CN114480437A - Protein A9 with binding capacity of arsenite and methyl arsenite, engineering strain containing protein gene and application - Google Patents

Abstract

The invention provides an arsenic binding protein A9, a recombinant vector containing an encoding gene of the arsenic binding protein A9 and a recombinant engineering strain containing the recombinant vector. The arsenic binding protein A9 can improve the application of the strain to arsenite and methyl arsenite resistance and improve the binding rate of the strain to arsenite and methyl arsenite. The arsenic binding protein a9 of the present invention achieves binding of arsenite and methyl arsenite in vitro. The recombinant engineering bacteria have strong resistance to arsenite (60 mu M) and methyl arsenite (3 mu M), the absorption efficiency of the recombinant engineering bacteria to the arsenite and the methyl arsenite is up to 89.71% and 84.95%, the absorption efficiency of the target protein A9 to the arsenite and the methyl arsenite is up to 89.08% and 88.27%, and the arsenic in a culture environment can be effectively removed. The arsenic binding protein of the invention provides a new bioremediation technology for arsenic pollution remediation of paddy fields and water body environments in the south of China.

Description

Protein A9 with binding capacity of arsenite and methyl arsenite, engineering strain containing protein gene and application

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of genetic engineering, in particular to an arsenic binding protein with binding capacity of arsenite and methyl arsenite from trichoderma asperellum, and also relates to a gene for coding the protein, a vector containing the gene, a recombinant engineering strain and application thereof.

[ background of the invention ]

Arsenic (As) is a metalloid widely existing in nature, and the background value of arsenic in soil in China is 11.2 mg/kg^-1The physicochemical property and the toxic action of the heavy metal in the soil are similar to those of other heavy metals, and the heavy metal is listed as one of eight kinds of heavy metals in the soil. The common arsenide in soil is arsenate (AsO)₄ ^3-) Sodium arsenite (AsO)₂ ^1-) Monomethylarsonium (MMA), and Dimethylarsinium (DMA). In a farmland flooding environment, arsenic mainly exists in the forms of arsenite (As) (III) and reduced organic arsenic (MMA (II I) and DMA (III)). In 2014, the former ministry of environmental protection and the former ministry of land resources release national soil pollution condition survey reports, and the reports indicate that the content of pollutants at soil sites of cultivated land in China exceeds the standard by 19.4%, wherein light and medium pollution accounts for 94.3% of the standard, wherein the standard exceeding rate of metalloid arsenic is 2.7%, and the pollution degree is mainly medium and low pollution.

In recent years, the treatment of arsenic pollution has been receiving attention. Some repairing technologies, such as phytoremediation, passivation, leaching and the like, can reduce the environmental risk of arsenic in soil to a certain extent, but in the long run, the economic investment is large, the repairing time is long, problems such as excessive repairing can cause negative effects on the soil structure, the fertility and the like, and the sustainability of agricultural development is not facilitated. The microbial material is used as a novel arsenic pollution repair material and mediates the accumulation and conversion of arsenic in the metabolic process. As Trichoderma asperellum in previous studies in the subject group, it was found that arsenic isolation by cell wall immobilization and vacuolar region had a significant effect on arsenic accumulation. At present, more and more researches prove that environmental microorganisms are successfully applied to the remediation of arsenic-polluted water or soil, the efficiency is improved, the cost is reduced, and good development potential is shown.

[ summary of the invention ]

The invention aims to overcome the defects of the prior art, provides a novel arsenic binding protein, further constructs a recombinant vector and a recombinant engineering strain containing the binding protein gene, and determines that the protein has good binding effect on arsenite and methyl arsenite after being expressed through experiments, so that the arsenic content in a culture environment can be effectively reduced.

