CN114605510A - Protein A10 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 A10, a recombinant vector containing a coding gene of the protein and an engineering strain containing the recombinant vector. The arsenic binding protein A10 can improve the resistance of the strain to arsenite and methyl arsenite, improve the accumulation efficiency of the strain to arsenite and methyl arsenite, and bind arsenite and methyl arsenite in vitro. The engineering bacteria have strong resistance to arsenite and methyl arsenite, the binding efficiency of arsenite and reducing monomethyl arsenic is as high as 92.24% and 76.69%, the binding efficiency of target protein A10 to arsenite and reducing monomethyl arsenic is as high as 96.70% and 83.33%, and arsenic in a culture environment can be effectively removed. The protein A10 with strong binding capacity to arsenite and methyl arsenite provides a new bioremediation material for the remediation of arsenic pollution in paddy fields and water body environments.

Description

Protein A10 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 common toxic heavy metal element in soil environments. The arsenic source in the soil is usually from mining of arsenic ore, sewage irrigation, agricultural chemicals and fertilizers for farmland application, and the like. Crops are planted in farmlands seriously polluted by over-standard arsenic, so that potential hazards can be brought to the safety of agricultural products, and the health of people can be harmed. As an important food crop in China, the growing environment of long-term flooding and the physiological characteristics of silicon and phosphorus preference of rice are main reasons for the sensitivity and accumulation of the rice to arsenic, and the accumulated methyl arsenic in rice grains is a main reason for the direct panicle disease of the rice. At present, the environmental arsenic remediation technology mainly comprises a biological remediation technology, a chemical remediation technology and a physical remediation technology. The microbial remediation mainly depends on the interaction of microbes and arsenic, including adsorption/analysis, precipitation/dissolution, oxidation/reduction, methylation/demethylation and other actions, and reduces the biological effectiveness of heavy metal pollutants, thereby achieving the purpose of remedying the environmental arsenic pollution and being a more green, economic and environment-friendly biological material. In the early research of the laboratory, a Trichoderma asperellum SM-12F1 strain with high resistance is discovered, and is separated from soil near a realgar mining area in Shimen county of Hunan province in 2009, and the resistance to arsenate can reach as high as 500 mg.L^-1The oxidation of arsenite, reduction of arsenate, methylation of inorganic arsenic and demethylation of organic arsenic can be mediated.

In a flooded farmland environment, arsenic is present mainly in the form of arsenite (As (III)) and reduced organic arsenic such as methyl arsenite [ MMAs (III) ], reduced dimethyl arsenic [ DMAs (III) ]. As (III) is not only absorbed by the plant root system through aquaporin, but also is the main form of transfer in the organism; after organic arsenic, particularly DMAs are absorbed by the root system of rice and accumulated on glumes, the rice spike disease is easy to occur, and accordingly, the yield reduction and even the dead yield of the rice are caused. Therefore, the detoxification mechanism of the microorganism to arsenic is known, a novel arsenic pollution remediation method and material are developed, theoretical basis and technical support are provided for arsenic pollution remediation in the environment, and the method has practical significance and economic effect.

[ 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 an engineering strain containing the binding protein gene, and finds that the protein has strong binding effect on arsenite and methyl arsenite after the protein is determined to be 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 A10, 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 A10, wherein the nucleic acid sequence of the coding gene of the protein is shown in SEQ ID NO. 2;

the arsenic-binding protein A10 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 A10.

And engineering strains containing the recombinant vector, and particularly preferably Escherichia coli.

The invention also provides application of the arsenic binding protein A10 in improving resistance of strains to arsenite and methyl arsenite, improving accumulation efficiency of strains to arsenite and methyl arsenite, and combining arsenite and methyl arsenite in vitro.

The protein A10 with strong binding capacity to arsenite and methyl arsenite is discovered for the first time, and a recombinant vector and engineering bacteria are constructed, so that the engineering bacteria are verified to have strong resistance to arsenite (60 mu M) and methyl arsenite (3 mu M). According to the invention

The binding efficiency of the engineering bacteria to arsenite and reductive monomethyl arsenic is as high as 92.24% and 76.69%, the binding efficiency of the target protein A10 to arsenite and reductive monomethyl arsenic is as high as 96.70% and 83.33%, and arsenic in a culture environment can be effectively removed.

The protein A10 with strong binding capacity to arsenite and methyl arsenite provides a new bioremediation material for the remediation of arsenic pollution in paddy fields and water body environments.

