 100-300rpm, taking supernatant to determine the residual amount of hexavalent chromium, wherein the removal rate of Cr (VI) is more than 90%.

Preferably, the Aspergillus niger liquid culture medium in the Cr (VI) reduction technology mainly comprises the following components: 0 to 10 percent of cane sugar and 0 to 0.6 percent of (NH)₄)₂SO₄，0％～0.2％MgSO₄·7H₂O，0％～0.2％KH₂PO₄0-0.1% of yeast extract.

Specifically, the preparation and detection are as follows:

example 1

Obtaining of over-expression chromium reductase gene chrB plasmid:

the plasmid pLH864 (FIG. 1) is obtained by transforming pLH454 (a patent previously granted by the present applicant, patent No.: ZL201810985901.9) vector. The gene chrB sequence fragment is transcribed under the control of an Aspergillus niger 3-glyceraldehyde phosphate dehydrogenase gene promoter PgpdA.

The specific acquisition process is as follows: carrying out PCR amplification on a chrB gene sequence fragment by taking the cDNA of the S469 strain as a template and chrB-F, chrB-R as a primer, wherein the nucleotide sequence is SEQ NO.1, the length is 606bp, the amino acid sequence is SEQ NO.2, and the length is 201 aa; the chrB gene sequence fragment and the starting vector pLH454 linearized by Kpn I are connected by using a Novozan C113-Clon express-MultiS One Step Cloning Kit, the connection product is transformed into escherichia coli JM109 competent cells, the competent cells are uniformly coated on an LB culture dish containing 100 mu g/mL kanamycin and cultured overnight at 37 ℃, single clones are picked up, and the plasmid pLH864 is obtained through single enzyme digestion verification (figure 2). The primers for amplifying the chrB gene sequence fragment were designed as chrB-F, chrB-R (Table 1).

Example 2

Acquisition of a chromium reductase gene chrB overexpression Aspergillus niger strain:

agrobacterium containing plasmid pLH864 was co-cultured with Aspergillus niger host strain S469 on IM plates, and the co-culture was then transferred to CM plates containing 200. mu.M cefotaxime, 100. mu.g/mL ampicillin, 100. mu.g/mL streptomycin, 250. mu.g/mL hygromycin B and cultured at 28 ℃ until single colonies were formed. Selecting a single clone to be transferred to a PDA plate containing hygromycin B, screening hygromycin B resistant transformants, extracting a genome for verification, collecting correct transformant spores, inoculating the correct transformant spores to a doxycycline plate containing 30 mu g/mL, inducing an hph resistance screening marker to be cut off from the genome, and obtaining a hygromycin sensitive chrB gene over-expression strain chrB/OE.

Example 3

Obtaining of over-expression sulfate transporter gene sulB plasmid:

the plasmid pLH969 (FIG. 3) was modified from pLH454 (a previously issued patent by the inventors: ZL201810985901.9) vector. The gene sulB sequence fragment is transcribed under the control of an Aspergillus niger 3-glyceraldehyde phosphate dehydrogenase gene promoter PgpdA.

The specific acquisition is as follows: carrying out PCR amplification on a sulB gene sequence fragment by taking S469 strain cDNA as a template and sulB-F, sulB-R as a primer, wherein the nucleotide sequence is SEQ NO.3, and the length is 2193 bp; the amino acid sequence is SEQ NO.4, and the length is 730 aa; the sequence fragment of the sulB gene is connected with an original vector pLH454 linearized by two enzyme digestion of EcoR I and BamH I by using a Novozan C113-Clon express-MultiS One Step Cloning Kit, a connecting product is transformed into escherichia coli JM109 competent cells, the competent cells are uniformly coated on an LB culture dish containing 100 mu g/mL kanamycin and cultured overnight at 37 ℃, a single clone is picked up, and the plasmid pLH969 is obtained by two enzyme digestion verification (figure 4). Primers were designed for amplification of the fragment of the sulB gene sequence as sulB-F, sulB-R (Table 1).

Example 4

Acquisition of sulfate transporter gene sulB overexpression Aspergillus niger strains:

agrobacterium containing plasmid pLH969 was co-cultured with spores of the A.niger chrB/OE strain of example 2 on IM plates, and the co-culture was then transferred to CM plates containing 200. mu.M cefotaxime, 100. mu.g/mL ampicillin, 100. mu.g/mL streptomycin, 250. mu.g/mL hygromycin B and cultured at 28 ℃ until single colonies were formed. Selecting a single clone to be transferred to a PDA plate containing hygromycin B, screening hygromycin B resistant transformants, extracting a genome for verification, collecting correct transformant spores, inoculating the correct transformant spores to a doxycycline plate containing 30 mu g/mL, inducing an hph resistance screening marker to be cut off from the genome, and obtaining a hygromycin sensitive chrB and sulB gene co-overexpression strain (chrB, sulB)/OE.

Example 5

Obtaining of over-expression superoxide dismutase gene sodA plasmid:

plasmid pLH1032 (FIG. 5) was engineered from the pLH509 (previously issued patent by the inventors: ZL201810985901.9) vector. The sequence fragment of the gene sodA is transcribed under the control of an Aspergillus niger pyruvate kinase gene promoter PpkiA.

The specific acquisition process is as follows: carrying out PCR amplification on a sodA gene sequence fragment by taking the S469 strain cDNA as a template and sodA-F, sodA-R as a primer, wherein the nucleotide sequence is SEQ NO.5, and the length is 465 bp; the amino acid sequence is SEQINO. 6, and the length is 154 aa; the sodA gene sequence fragment and an original vector pLH509 linearized by two enzyme digestion of EcoR I and Kpn I are connected by using a Novozan C113-Clon express-MultiS One Step Cloning Kit, a connection product is transformed into escherichia coli JM109 competent cells, the competent cells are evenly spread in an LB culture dish containing 100 mu g/mL kanamycin and are cultured overnight at 37 ℃, a single clone is picked up, and the plasmid pLH1032 is obtained by two enzyme digestion verification (figure 6). Amplification of sodA Gene sequence fragment the primers were designed as sodA-F, sodA-R (Table 1).