In order to achieve the aim, the invention provides an arsenic binding protein A9, wherein the nucleic acid sequence of the coding gene of the protein is shown as SEQ ID NO. 1; the invention provides an arsenic binding protein A9, wherein the nucleic acid sequence of the coding gene of the protein is shown in SEQ ID NO. 2;

the arsenic-binding protein A9 of the present invention is derived from Trichoderma asperellum, particularly Trichoderma asperellum SM-12F1(Arsenite Resistance, Accumulation, and mutation Properties of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1, Xibai Zeng et al), Trichoderma asperellum SM-12F1 is also described in the Chinese patent application CN 201210361866.6 written as Trichoderma asperellum, which was preserved in the China Committee for culture Collection of microorganisms under the accession number CGMCC No. 3186.

Based on the above, the present invention also provides a recombinant vector containing the gene encoding arsenic-binding protein A9.

And recombinant engineered strains containing the recombinant vectors, and particularly preferably Escherichia coli.

The invention also provides application of the arsenic binding protein A9 in improving resistance of strains to arsenite and methyl arsenite and application in improving binding rate of strains to arsenite and methyl arsenite.

The invention also provides application of the arsenic binding protein A9 in vitro binding of arsenite and methyl arsenite.

The invention discovers a protein (A9) with a binding effect on arsenite and methyl arsenite for the first time, further constructs a recombinant vector and recombinant engineering bacteria, and confirms that the recombinant engineering bacteria has strong resistance to arsenite (60 mu M) and methyl arsenite (3 mu M) through experiments. The recombinant engineering bacteria have the absorption efficiency of 89.71% and 84.95% for arsenite and methyl arsenite, the absorption efficiency of 9 for arsenite and methyl arsenite is 89.08% and 88.27%, and arsenic in a culture environment can be effectively removed.

The arsenic binding protein of the invention provides a novel bioremediation technology for remedying arsenic pollution (mainly reducing arsenic) in paddy fields and water body environment in the south of China.

Compared with the prior art, the engineering bacteria disclosed in the Chinese patent application CN 111996207A can only remove one form of arsenic, and the removal rate is not higher than 50%, so that the arsenic binding protein A9 and the arsenic binding capacity of the related recombinant engineering strains are obviously higher than the record of the prior art.

[ description of the drawings ]

FIG. 1 is a purified electrophoretogram of arsenic binding protein A9;

FIG. 2 is a graph showing the effect of arsenic binding gene a9 on the resistance of E.coli to arsenite;

FIG. 3 is a graph showing the effect of arsenic binding gene a9 on the resistance of E.coli to methyl arsenite;

FIG. 4 shows the accumulation of arsenite by recombinant strain BL21(pET30a-a 9);

FIG. 5 shows the accumulation of methyl arsenite by recombinant strain BL21(pET30a-a 9);

FIG. 6 shows the binding efficiency of arsenic binding protein A9 to arsenite;

FIG. 7 is a graph showing the binding efficiency of arsenic binding protein A9 to methyl arsenite.

[ detailed description ] embodiments

The following examples serve to illustrate the technical solution of the present invention without limiting it.

In the present invention, "%" used for specifying concentrations is, unless otherwise specified, "%" used for specifying the ratio of amounts ": all the terms "are mass ratios.

The present invention relates to the following media and chemicals:

PGP liquid medium: 100g of potato (cut into 1cm potato blocks, boiled for about 30min), 10g of glucose and 2.5g of peptone, the volume is adjusted to 1000ml after boiling, and the potato is sterilized at 121 ℃ for 20 min.

LB culture medium: 5g of yeast extract, 10g of tryptone and 10g of sodium chloride, and the volume is adjusted to 1L (pH 7), and the mixture is sterilized at 121 ℃ for 20 min.

ST10^-1Culture medium: 0.05g of yeast powder, 0.5g of peptone and 5g of glucose, and the volume is fixed to 1L, and the mixture is sterilized at 121 ℃ for 20 min.

ST medium: 0.5g of yeast phenol, 5g of peptone and 5g of glucose, diluting to 1L, and sterilizing at 121 ℃ for 20 min.