Compared with the prior art, the binding protein is found in fungus bodies for the first time, and has high binding capacity to reduced inorganic arsenic and organic arsenic. For example, compared with the technical scheme disclosed in the Chinese patent application CN 111996207A, the recombinant strain disclosed by the invention not only relates to the removal of arsenic in one form, but also has a removal rate remarkably higher than that of the prior art.

[ description of the drawings ]

FIG. 1 is a diagram of the purification electrophoresis of arsenic binding protein A10;

FIG. 2 is a graph showing the effect of arsenic binding gene a10 on the resistance of E.coli to arsenite;

FIG. 3 is a graph showing the effect of arsenic binding gene a10 on the resistance of E.coli to methyl arsenite;

FIG. 4 shows the accumulation of arsenite by recombinant strain BL21(pET30a-a 10);

FIG. 5 shows the accumulation of methyl arsenite by recombinant strain BL21(pET30a-a 10);

FIG. 6 shows the binding efficiency of arsenic binding protein A10 to arsenite;

FIG. 7 is a graph showing the binding efficiency of arsenic binding protein A10 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 invention relates to the following culture media and chemical reagents:

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: yeast extract 5g, tryptone 10g, sodium chloride 10g, constant volume to 1L (pH 7), 121 deg.C sterilization for 20 min.

ST10^-1Culture medium: 0.05g of yeast powder, 0.5g of peptone and 5g of glucose, and the constant volume is 1L,

sterilizing at 121 deg.C 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.

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 NaS₂O₃，66 mM 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 DNA extraction of Trichoderma asperellum SM-12F1 Trichoderma asperellum

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, the target DNA band was detected by 10% agarose gel electrophoresis, and the purified DNA concentration was confirmed by Nanodrop detection.

A genomic DNA insert of 20kb in size of SMRTbell was constructed using an SMRTbell Template preparation kit (SMRTbell Template Kits, Pacific Biosciences). 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 Hiseq 2000, respectively.

Then the gene prediction and annotation are carried out on the genome assembled by the 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 organisational Groups of proteins (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional annotation using alignment tools such as BLAST, Diamond, HMMER, etc. The results are annotated with 1 dioxygenase a10 associated with 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 amplification system of the target gene a10 comprises the following steps:

(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 cleavage 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 TOP10 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 sequencing the size of a target band.

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.

TOP10 strain with the same sequencing result was cultured, and its 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 10).

1.2.5 expression and purification of arsenite and Methylarsenate binding proteins

The recombinant expression strain BL21(pET30a-a10) 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 is finished, centrifuging at 4 ℃ and 8000rpm for 10min, collecting escherichia coli thalli, resuspending the thalli by using Tris-HCl buffer solution of 20mmol/L, crushing by using an ultrasonic crusher, purifying arsenic aggregate protein A10 by using a nickel ion affinity chromatography column on crushed supernatant, and detecting the purification effect by SDS-PAGE protein electrophoresis.

As shown in figure 1, the expression of the arsenic binding protein in Escherichia coli shows that the crushed supernatant has a specific protein band at 19.2 kDa, 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-a10) obtained in example 1 was cultured in LB medium (50 mg. L)^{- 1}Kana), inoculated in LB medium at an inoculum size of 1% and grown to an OD value of 0.6-0.8, added with 0.4mM IPTG, and cultured at 16 ℃ for 18 hours. Centrifuging to remove supernatant, eluting thallus with ST culture medium of the same volume, adding ST culture medium of the same volume for resuspension, inoculating into 30ml ST culture medium according to the inoculation amount of 2%, simultaneously adding antibiotics, adding 0 μ M, 10 μ M, 20 μ M, 40 μ M and 60 μ M of As (III), taking 1ml of bacterial liquid in 0, 6, 10, 14, 22 and 24 hours, and determining 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 is measured for growth of the bacteria at 600nm wavelength by an ultraviolet spectrophotometer.

The experimental result is shown in FIG. 2, after the arsenic binding gene a10 is expressed in arsenic-sensitive Escherichia coli, the resistance of the recombinant expression strain pET30a-a10 to arsenite can reach 60 mu M, and the growth condition of the recombinant strain pET30a-a10 in 60 mu M As (III) is better than that of the unloaded strain pET30a grown in 10 mu M As (III), which shows that the arsenic binding gene a10 can obviously improve the resistance of Escherichia coli to arsenite.

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

The expression strain BL21(pET30a-a10) 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. mu.M, 1. mu.M, 3. mu.M, 9. mu.M MMA (III), 1ml of the bacterial suspension was collected at 0, 2, 4, 6, 12, 14, 24 hours, respectively, and the OD value was measured with a spectrophotometer.