Example 6

Acquisition of superoxide dismutase gene sodA overexpression Aspergillus niger strains:

agrobacterium containing plasmid pLH1032 was co-cultured with spores of Aspergillus niger (chrB, sulB)/OE strain of example 4 on IM plates, and the co-culture was then transferred to CM plates containing 200. mu.M cefotaxime, 100. mu.g/mL ampicillin, 100. mu.g/mL streptomycin, 250. mu.g/mL hygromycin B and cultured at 28 ℃ until single colonies were formed. Selecting a single clone to be transferred to a PDA plate containing hygromycin B, screening hygromycin B resistant transformants, extracting a genome for verification, collecting correct transformant spores, inoculating the correct transformant spores to a plate containing 30 mu g/mL doxycycline, inducing an hph resistance screening marker to be cut off from the genome, and obtaining a hygromycin sensitive sodA gene over-expression strain (chrB, sulB, sodA)/OE.

Example 7

The effect of Cr (VI) on ROS levels in Aspergillus niger strain S469 and (chrB, sulB, sodA)/OE cells was qualitatively and quantitatively analyzed.

Microscopic observation and active oxygen quantitative analysis:

(1) inoculating 2X 10 in Aspergillus niger liquid culture medium⁶Culturing Aspergillus niger spores at 10-40 deg.C and 100-300rpm for 36-72 hr, and adding 0-50 μ g/mL hexavalent chromium for treatment for 5-12 hr;

(2) the mycelia were collected by centrifugation at 3000rpm at 4 ℃. Washing mycelium with PBS for 3 times, incubating with 10mM DCFH-DA (active oxygen determination reagent 6-carboxyl-2, 7-dichlorodihydrofluorescein diacetate) at 37 deg.C in dark for 20-30min, and reversing and mixing at intervals of 3-5 min;

(3) after incubation, the mycelium was washed 3-5 times with PBS.

(4) A portion of the treated mycelia was mounted on a glass slide, and the slide was sectioned, observed with a fluorescence microscope, and photographed and recorded (FIG. 7).

(5) PBS resuspended DCFH-DA labeled bacteria, and aliquoted into 96-well plates (200. mu.L per well), three replicates. The 96-well plate was placed in a multifunctional microplate reader imaging system and the fluorescence intensity was measured (FIG. 8).

Example 8

The technology for reducing Cr (VI) in water by a biological method comprises the following steps:

collecting the above Aspergillus niger (c)hrB spores of SulB, sodA)/OE strain at 2X 10⁶Inoculating the strain/mL into 50mL Aspergillus niger liquid culture medium, culturing for 36-72h under the conditions of 10-40 ℃, 100-300rpm, filtering by using filter cloth to harvest the strain, washing the strain for 2-4 times by using PBS buffer solution, transferring the strain into PBS (phosphate buffer solution) and yeast extract culture medium containing 0-100mg/L Cr (VI), incubating for 96h under the conditions of 10-40 ℃ and 100-300rpm, sampling at intervals of 12000rpm, centrifuging for 10-20min, and determining the concentration change of Cr (VI) in the supernatant by using a dibenzoyl dihydrazide spectrophotometry (GB/T7467-1987), wherein the removal rate of Cr (VI) is more than 99% (figure 9).

Preferably, the Aspergillus niger liquid culture medium in the Cr (VI) reduction technology mainly comprises the following components: 0 to 10 percent of cane sugar and 0 to 0.6 percent of (NH)₄)₂SO₄，0％～0.2％MgSO₄·7H₂O，0％～0.2％KH₂PO₄0-0.1% of yeast extract.

Composition of PBS buffer: 0 to 1.5 percent of NaCl, 0 to 0.1 percent of KCl and 0 to 0.5 percent of Na₂HPO₄，0％～0.1％KH₂PO₄。

Example 9

The technology for efficiently reducing Cr (VI) strain to reduce Cr (VI) -containing wastewater of certain electroplating enterprises comprises the following steps:

a certain amount of Cr (VI) -containing wastewater is taken from a total wastewater discharge port of a workshop taken from certain Tianjin electroplating enterprises, and the Cr (VI) concentration is determined to be 83.6 mg/L. Spores of the A.niger (chrB, sulB, sodA)/OE strain of example 6 were harvested at 2X 10⁶Inoculating the strain/mL into 50mL of Aspergillus niger liquid culture medium, culturing at 10-40 ℃ under the condition of 100-300rpm for 36-72h, filtering by using filter cloth to obtain thallus, transferring the thallus to the Cr (VI) -containing wastewater taken from electroplating enterprises, and setting a control group without adding Aspergillus niger thallus. Incubating for 96h under the conditions of 10-40 ℃, 100-300rpm, sampling at 12000rpm at regular intervals, centrifuging for 10-20min, and determining the concentration change of Cr (VI) in the supernatant by using a dibenzoyl dihydrazide spectrophotometry (GB/T7467-1987). As shown in FIG. 10, the Cr (VI) concentration in the wastewater without the Aspergillus niger mycelia was not significantly changed. The concentration of Cr (VI) in the waste water added with the Aspergillus niger mycelium is reduced to 5.02mg/L,the reduction rate reaches 94 percent, and the hexavalent chromium in the wastewater can be quickly and effectively removed (figure 10).