Chemical reagents: sodium arsenite (As (III)) NaAsO₂(ii) a Sodium methyl arsenite (mma (iii)) was reduced by sodium methyl arsenite, reduction system: 0.2mM methyl arsenate MMA (V), 27mM NaS₂O₃，66mM NaS₂O₅，82mM H₂S0₄，pH＝6。

Antibiotics: kanamycin sulfate (Kana); inducing expression: isopropyl-beta-D-thiogalactoside (IPTG).

The invention relates to the following primers:

TABLE 1 primer information

Example 1 construction of arsenic-binding Gene vectors, expression strains and purification of arsenic-binding proteins

1.1 obtaining of dioxygenases associated with binding to arsenic proteins

Trichoderma asperellum SM-12F1 was taken, Fungal DNA was extracted according to the DNA Kit (Quick-DNATM Fungal/Bacterial Midiprep Kit, Zymo Research, Orange, CA, USA) extraction procedure, target DNA bands were detected by 10% agarose gel electrophoresis, and Nanodrop detection was used to confirm that the purified DNA concentration reached the standard.

The SMRTbell Template preparation kit (SMRTbell Template Kits, Pacific Biosciences) was used to construct a genomic DNA with an insert size of 20kb SMRTbell. The DNA sample is cut into fragments smaller than 400bp by using an Illumina TruSeq Nano DNA Library Prep kit. The 20kb and 400bp libraries were sequenced using the PacBio RS II platform and Illumina Hiseq2000, respectively.

Gene prediction and annotation were then performed on the genome assembled from Trichoderma asperellum SM-12F 1. The coding sequence CDS was predicted using Augusts v3.3.2 and GeneMark-ES v 4.46. The obtained Genes were submitted to databases such as Cluster of organizations group of proteins (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomics (KEGG) for functional annotation using sequence alignment tools such as BLAST, Diamond, HMMER, etc. The results note a dioxygenase a9 associated with arsenic protein binding.

1.2 amplification of arsenic-binding protein coding Gene and construction of expression Strain

1.2.1 PCR amplification, digestion, and recovery of arsenic-binding protein coding Gene

The Trichoderma asperellum SM-12F1 DNA gene is used as a template after being inverted, and a target gene is obtained by a primer for amplifying an arsenic binding protein coding gene.

(1) The target gene a9 amplification system comprises:

(2) target gene amplification procedure:

95℃5min；

30sec at 95 ℃, 30sec at 65 ℃, 1 ℃/cycle at-1 ℃, 1min at 72 ℃ and 14 cycles;

30sec at 95 ℃, 30sec at 62 ℃, 1min at 72 ℃, 10min at 16 ℃ and 32 cycles.

And (3) carrying out 1% agar gel electrophoresis on the amplified PCR system, and cutting and recovering to obtain the target fragment.

(3) The EcoR I and Hind III are subjected to double enzyme digestion, and the enzyme digestion system is as follows:

opening a water bath kettle in advance, setting the temperature to be 37 ℃, completing the immersion of the enzyme digestion system, and reacting for 12-16 hours. Then 5. mu.L of the DNA was subjected to 1% agarose gel assay and recovered using a common DNA product purification kit.

1.2.2 digestion and recovery of vectors

The plasmid pET-30a (+) was also digested simultaneously with EcoR I and Hind III, as follows:

the enzyme cutting condition and the recovery process are the same as before.

1.2.3 ligation of the target Gene to the vector

The DNA concentrations of the target gene and the vector were determined by microplate quantification using the following ligation system:

the above system was mixed well and placed in a metal bath at 16 ℃ for reaction overnight.

1.2.4 construction and Positive identification of recombinant expression strains

The recombinant plasmid is introduced into TOP 10 competent cells of escherichia coli, after culture, a bacterium solution is used as a PCR template to identify positive clones, and T7 and T7ter are used as primers to amplify. And (3) carrying out 1% agarose gel electrophoresis detection on the PCR product, and taking the size of a target band for sequencing.

The amplification system was as follows:

the PCR reaction amplification program is as follows:

95℃5min；

30sec at 95 ℃, 30sec at 55 ℃, 1min at 72 ℃ for 30sec, 29 cycles; 72 ℃ for 10min and 16 ℃ for 10 min.