As shown in FIG. 3, after expressing the arsenic-binding gene a10 in arsenic-sensitive E.coli, the resistance of the expression strain pET30a-a10 to methyl arsenite was as high as 3. mu.M in the MMA (III) resistance test; while the empty-load strain pET30a was only 1 μ M resistant, which was a significant improvement of about 3-fold. After the arsenic binding gene a10 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 expression strain BL21(pET30a-a10) 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-a10)

The expression strain BL21(pET30a-a10) was cultured in LB medium (50 mg. L)^-1Kana) overnight, inoculated in LB medium at an inoculum size of 1% to OD₆₀₀When the value was 0.6 to 0.8, 0.4mM IPTG was added and the mixture was cultured 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 1h of incubation.

An empty strain (pET30a) was also set as a control, with three replicates per set of experiments. 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 is that freeze-dried Escherichia coli is weighed to 0.01g, added into a cracking tube containing 0.5g of glass beads (0.01-0.1 mm), and 600. mu.L of ultrapure water is added, and placed into Fastprep-24(MB biomedicals) to 6.5 m.s^-1Crushing for 60 s; centrifuge at 12000rpm for 1Min, aspirate the supernatant, add to a new tube containing glass strain, and continue disruption. Until the supernatant was clear and transparent. The supernatant was filtered through a 0.22. mu.M filter and 10% H was added₂O₂And the arsenic form is determined after the mixture is placed for 30 min.

The experimental results are shown in FIG. 4, and the expression strain BL21 is 50 mu M As (III) extracellular arsenic concentration (arsenic concentration in Medium) of the No-load Strain (pET30a) after 1 hour of stress in Medium was 3125.51. mu.g.kg^-1And the extracellular arsenic concentration of the expression strain pET30a-a10 is as low as 291 mu g kg^-192.24 percent of arsenite in the culture medium is removed; meanwhile, in the control strain of 0.01g, the same phenomenon was found: arsenic accumulated in cells of the recombinant expression strain pET30a-a10 is as high as 1993.93 mu g kg^-1Much higher than 334.25. mu.g.kg of the unloaded strain (pET30a)^-1. In conclusion, the expression strain containing the arsenic binding gene a10 can remarkably remove arsenite in a stress environment by accumulating the arsenite from the outside to the inside within 1 hour, and the removal rate is as high as 92.24%.

3.2 efficiency of absorption of Methylarsenate by recombinant expression Strain

The expression strain BL21(pET30a-a10) 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 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-30 a) 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. 5, the extracellular As concentration decreased to 29.76. mu.g.kg after the recombinant expression strain pET30a-a10 was stressed in 2. mu.M MMA (III) medium for 1 hour^-1Much lower than 94.52. mu.g.kg of the unloaded strain^-1(ii) a Meanwhile, the intracellular arsenic content of 0.01g of the expression strain pET30a-a10 is not 329 microgram kg^-1Is far higher than 180.02 mug.kg of the unloaded bacteria^-1. The expression strain pET30a-a10 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 79.69%.

In conclusion, the results in example 3 show that the recombinant expression strain pET30a-a10 has high accumulation of arsenite and high accumulation of methyl arsenite, and the gene a10 has the capacity of accumulating 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 A10 of interest on arsenite and methyl arsenite

4.1 in vitro Activity assay of protein A10 of interest on arsenite

Reaction system: 100mM MOPS buffer (pH 7.0), 1mM cysteine, 50. mu.M As (III), 10. mu.M protein of interest A10. 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 an enzyme activity reaction system before oxidation; the other part was charged with 10% by volume of H₂O₂Stopping reaction, centrifugally extracting supernatant, and measuring the arsenic form by using HPLC-ICP-MS, wherein the part of arsenic is the arsenic content released by an enzyme activity system due to protein denaturation after the part of arsenic is oxidized.

As shown in FIG. 6, the concentration of inorganic arsenic in the reaction system was 123.54. mu.g/kg, after the reaction of the purified target protein A10 with arsenite for 30Min^-1Is far lower than 3447 mu g/kg of blank group CT reaction system^-1About 96.70% of the arsenite is bound. And H is added₂O₂Then, the protein structure was destroyed to release arsenic, and the concentration of arsenic in reaction system A10 was 3124. mu.g.kg^-1It indicates the binding ability of the target protein to arsenic.

4.1 in vitro activity assay of protein A10 of interest on methyl arsenite.