Table 1 primer sequences used in the examples

Wherein, (1) the LB culture medium comprises the following components:

0 to 2 percent of tryptone, 0 to 1 percent of yeast extract, 0 to 2 percent of NaCl, the pH value is adjusted to 7.0 to 7.2, and 1.5 percent (W/V) of agar powder is added into a solid culture medium. Sterilizing at 121 deg.C for 20 min. After sterilization, kanamycin was added to a final concentration of 100. mu.g/mL when cooled to about 50-60 ℃.

(2) The PDA culture medium comprises the following components: 100-500g of potatoes are cut into small pieces, 500-3000mL of water is added for boiling for 20-40min, and the clear liquid is filtered by double-layer gauze. Then 10-50g of glucose is added to be completely dissolved, and water is added to the mixture until the volume is 500-3L. Solid culture of 1.5% (W/V) agar powder. Autoclaving at 121 deg.C for 20 min.

(3) The IM medium comprises the following components:

adding 15-20 g of agar powder, adding water to 905.7mL, sterilizing at 121 ℃ for 20min, heating by microwave until the agar is completely dissolved, and adding: 0 to 0.1 percent of K buffer, 0 to 5 percent of MN buffer, 0 to 0.2 percent of 1 percent of CaCl₂·2H₂O，0％～2％0.01％FeSO₄，0％～1％IM Trace elements，0％～1％20％NH₄NO₃0-2% of 50% of glycerol, 0-6% of 1M MES and 0-1% of 20% of glucose.

Preparation of required reagents in the IM medium:

1) k buffer: 1.25M K₂HPO₄Adding into 1.25M KH₂PO₄Resulting in a pH of 4.8.

(a)：1.25M KH₂PO₄：KH₂PO₄17.01g, adding deionized water to make the volume to 100mLAnd sterilizing at 121 ℃ for 20 min.

(b)：1.25M K₂HPO₄：K₂HPO₄21.77g, adding deionized water to 100mL, and sterilizing at 121 ℃ for 20 min.

2)MN buffer：0％～0.6％MgSO₄·7H₂O, 0 to 0.3 percent of NaCl, adding deionized water to dissolve, and sterilizing for 20min at 121 ℃.

3)1％CaCl₂：CaCl₂·2H₂O1g, adding deionized water to 100mL, and sterilizing at 121 ℃ for 20 min.

4)0.01％FeSO₄：FeSO₄·7H₂0.01g of O, adding deionized water to the solution until the volume is 100mL, and sterilizing the solution for 20min at 121 ℃.

5)IM Trace elements：0％～0.05％ZnSO₄·7H₂O，0％～0.05％CuSO₄·5H₂O，0％～0.05％H₃BO₃，0％～0.05％MnSO₄·H₂O，0％～0.05％Na₂MoO₄·2H₂And O, adding deionized water to dissolve, and sterilizing for 20min at 121 ℃.

6)20％NH₄NO₃: addition of NH₄NO₃20g, adding deionized water to 100mL, and sterilizing at 121 ℃ for 20 min.

7) 50% of glycerin: adding 50mL of glycerol, adding deionized water to the volume of 100mL, and sterilizing at 121 ℃ for 20 min.

8)1M MES: 19.524g of MES, adding deionized water to the solution until the volume is 100mL, adding NaOH to adjust the pH value to 5.5, and filtering and sterilizing the solution. Storing in dark for one month, or subpackaging and storing at-20 deg.C.

9) 20% glucose: glucose 20g, ddH added₂And (4) metering the volume of O to 100mL, and sterilizing at 115 ℃ for 20 min.

(4) The CM medium comprises the following components:

15-20 g of agar powder, adding water to 897mL, and sterilizing at 121 ℃ for 20 min. After the agar is completely dissolved, adding the following components: 0 to 5 percent of ASP + N, 0 to 5 percent of 50 percent of glucose, 0 to 0.5 percent of 1M MgSO₄0-0.5% of CM Trace elements, 0-5% of 10% of casein hydrolysate and 0-10% of yeast extract.

Preparation of required reagents in the CM medium:

1)ASP+N：0％～0.5％KCl(350mM)，0％～1.5％KH₂PO₄(550mM)，0％～5％NaNO₃(3.5M), adding deionized water to dissolve, pH5.5(5MKOH), and sterilizing at 121 deg.C for 20 min.

2) 50% glucose: glucose 50g, add ddH₂And (4) metering the volume of O to 100mL, and sterilizing at 115 ℃ for 20 min.

3)1M MgSO₄：MgSO₄24.648g, add ddH₂And (4) metering the volume of O to 100mL, and sterilizing at 121 ℃ for 20 min.

4)CM Trace elements：0％～0.5％ZnSO₄·7H₂O(76mM)，0％～0.3％H₃BO₃(178mM)，0％～0.1％MnCl₂·4H₂O，0％～0.1％FeSO₄·7H₂O，0％～0.05％CoCl₂·6H₂O，0％～0.05％CuSO₄·5H₂O，0％～0.04％Na₂MoO₄·2H₂O, 0 to 1 percent of EDTA, adding deionized water to dissolve, and sterilizing for 20min at 121 ℃.

5) 10% casein hydrolysate: casein hydrolysate 10g, ddH₂And (4) metering the volume of O to 100mL, and sterilizing at 121 ℃ for 20 min.

6) 10% yeast extract: 10g of yeast extract, ddH was added₂And (4) metering the volume of O to 100mL, and sterilizing at 121 ℃ for 20 min.

(5) MM medium composition described above: 0 to 5 percent of Vogel' sSalts, 0 to 3 percent of glucose, 0 to 3 percent of agar and distilled water. Sterilizing at 121 deg.C for 20 min.