After culturing the TOP 10 strain with the same sequencing result, the plasmid was extracted and introduced into BL21(DE3) for expression. And (3) amplifying by using T7 and T7ter as primers, detecting a PCR product by using 1% agarose gel electrophoresis, and sequencing by taking the size of a target band.

The transformant which was correctly sequenced was designated BL21(pET30a-a 9).

1.2.5 expression and purification of arsenite and Methylarsenate binding proteins

The recombinant expression strain BL21(pET30a-a9) is cultured in LB culture medium to OD₆₀₀After 0.6 to 0.8 nm, 0.4mM IPTG (isopropyl-. beta. -D-thiogalactoside) was added and incubated at 16 ℃ overnight at 200 rpm. After the culture, the cells were centrifuged at 8000rpm for 10min at 4 ℃ to collect Escherichia coli cells, and 20 mmol/L of the cells were added^-1The Tris-HCl buffer solution is used for resuspending the thalli, an ultrasonic crusher is used for crushing, the crushed supernatant is purified by a nickel ion affinity chromatographic column to arsenic aggregate protein A9, and the SDS-PAGE protein electrophoresis detects the purification effect.

As shown in figure 1, the expression of arsenic binding protein in Escherichia coli shows that the broken supernatant has a specific protein band at 21.8kDa (A9), which indicates that the arsenic binding gene realizes soluble expression in Escherichia coli and has better expression.

Example 2 resistance test

2.1 arsenic binding genes increase resistance of E.coli to arsenite

The recombinant expression strain BL21(pET30a-a9) obtained in example 1 was cultured in LB medium (50 mg. L)^-1Kana) was cultured overnight, and then inoculated into LB medium at an inoculum size of 1% to grow to an OD of 0.6 to 0.8, 0.4mM IPTG was added thereto, and the culture was carried out at 16 ℃ for 18 hours. After centrifugation to remove the supernatant, the cells were eluted with an equal volume of ST medium, and then an equal volume of ST medium was addedSuspending, inoculating into 30ml ST medium at 2% inoculation amount, and adding 50 mg.L^-1Kanamycin antibiotic and 0 μ M, 10 μ M, 20 μ M, 40 μ M, 60 μ M As (III), taking 1ml of bacterial liquid in 0, 6, 10, 14, 22, 24h, and measuring OD value; while an empty-carrying strain BL21(pET30a) was set as a control, three replicates were set for each set of experiments, and cultured at 30 ℃ and 200 rpm. The bacterial liquid was measured for growth of the cells at a wavelength of 600nm using an ultraviolet spectrophotometer.

As shown in FIG. 2, after the arsenic binding gene is expressed in arsenic-sensitive Escherichia coli, the resistance of the recombinant expression strain pET30a-a9 to arsenite can reach as high as 60 mu M (OD)₆₀₀A value of about 0.6), and the recombinant strain pET30a-a9 grew better in 60 μ M As (III) than in 10 μ M As (III) in the case of the unloaded expression strain pET30a, indicating that the arsenic-binding gene a9 can significantly improve the resistance of the expression strain to arsenite.

2.2 arsenic binding genes to increase resistance of E.coli to methyl arsenite

The recombinant expression strain BL21(pET30a-a9) obtained in example 1 was cultured in LB medium (50 mg. L)^-1Kana) overnight, inoculated in LB medium at an inoculum size of 1% to OD₆₀₀When the value is 0.6-0.8, 0.4mM IPTG is added and the culture is carried out at 16 ℃ for 18 hours. Centrifuging to remove supernatant, eluting thallus with equal volume of ST culture medium, adding equal volume of ST culture medium for resuspension, inoculating into 30ml of ST culture medium at 2% inoculation amount, and adding 50 mg.L^-1Kana, 0 μ M, 1 μ M, 3 μ M, 9 μ M MMA (III), collecting 1ml of the bacterial solution at 0, 2, 4, 6, 12, 14, 24h, and measuring OD with spectrophotometer₆₀₀The value is obtained.