Reaction system: 100mM MOPS buffer (pH 7.0), 1mM cysteine, 2. mu.M MAs (III), 10. mu.M protein of interest A10. Blank CT is treatment without added protein. The termination reaction and the treatment of the sample to be tested are the same as in example 4.1.

As shown in FIG. 7, the reaction system of the target protein A10 contained only a small amount of organic arsenic at a concentration of 7.52. mu.g/kg after 30Min hours of reaction between the purified target protein A10 and methyl arsenite^-1The arsenic concentration of the blank group CT reaction system is 142.56 mug.kg^-1About 83.33% of the methyl arsenite is bound by the protein; when addingAfter the oxidizing agent is added to destroy the protein structure, a large amount of organic arsenic is released, and the concentration of the arsenic in the A10 reaction system is 132.57 mug/kg^-1The concentration of the blank group is 143.6 mug/kg without obvious difference before and after oxidation^-1. The target protein A10 is shown to have a remarkable binding effect on methyl arsenite.

In conclusion, the target protein A10 has significant binding effect on arsenite and methyl arsenite, and the binding rates are 96.70% and 83.33%, respectively.

The invention obtains the arsenic binding protein A10 from Trichoderma asperellum SM-12F1 of Trichoderma asperellum, and experiments prove that when the gene is expressed in arsenic-sensitive Escherichia coli BL21, the resistance of the Escherichia coli to arsenite and organic arsenic can be obviously improved. 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 A10 with arsenite and methyl arsenite binding capacity, engineering strain containing protein gene and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 441

<212> DNA

<213> arsenic-binding protein A10 of Trichoderma asperellum (Trichoderma asperellum)

<400> 1

atgtcatcta acaatactga ggtgcccact gaggaatgtt acggccagat gtgctggctc 60

accgtccctg ttgtagatat cgacagggcc aaggcctttt acgccgagat cttcaattgg 120

gagatcagcc ccgaaggcgt cgccaacgac cggcccggcg ttaaggagct ttattacttc 180

aatcgcggaa aaacgctgca tggcgctttt tgcgtgatgg aagatggatt ccacgtcatc 240

aaccaatcaa tgggttctac agatgccata tccgttcatc cgagcttcta cgtcaagaac 300

tgcaaggata cactcgagga agttgaaaga cttggtggca agaagcacct gcataagacg 360

gagattggcg gggacatggg ccattatgct cgattcatcg atactgaggg caacttgatc 420

ggaatctggt ccaaaatctg a 441

<210> 2

<211> 146

<212> PRT

<213> arsenic-binding protein A10 of Trichoderma asperellum (Trichoderma asperellum)

<400> 2

Met Ser Ser Asn Asn Thr Glu Val Pro Thr Glu Glu Cys Tyr Gly Gln

1 5 10 15

Met Cys Trp Leu Thr Val Pro Val Val Asp Ile Asp Arg Ala Lys Ala

20 25 30

Phe Tyr Ala Glu Ile Phe Asn Trp Glu Ile Ser Pro Glu Gly Val Ala

35 40 45

Asn Asp Arg Pro Gly Val Lys Glu Leu Tyr Tyr Phe Asn Arg Gly Lys

50 55 60

Thr Leu His Gly Ala Phe Cys Val Met Glu Asp Gly Phe His Val Ile

65 70 75 80

Asn Gln Ser Met Gly Ser Thr Asp Ala Ile Ser Val His Pro Ser Phe

85 90 95

Tyr Val Lys Asn Cys Lys Asp Thr Leu Glu Glu Val Glu Arg Leu Gly

100 105 110

Gly Lys Lys His Leu His Lys Thr Glu Ile Gly Gly Asp Met Gly His

115 120 125

Tyr Ala Arg Phe Ile Asp Thr Glu Gly Asn Leu Ile Gly Ile Trp Ser

130 135 140

Lys Ile

145

Claims (9)

1. An arsenic binding protein A10, the nucleic acid sequence of the coding gene of the protein is shown in SEQ ID NO. 1.

2. The arsenic-binding protein A10 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 a10, according to claim 1, wherein the arsenic binding protein a10 is derived from trichoderma asperellum.

4. A recombinant vector comprising a gene encoding arsenic binding protein a10 according to claim 1.

5. A recombinant 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 A10 of claim 1 to increase the resistance of a strain to arsenite and methyl arsenite.

8. Use of the arsenic binding protein A10 of claim 1 to increase the efficiency of arsenite and methyl arsenite uptake by a strain.

9. Use of the arsenic binding protein a10 of claim 1 to bind arsenite and methyl arsenite in vitro.

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