Preparation of required reagents in the MM medium:

1) vogel's 50 × salts: 0 to 20 percent of sodium citrate and 0 to 40 percent of KH₂PO₄，0％～20％NH₄NO₃，0％～3％MgSO₄·7H₂O，0％～0.2％CaCl₂·2H₂.0 to 1 percent of trace element solution, 0 to 0.5 percent of biotin solution, and distilled water for dissolving, adding 0 to 0.1 percent of chloroform as a preservative, and preserving at room temperature.

2) Micro-meterMeasuring element solution: 0 to 0.1 percent of citric acid H₂O，0％～0.1％ZnSO₄·7H₂O，0％～0.2％Fe(NH₄)₂(SO₄)₂·6H₂O，0％～0.05％CuSO₄·5H₂O，0％～0.01％MnSO₄·H₂O，0％～0.01％H₃BO₃，0％～0.1％Na₂MoO₄·2H₂Dissolving O in distilled water, adding 0-0.1% chloroform as preservative, and storing at room temperature.

3) Biotin solution: 0 to 0.5 percent of biotin, and is dissolved by distilled water and stored at the temperature of minus 20 ℃.

Sequence listing

Seq No. 1: nucleotide sequence 606bp of chrB

ATGCCTCCCTCTATCGGCCTCATAATCTGCAGCCAACGCACTCCACGCGCAGGCCCTCAAATCGCCACCTTCATTCACAATACTATTCGTGAATCCTACCCCCCCGAAACAGCCACAATTACGACCATTGACCTAGCAAAATGGAACCTTCCACTCTACAATGAGTCGGGGATGCCATCGTTCATCAACTCAGCGGACGAGTACGAGCACGAGCACACAAAGGCCTGGTCACGGGAGATATCGCGCCACGAAGCGTTTATTTTCGTCACACCGCAGTATAACTGGGGGTATCCCGCAAGCGTGAAGAATGCGATTGATTACTTGTTCCATGAGTGGAAGGGAAAGGCGGCGTTGGTGGTGAGCTATGGGGGGCATGGGGGCGGGAAGGCGGCGGCGCAATTGAGGCAGGTGTTGCAGGGGGTGAGGATGAGGCCATTGGAGAGGATGGTCGAGCTGAGGTTTCCGGAGATCGAGGAAGTTAAGAGGGCCGCTAAGGGGGAGGATCTGGGACTGAATGGTGAGGGAGGGTATTGGGCTGGGGAGAGGGAGGGGATTAAGGCTGCGTTTGGGGAATTGATTGCTGCGCTGGAGGGAGAGGCTGAATAG

Seq No. 2: amino acid sequence 201aa of chrB

MPPSIGLIICSQRTPRAGPQIATFIHNTIRESYPPETATITTIDLAKWNLPLYNESGMPSFINSADEYEHEHTKAWSREISRHEAFIFVTPQYNWGYPASVKNAIDYLFHEWKGKAALVVSYGGHGGGKAAAQLRQVLQGVRMRPLERMVELRFPEIEEVKRAAKGEDLGLNGEGGYWAGEREGIKAAFGELIAALEGEAE

Seq No. 3: nucleotide sequence 2193bp of sulB

ATGACGGATCCCAATCCGCAGGGCGGACACCTCGAACACCGCTCTTTGCGGGATTGGCTTGCCAACTTCTTCCGAGCACCTTTGTCAAACAGTTCTGCAGATTCTCCGCCTGAACGGATTCCCGAGACCTTAGAACCGAATGATAGAACCCGCTTGTTGAAATTGGACGATGGTCAAGGTCCCGCATACGGTACAAGAGATGGTTTATCGACGCCGCAGGTTGACGGTGGGCCTGAAGGACATATTCGGAACGGGAATGGAAGCCCGACGTTCGTACAAGGGCCGGAGAACCTGCCTCCCAGGACTGGGTCAGGCCTGGCATGGCCTGTGAAGAACCATAAGACTATGTACCTCTCATACTACATCCCGTTCTTCAACTGGATCACACAATATCGCTGGTCTTTCCTCCGAGGCGACTTAATTGCAGCCTTGACAATCGCGTCAATCTACATTCCCATGGCCCTATCTTTGGCTTCGAACCTCGCCCATGCACCAGCCATCAACGGCCTTTACTCATTCGTTTTCATGCCACTGCTTTATGCAATTCTAGGAAGCAGTCCTCTGCTCGTTGTTGGTCCGGAAGCAGCTGGGTCGCTATTGACGGGAACAATCGTTAAAACTAGCGTCAAACAAGGCCACTCGCAGGAGGATGATGAGGCCGCAAGCGCAATGGTTGTAGGCATAGCCACTGCCATGGCAGGAGCAATGATACTAGTCGCTGGACTGACTAGGCTGGGTTTCCTGGACAATGTACTCAGTCGGCCTTTTTTGAGAGGCTTCATTACTGCCATTGGCTTTGTCATCTTTGTTGACCAGCTCATCCCGGAGGTTGGACTGGCAGACTTGGCCAAAGAGGCCGGTGTCAGTCATGGCAGCACAGTGCAGAAGCTTGTCTTTCTTGCTGGGAATATCAAAAGCTGTCATGGTCTTACAGCTGCTGTATCCTTCGGAAGTTTTGCAATCATTATGCTCTTTCGAACAATCAAAAAAGCACTAGAACCACGATACCCTCAGGTCGTATACTTCCCCGACCGTATCCTTGTCGTGATCCTCTCGGCCATTCTGACCTGGCGTCTTGGTTGGTACGAGCAGGGGCTGGAAGTTCTGGGGTCGGTTCAGAACACTGCCACTGGGCTCTTCGCCTTCAGGTGGCCCTTCCAAATCAAACATCTGAAGCATGTGCGCACCGCCATGAGCACCTCCTTCATCATTGCTATCCTTGGCTTCTTCGAGTCCTCGGTTGCTGCCAAGGGGCTCGGGGAGAGACGAGACGGGGTCCAAGGCATGTCTGTCAGTGCGAATAGGGAGATGGTTGCTCTGGGCGTTGCTAATGTGGTTGGGGGCTGCTTTATGGCTCTCCCTGCCTTTGGTGGATACGGACGGAGCAAAGTCAACGCCTCTACGGGCGCTCGCTCTCCTATGAGCAGTATCTTCCTCAGTATTATCACCTTCATTGTTATTATGGTGCTCTTGCCGTACCTATACTATCTTCCTAAAGCTGTACTATGCTCTACGATATCGGTCGTCGCATACAGTCTTATAGAGGAATGTCCTCACGACGTTGCCTTCTTCATCCGGCTGCGTGGTTGGACAGAGCTTGCATTGATGCTTCTCATCTTCGTCTCCACTATATTCTACTCGCTGGAGCTGGGTATCGCGCTTGGCATTGGGCTCTCAATCCTGATCCTTATTCGCCACTCCACCCAGCCTCGCATACAGATTCTCGGGAAAATAGCTGGAACACCCGACCAATACGATAACGCCGAAATGCATGCGGAAAATGTGGAGCTCATTGACGGTGCCCTTGTTGTTAAGATCCCCGAGCCGCTCACCTTCGCCAACACTGGCGATCTCAAGAATCGCTTGCGCCGCCTAGAGTTCTACGGCTCAAATCGCGCACATCCGTCCCTTCCACGGTTGCGCCCCCCGGAATCAAATAAGAATGTTATCTTCGACGTTCACGGTGTTACGAGCATTGATGGCTCCGGTACGCAGGTCCTCTCCGAGATCGTTAATGATTACATCAATCTCGGAGTAAGCGTGTATTTCTGTCGGCTTTCGAACCGCAGCGTCTTCCGCATGTTCGAAAGAAGCGGCATAGTCGACCGGTGTGGAGGCATGTCTCATTTTGTCACCGGTGTTGATGAGGCGCTTCGGCTCGCTGAGTCGGAAGGCCATGCATCGGAACCTTGA