As shown in FIG. 3, after expressing the arsenic-binding gene a9 in arsenic-sensitive E.coli, the resistance of the recombinant expression strain pET30a-a9 to methyl arsenite was as high as 3. mu.M in the MMA (III) resistance test; the resistance of the unloaded bacteria pET30a is only 1 mu M, and is obviously improved by about 3 times. After the arsenic binding gene a9 is expressed in arsenic-sensitive escherichia coli, the resistance of an expression strain methyl arsenite is obviously improved.

In conclusion, the results in example 2 show that the recombinant expression strain BL21(pET30a-a9) has high resistance to both arsenite and methyl arsenite, and is the gene which is found to have resistance to two kinds of arsenic reducibility in eukaryotic microorganisms for the first time.

Example 3 binding efficiency of recombinant expression strains to arsenite and methyl arsenite

3.1 efficiency of arsenite absorption by recombinant expression strain BL21(pET30a-a9)

The recombinant expression strain BL21(pET30a-a9) is put in LB culture medium (50 mg. L)^-1Kana) overnight, inoculated in LB medium at an inoculum size of 1% to OD₆₀₀When the value is 0.6-0.8, 0.4mM IPTG is added and the culture is carried out at 25 ℃ for 6 hours. After centrifugation of the supernatant, an equal volume of ST10 was used^-1After the cells were eluted from the medium, an equal volume of ST10 was added^-1Resuspension of the medium (50 mg. L)^-1Kana), and 50 μ M As (III) was added, and samples were taken after 1 hour of incubation.

While the empty strain BL21(pET30a) was set as a control, three replicates were set for each experiment.

Measuring the arsenic form by a supernatant filter membrane through HPLC-ICP-MS, wherein the result is extracellular arsenic; the bacterial pellet is eluted by PBS and then freeze-dried, then arsenic is extracted by water, and the form of the arsenic is measured, and the result is intracellular arsenic. The specific method comprises the following steps: 0.01g of the lyophilized Escherichia coli was weighed, added to a lysis tube containing 0.5g of glass beads (0.01 to 0.1mm), and 600. mu.L of ultrapure water was added and placed in Fastprep-24(MB biomedicals) at 6.5 m.s^-1Crushing for 60 s; centrifuging at 12000rpm for 1min, sucking supernatant, adding into a new tube containing glass strain, and further crushing. Until the supernatant was clear and transparent. The supernatant was filtered through a 0.22. mu.M filter and 10% H was added₂O₂The arsenic form was measured after 30 Min.

As shown in FIG. 4, the extracellular arsenic concentration (arsenic concentration in the medium) of the empty-load strain (pET30a) was 3125.51. mu.g/kg after the strain BL21 was stressed in 50. mu.M As (III) medium for 1 hour^-1And the extracellular arsenic concentration of the expression strain pET30a-a9 is as low as 386 mu g kg^-1Namely, 89.71% of arsenate in the culture medium is removed by the expression strain pET30a-a 9; meanwhile, the same phenomenon was observed in 0.01g of dried yeast: expression strain pET30a-a9 has high arsenic content accumulated in cellsUp to 1000.24 mug/kg^-1Much higher than 334.25 mug.kg in cells of an unloaded strain (pET30a)^-1。

In conclusion, the recombinant expression strain containing the arsenic binding gene a9 can remarkably remove arsenite in a stress environment by accumulating the arsenite from the extracellular part to the intracellular part within 1 hour, and the removal rate is as high as 89.71%.

3.2 efficiency of absorption of methyl arsenite by recombinant expression strain BL21(pET30a-a9)

The recombinant expression strain BL21(pET30a-a9) is put in LB culture medium (50 mg. L)^-1Kana) overnight, inoculated in LB medium at an inoculum size of 1% to OD₆₀₀When the value is 0.6-0.8, 0.4mM IPTG is added and the culture is carried out at 25 ℃ for 6 hours. After centrifugation of the supernatant, an equal volume of ST10 was used^-1After the cells were eluted from the medium, an equal volume of ST10 was added^-1The medium was resuspended, 2. mu.M MMA (III) was added, respectively, and the culture was sampled after 1 h. While the empty strain BL21(pET-30a) was set as a control, three replicates were set for each set of experiments. For intracellular and extracellular arsenic determination, see example 3, step 3.1.