Seq No. 4: amino acid sequence 730aa of sulB

MTDPNPQGGHLEHRSLRDWLANFFRAPLSNSSADSPPERIPETLEPNDRTRLLKLDDGQGPAYGTRDGLSTPQVDGGPEGHIRNGNGSPTFVQGPENLPPRTGSGLAWPVKNHKTMYLSYYIPFFNWITQYRWSFLRGDLIAALTIASIYIPMALSLASNLAHAPAINGLYSFVFMPLLYAILGSSPLLVVGPEAAGSLLTGTIVKTSVKQGHSQEDDEAASAMVVGIATAMAGAMILVAGLTRLGFLDNVLSRPFLRGFITAIGFVIFVDQLIPEVGLADLAKEAGVSHGSTVQKLVFLAGNIKSCHGLTAAVSFGSFAIIMLFRTIKKALEPRYPQVVYFPDRILVVILSAILTWRLGWYEQGLEVLGSVQNTATGLFAFRWPFQIKHLKHVRTAMSTSFIIAILGFFESSVAAKGLGERRDGVQGMSVSANREMVALGVANVVGGCFMALPAFGGYGRSKVNASTGARSPMSSIFLSIITFIVIMVLLPYLYYLPKAVLCSTISVVAYSLIEECPHDVAFFIRLRGWTELALMLLIFVSTIFYSLELGIALGIGLSILILIRHSTQPRIQILGKIAGTPDQYDNAEMHAENVELIDGALVVKIPEPLTFANTGDLKNRLRRLEFYGSNRAHPSLPRLRPPESNKNVIFDVHGVTSIDGSGTQVLSEIVNDYINLGVSVYFCRLSNRSVFRMFERSGIVDRCGGMSHFVTGVDEALRLAESEGHASEP

Seq No. 5: nucleotide sequence 465bp of sodA

ATGGTCAAGGCTGTCGCTGTTATCCGTGGAGACTCTAAGGTCTCCGGCACTGTCACTTTCGAGCAGGCCAACGAGAACACCCCCACCACCATCTCCTGGAACATCACTGGCCACGACGCCAACGCTGAGCGTGGCTTCCACGTCCACCAGTTCGGTGACAACACCAACGGCTGCACCTCCGCTGGCCCTCACTTCAACCCCTTCGGCAAGACCCACGGTGCTCCCGAGGACGACGAGCGTCACGTCGGTGACCTTGGCAACTTCAAGACCGATGCCGAGGGTAACGCCGTTGGTTCCAAGCAGGACAAGCTGGTGAAGCTCATCGGTGCTGAGAGCGTCCTGGGCCGGACCCTGGTCGTCCACGCTGGTACTGATGACCTTGGCCGTGGTGGCAACGAGGAGTCCAAGAAGACCGGTAACGCTGGTCCTCGTCCCGCTTGCGGTGTCATTGGCATTGCTGCTTAA

Seq No. 6: amino acid sequence 154aa of sodA

MVKAVAVIRGDSKVSGTVTFEQANENTPTTISWNITGHDANAERGFHVHQFGDNTNGCTSAGPHFNPFGKTHGAPEDDERHVGDLGNFKTDAEGNAVGSKQDKLVKLIGAESVLGRTLVVHAGTDDLGRGGNEESKKTGNAGPRPACGVIGIAA

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

<110> Tianjin science and technology university

<120> hexavalent chromium reducing strain, method and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 606

<212> DNA/RNA

<213> nucleotide sequence of chrB (Unknown)

<400> 1

atgcctccct ctatcggcct cataatctgc agccaacgca ctccacgcgc aggccctcaa 60

atcgccacct tcattcacaa tactattcgt gaatcctacc cccccgaaac agccacaatt 120

acgaccattg acctagcaaa atggaacctt ccactctaca atgagtcggg gatgccatcg 180

ttcatcaact cagcggacga gtacgagcac gagcacacaa aggcctggtc acgggagata 240

tcgcgccacg aagcgtttat tttcgtcaca ccgcagtata actgggggta tcccgcaagc 300

gtgaagaatg cgattgatta cttgttccat gagtggaagg gaaaggcggc gttggtggtg 360

agctatgggg ggcatggggg cgggaaggcg gcggcgcaat tgaggcaggt gttgcagggg 420

gtgaggatga ggccattgga gaggatggtc gagctgaggt ttccggagat cgaggaagtt 480

aagagggccg ctaaggggga ggatctggga ctgaatggtg agggagggta ttgggctggg 540

gagagggagg ggattaaggc tgcgtttggg gaattgattg ctgcgctgga gggagaggct 600

gaatag 606

<210> 2

<211> 201

<212> PRT

<213> amino acid sequence of chrB (Unknown)

<400> 2

Met Pro Pro Ser Ile Gly Leu Ile Ile Cys Ser Gln Arg Thr Pro Arg

1 5 10 15

Ala Gly Pro Gln Ile Ala Thr Phe Ile His Asn Thr Ile Arg Glu Ser

20 25 30

Tyr Pro Pro Glu Thr Ala Thr Ile Thr Thr Ile Asp Leu Ala Lys Trp

35 40 45

Asn Leu Pro Leu Tyr Asn Glu Ser Gly Met Pro Ser Phe Ile Asn Ser

50 55 60

Ala Asp Glu Tyr Glu His Glu His Thr Lys Ala Trp Ser Arg Glu Ile

65 70 75 80

Ser Arg His Glu Ala Phe Ile Phe Val Thr Pro Gln Tyr Asn Trp Gly

85 90 95

Tyr Pro Ala Ser Val Lys Asn Ala Ile Asp Tyr Leu Phe His Glu Trp

100 105 110

Lys Gly Lys Ala Ala Leu Val Val Ser Tyr Gly Gly His Gly Gly Gly

115 120 125

Lys Ala Ala Ala Gln Leu Arg Gln Val Leu Gln Gly Val Arg Met Arg

130 135 140

Pro Leu Glu Arg Met Val Glu Leu Arg Phe Pro Glu Ile Glu Glu Val

145 150 155 160

Lys Arg Ala Ala Lys Gly Glu Asp Leu Gly Leu Asn Gly Glu Gly Gly

165 170 175

Tyr Trp Ala Gly Glu Arg Glu Gly Ile Lys Ala Ala Phe Gly Glu Leu

180 185 190

Ile Ala Ala Leu Glu Gly Glu Ala Glu

195 200

<210> 3

<211> 2193

<212> DNA/RNA

<213> nucleotide sequence of sulB (Unknown)