As shown in FIG. 6, the extracellular As concentration was 22.06. mu.g.kg after the recombinant expression strain pET30a-a9 was stressed in 2. mu.M MMA (III) medium for 1 hour^-1The concentration of the extracellular organic arsenic is far lower than that of an unloaded strain BL21(pET-30a) and is 94.52 mu g kg^-1. Meanwhile, the intracellular arsenic content of 0.01g of expression strain pET30a-a9 is as high as 296 mu g kg^-1Much higher than 180.02 mug. kg of the unloaded strain BL21(pET-30a)^-1. The expression strain pET30a-a9 can accumulate a large amount of MMA (III) from an arsenic environment within 1 hour, so that the MMA (III) in the environment can be removed, and the removal rate is as high as 84.95%.

In conclusion, the results in example 3 show that the recombinant expression strain pET30a-a9 has high accumulation of arsenite and high accumulation of methyl arsenite, and the gene a9 has the accumulation capacity of various reduced arsenic, so that the recombinant engineering strain containing the gene is a biological material suitable for repairing various arsenic pollutions.

EXAMPLE 4 in vitro Activity experiment of protein A9 of interest on arsenite and methyl arsenite

4.1 in vitro Activity assay of protein A9 of interest on arsenite

An enzyme activity reaction system: 100mM MOPS buffer (pH 7.0), 1mM cysteine, 50. mu.M As (III), 10. mu.M of the protein of interest. Blank CT is treatment without added protein. After 30Min of stationary reaction at 30 ℃, a part of arsenic is directly filtered by a filter membrane to measure the form of the arsenic, and the part of arsenic is the arsenic content in the pre-oxidation enzyme active reaction system; the other part was charged with 10% by volume of H₂O₂Stopping reaction, and centrifugally extracting supernatant, wherein the part of arsenic is the arsenic content in the enzyme activity system after oxidation. Arsenic morphology was determined by HPLC-ICP-MS.

As shown in FIG. 6, the concentration of the A9 reaction system was 374.49. mu.g/kg after the purified target protein A9 reacted with arsenite for 30min^-1Is far lower than that of a CT blank reaction system of 3447 mu g/kg^-1About 89.08% of the arsenite was bound. When the oxidizing agent was added, the protein structure was destroyed and a large amount of arsenic was released into the reaction system, at which time the arsenic content in the solution of reaction system A9 was 3444. mu.g.kg^-1。

4.2 in vitro Activity assay of protein A9 of interest on Methylarsonate

An enzyme activity reaction system: 100mM MOPS buffer (pH 7.0), 1mM cysteine, 2. mu.M MAs (III), 10. mu.M of the protein of interest. Blank CT is treatment without added protein. The termination reaction and the treatment of the sample to be tested are the same as in step 4.1 of example 4.

As shown in FIG. 7, the reaction system of the purified target protein A9 with methyl arsenite reacted for 30Min showed only a small amount of organic arsenic in the target protein A9, and the concentration was 17.76. mu.g/kg^-1The arsenic concentration in the CT blank reaction system is 142.56 mug.kg^-1About 88.27% of the methyl arsenate is immobilized by the protein; when oxidant is added to destroy protein structure, great amount of organic arsenic is released and the arsenic concentration is 143.39 microgram kg^-1. The target protein A9 is shown to have a remarkable binding effect on methyl arsenite.

It can be seen that the target protein A9 has significant binding effect on arsenite and methyl arsenite, and the binding rates are 89.08% and 88.27% respectively.

In conclusion, the invention obtains the arsenic binding protein A9 from Trichoderma asperellum SM-12F1 of Trichoderma asperellum, and experiments prove that the resistance of Escherichia coli to arsenite and organic arsenic can be obviously improved when the gene a9 is expressed in the Escherichia coli BL21 sensitive to arsenic. In addition, the arsenic binding protein also has the effect of binding arsenite and organic arsenic in vitro, thereby having obvious application prospect and environmental protection significance.