<400> 3

atgacggatc ccaatccgca gggcggacac ctcgaacacc gctctttgcg ggattggctt 60

gccaacttct tccgagcacc tttgtcaaac agttctgcag attctccgcc tgaacggatt 120

cccgagacct tagaaccgaa tgatagaacc cgcttgttga aattggacga tggtcaaggt 180

cccgcatacg gtacaagaga tggtttatcg acgccgcagg ttgacggtgg gcctgaagga 240

catattcgga acgggaatgg aagcccgacg ttcgtacaag ggccggagaa cctgcctccc 300

aggactgggt caggcctggc atggcctgtg aagaaccata agactatgta cctctcatac 360

tacatcccgt tcttcaactg gatcacacaa tatcgctggt ctttcctccg aggcgactta 420

attgcagcct tgacaatcgc gtcaatctac attcccatgg ccctatcttt ggcttcgaac 480

ctcgcccatg caccagccat caacggcctt tactcattcg ttttcatgcc actgctttat 540

gcaattctag gaagcagtcc tctgctcgtt gttggtccgg aagcagctgg gtcgctattg 600

acgggaacaa tcgttaaaac tagcgtcaaa caaggccact cgcaggagga tgatgaggcc 660

gcaagcgcaa tggttgtagg catagccact gccatggcag gagcaatgat actagtcgct 720

ggactgacta ggctgggttt cctggacaat gtactcagtc ggcctttttt gagaggcttc 780

attactgcca ttggctttgt catctttgtt gaccagctca tcccggaggt tggactggca 840

gacttggcca aagaggccgg tgtcagtcat ggcagcacag tgcagaagct tgtctttctt 900

gctgggaata tcaaaagctg tcatggtctt acagctgctg tatccttcgg aagttttgca 960

atcattatgc tctttcgaac aatcaaaaaa gcactagaac cacgataccc tcaggtcgta 1020

tacttccccg accgtatcct tgtcgtgatc ctctcggcca ttctgacctg gcgtcttggt 1080

tggtacgagc aggggctgga agttctgggg tcggttcaga acactgccac tgggctcttc 1140

gccttcaggt ggcccttcca aatcaaacat ctgaagcatg tgcgcaccgc catgagcacc 1200

tccttcatca ttgctatcct tggcttcttc gagtcctcgg ttgctgccaa ggggctcggg 1260

gagagacgag acggggtcca aggcatgtct gtcagtgcga atagggagat ggttgctctg 1320

ggcgttgcta atgtggttgg gggctgcttt atggctctcc ctgcctttgg tggatacgga 1380

cggagcaaag tcaacgcctc tacgggcgct cgctctccta tgagcagtat cttcctcagt 1440

attatcacct tcattgttat tatggtgctc ttgccgtacc tatactatct tcctaaagct 1500

gtactatgct ctacgatatc ggtcgtcgca tacagtctta tagaggaatg tcctcacgac 1560

gttgccttct tcatccggct gcgtggttgg acagagcttg cattgatgct tctcatcttc 1620

gtctccacta tattctactc gctggagctg ggtatcgcgc ttggcattgg gctctcaatc 1680

ctgatcctta ttcgccactc cacccagcct cgcatacaga ttctcgggaa aatagctgga 1740

acacccgacc aatacgataa cgccgaaatg catgcggaaa atgtggagct cattgacggt 1800

gcccttgttg ttaagatccc cgagccgctc accttcgcca acactggcga tctcaagaat 1860

cgcttgcgcc gcctagagtt ctacggctca aatcgcgcac atccgtccct tccacggttg 1920

cgccccccgg aatcaaataa gaatgttatc ttcgacgttc acggtgttac gagcattgat 1980

ggctccggta cgcaggtcct ctccgagatc gttaatgatt acatcaatct cggagtaagc 2040

gtgtatttct gtcggctttc gaaccgcagc gtcttccgca tgttcgaaag aagcggcata 2100

gtcgaccggt gtggaggcat gtctcatttt gtcaccggtg ttgatgaggc gcttcggctc 2160

gctgagtcgg aaggccatgc atcggaacct tga 2193

<210> 4

<211> 730

<212> PRT

<213> amino acid sequence of sulB (Unknown)

<400> 4

Met Thr Asp Pro Asn Pro Gln Gly Gly His Leu Glu His Arg Ser Leu

1 5 10 15

Arg Asp Trp Leu Ala Asn Phe Phe Arg Ala Pro Leu Ser Asn Ser Ser

20 25 30

Ala Asp Ser Pro Pro Glu Arg Ile Pro Glu Thr Leu Glu Pro Asn Asp

35 40 45

Arg Thr Arg Leu Leu Lys Leu Asp Asp Gly Gln Gly Pro Ala Tyr Gly

50 55 60

Thr Arg Asp Gly Leu Ser Thr Pro Gln Val Asp Gly Gly Pro Glu Gly

65 70 75 80

His Ile Arg Asn Gly Asn Gly Ser Pro Thr Phe Val Gln Gly Pro Glu

85 90 95

Asn Leu Pro Pro Arg Thr Gly Ser Gly Leu Ala Trp Pro Val Lys Asn

100 105 110

His Lys Thr Met Tyr Leu Ser Tyr Tyr Ile Pro Phe Phe Asn Trp Ile

115 120 125

Thr Gln Tyr Arg Trp Ser Phe Leu Arg Gly Asp Leu Ile Ala Ala Leu

130 135 140

Thr Ile Ala Ser Ile Tyr Ile Pro Met Ala Leu Ser Leu Ala Ser Asn

145 150 155 160

Leu Ala His Ala Pro Ala Ile Asn Gly Leu Tyr Ser Phe Val Phe Met

165 170 175

Pro Leu Leu Tyr Ala Ile Leu Gly Ser Ser Pro Leu Leu Val Val Gly

180 185 190

Pro Glu Ala Ala Gly Ser Leu Leu Thr Gly Thr Ile Val Lys Thr Ser

195 200 205

Val Lys Gln Gly His Ser Gln Glu Asp Asp Glu Ala Ala Ser Ala Met

210 215 220

Val Val Gly Ile Ala Thr Ala Met Ala Gly Ala Met Ile Leu Val Ala

225 230 235 240

Gly Leu Thr Arg Leu Gly Phe Leu Asp Asn Val Leu Ser Arg Pro Phe

245 250 255

Leu Arg Gly Phe Ile Thr Ala Ile Gly Phe Val Ile Phe Val Asp Gln

260 265 270

Leu Ile Pro Glu Val Gly Leu Ala Asp Leu Ala Lys Glu Ala Gly Val

275 280 285

Ser His Gly Ser Thr Val Gln Lys Leu Val Phe Leu Ala Gly Asn Ile

290 295 300

Lys Ser Cys His Gly Leu Thr Ala Ala Val Ser Phe Gly Ser Phe Ala

305 310 315 320

Ile Ile Met Leu Phe Arg Thr Ile Lys Lys Ala Leu Glu Pro Arg Tyr

325 330 335

Pro Gln Val Val Tyr Phe Pro Asp Arg Ile Leu Val Val Ile Leu Ser

340 345 350

Ala Ile Leu Thr Trp Arg Leu Gly Trp Tyr Glu Gln Gly Leu Glu Val

355 360 365

Leu Gly Ser Val Gln Asn Thr Ala Thr Gly Leu Phe Ala Phe Arg Trp

370 375 380

Pro Phe Gln Ile Lys His Leu Lys His Val Arg Thr Ala Met Ser Thr

385 390 395 400

Ser Phe Ile Ile Ala Ile Leu Gly Phe Phe Glu Ser Ser Val Ala Ala

405 410 415

Lys Gly Leu Gly Glu Arg Arg Asp Gly Val Gln Gly Met Ser Val Ser

420 425 430

Ala Asn Arg Glu Met Val Ala Leu Gly Val Ala Asn Val Val Gly Gly

435 440 445

Cys Phe Met Ala Leu Pro Ala Phe Gly Gly Tyr Gly Arg Ser Lys Val

450 455 460

Asn Ala Ser Thr Gly Ala Arg Ser Pro Met Ser Ser Ile Phe Leu Ser

465 470 475 480

Ile Ile Thr Phe Ile Val Ile Met Val Leu Leu Pro Tyr Leu Tyr Tyr

485 490 495

Leu Pro Lys Ala Val Leu Cys Ser Thr Ile Ser Val Val Ala Tyr Ser

500 505 510

Leu Ile Glu Glu Cys Pro His Asp Val Ala Phe Phe Ile Arg Leu Arg

515 520 525

Gly Trp Thr Glu Leu Ala Leu Met Leu Leu Ile Phe Val Ser Thr Ile

530 535 540

Phe Tyr Ser Leu Glu Leu Gly Ile Ala Leu Gly Ile Gly Leu Ser Ile

545 550 555 560

Leu Ile Leu Ile Arg His Ser Thr Gln Pro Arg Ile Gln Ile Leu Gly

565 570 575

Lys Ile Ala Gly Thr Pro Asp Gln Tyr Asp Asn Ala Glu Met His Ala

580 585 590

Glu Asn Val Glu Leu Ile Asp Gly Ala Leu Val Val Lys Ile Pro Glu

595 600 605

Pro Leu Thr Phe Ala Asn Thr Gly Asp Leu Lys Asn Arg Leu Arg Arg

610 615 620

Leu Glu Phe Tyr Gly Ser Asn Arg Ala His Pro Ser Leu Pro Arg Leu

625 630 635 640

Arg Pro Pro Glu Ser Asn Lys Asn Val Ile Phe Asp Val His Gly Val

645 650 655

Thr Ser Ile Asp Gly Ser Gly Thr Gln Val Leu Ser Glu Ile Val Asn

660 665 670

Asp Tyr Ile Asn Leu Gly Val Ser Val Tyr Phe Cys Arg Leu Ser Asn

675 680 685

Arg Ser Val Phe Arg Met Phe Glu Arg Ser Gly Ile Val Asp Arg Cys

690 695 700

Gly Gly Met Ser His Phe Val Thr Gly Val Asp Glu Ala Leu Arg Leu

705 710 715 720

Ala Glu Ser Glu Gly His Ala Ser Glu Pro

725 730

<210> 5

<211> 465

<212> DNA/RNA

<213> nucleotide sequence of sodA (Unknown)

<400> 5

atggtcaagg ctgtcgctgt tatccgtgga gactctaagg tctccggcac tgtcactttc 60

gagcaggcca acgagaacac ccccaccacc atctcctgga acatcactgg ccacgacgcc 120

aacgctgagc gtggcttcca cgtccaccag ttcggtgaca acaccaacgg ctgcacctcc 180

gctggccctc acttcaaccc cttcggcaag acccacggtg ctcccgagga cgacgagcgt 240

cacgtcggtg accttggcaa cttcaagacc gatgccgagg gtaacgccgt tggttccaag 300

caggacaagc tggtgaagct catcggtgct gagagcgtcc tgggccggac cctggtcgtc 360

cacgctggta ctgatgacct tggccgtggt ggcaacgagg agtccaagaa gaccggtaac 420

gctggtcctc gtcccgcttg cggtgtcatt ggcattgctg cttaa 465

<210> 6

<211> 154

<212> PRT

<213> amino acid sequence of sodA (Unknown)

<400> 6

Met Val Lys Ala Val Ala Val Ile Arg Gly Asp Ser Lys Val Ser Gly

1 5 10 15

Thr Val Thr Phe Glu Gln Ala Asn Glu Asn Thr Pro Thr Thr Ile Ser

20 25 30

Trp Asn Ile Thr Gly His Asp Ala Asn Ala Glu Arg Gly Phe His Val

35 40 45

His Gln Phe Gly Asp Asn Thr Asn Gly Cys Thr Ser Ala Gly Pro His

50 55 60

Phe Asn Pro Phe Gly Lys Thr His Gly Ala Pro Glu Asp Asp Glu Arg

65 70 75 80

His Val Gly Asp Leu Gly Asn Phe Lys Thr Asp Ala Glu Gly Asn Ala

85 90 95

Val Gly Ser Lys Gln Asp Lys Leu Val Lys Leu Ile Gly Ala Glu Ser

100 105 110

Val Leu Gly Arg Thr Leu Val Val His Ala Gly Thr Asp Asp Leu Gly

115 120 125

Arg Gly Gly Asn Glu Glu Ser Lys Lys Thr Gly Asn Ala Gly Pro Arg

130 135 140

Pro Ala Cys Gly Val Ile Gly Ile Ala Ala

145 150

<210> 7

<211> 36

<212> DNA/RNA

<213> chrB-F(Unknown)

<400> 7

atggaattcg agctcggtac ccctccctct atcggc 36

<210> 8

<211> 36

<212> DNA/RNA

<213> chrB-R(Unknown)

<400> 8

agtggatccc tgcagggtac cctattcagc ctctcc 36

<210> 9

<211> 35

<212> DNA/RNA

<213> sulB-F(Unknown)

<400> 9

acatctaaac aatggaattc acggatccca atccg 35

<210> 10

<211> 35

<212> DNA/RNA

<213> sulB-R(Unknown)

<400> 10

cagtaacgtt aagtggatcc tcaaggttcc gatgc 35

<210> 11

<211> 42

<212> DNA/RNA

<213> sodA-F(Unknown)

<400> 11

tcatccgtca agatggaatt cgtcaaggct gtcgctgtta tc 42

<210> 12

<211> 44

<212> DNA/RNA

<213> sodA-R(Unknown)

<400> 12

tccagatctc tgcagggtac cttaagcagc aatgccaatg acac 44