Sequence listing

<110> research institute of agricultural environment and sustainable development of Chinese academy of agricultural sciences

<120> protein A9 with arsenite and methyl arsenite binding capacity, engineering strain containing protein gene and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 2

<211> 513

<212> DNA/RNA

<213> arsenic-binding protein A9 of Trichoderma asperellum (Trichoderma asperellum)

<400> 2

atgcccgcaa catcactcca gcctcgaggg cttaaccatt tcgcttacgc tacactgaat 60

atggacgaaa cccaccaatt ctggactgaa gttatgggtt gcaagttttt gggtgcatat 120

gcttttaagg aatccggtca tgacaagcct attccagaca gctacgtgca tagtctgtat 180

ggcatgtctg acggctcggc tttggctttt ttcgaacttg gaaaaggcta tgagaagaaa 240

gacgacggca tccccacata cacaaagcat cttgctctga cttgtgatag caaggagcag 300

gtgaaacaat ggcacgaaca ttttagtgca cacgggctcg atgttatagg agaaattgat 360

cacgaaggaa tgtggctttc aatttacgtg actgaccctt ccggtctcat aatcgaactt 420

acgtaccaat cgcacacttt cgacgaaaac gatgccatgg aggggctaaa ggtcttgaaa 480

cagtggcgga aggacaaagt caccacaaag tag 513

<210> 2

<211> 170

<212> PRT

<213> arsenic-binding protein A9 of Trichoderma asperellum (Trichoderma asperellum)

<400> 2

Met Pro Ala Thr Ser Leu Gln Pro Arg Gly Leu Asn His Phe Ala Tyr

1 5 10 15

Ala Thr Leu Asn Met Asp Glu Thr His Gln Phe Trp Thr Glu Val Met

20 25 30

Gly Cys Lys Phe Leu Gly Ala Tyr Ala Phe Lys Glu Ser Gly His Asp

35 40 45

Lys Pro Ile Pro Asp Ser Tyr Val His Ser Leu Tyr Gly Met Ser Asp

50 55 60

Gly Ser Ala Leu Ala Phe Phe Glu Leu Gly Lys Gly Tyr Glu Lys Lys

65 70 75 80

Asp Asp Gly Ile Pro Thr Tyr Thr Lys His Leu Ala Leu Thr Cys Asp

85 90 95

Ser Lys Glu Gln Val Lys Gln Trp His Glu His Phe Ser Ala His Gly

100 105 110

Leu Asp Val Ile Gly Glu Ile Asp His Glu Gly Met Trp Leu Ser Ile

115 120 125

Tyr Val Thr Asp Pro Ser Gly Leu Ile Ile Glu Leu Thr Tyr Gln Ser

130 135 140

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

145 150 155 160

Gln Trp Arg Lys Asp Lys Val Thr Thr Lys

165 170

Claims (9)

1. An arsenic binding protein A9, the nucleic acid sequence of the coding gene of the protein is shown in SEQ ID NO. 1.

2. The arsenic-binding protein A9 according to claim 1, wherein the amino acid sequence of the gene encoding it is as shown in SEQ ID No. 2.

3. The arsenic binding protein a9, according to claim 1, wherein the arsenic binding protein a9 is derived from trichoderma asperellum.

4. A recombinant vector comprising a gene encoding arsenic binding protein a9 according to claim 1.

5. An engineered strain comprising the recombinant vector of claim 4.

6. The recombinant engineered strain of claim 5, wherein the strain is E.

7. Use of the arsenic binding protein A9 of claim 1 to increase the resistance of a strain to arsenite and methyl arsenite.

8. Use of the arsenic binding protein A9 of claim 1 to increase the absorption of arsenite and methyl arsenite by a strain.

9. Use of the arsenic binding protein a9 of claim 1 to bind arsenite and methyl arsenite in vitro.

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