CN114605510B - Protein A10 with arsenite and methyl arsenite binding capacity, engineering strain containing protein gene and application - Google Patents
Protein A10 with arsenite and methyl arsenite binding capacity, engineering strain containing protein gene and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C02F2101/00—Nature of the contaminant
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- C02F2101/103—Arsenic compounds
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- C02F2101/00—Nature of the contaminant
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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 of the invention 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 combine arsenite and methyl arsenite in vitro. The engineering bacteria have strong resistance to arsenite and methyl arsenite, have high binding efficiency of 92.24 percent and 76.69 percent on arsenite and reducing monomethyl arsenic, have high binding efficiency of 96.70 percent and 83.33 percent on arsenite and reducing monomethyl of target protein A10, and can effectively remove arsenic in a culture environment. The protein A10 with strong binding capacity to arsenite and methyl arsenite provides a novel bioremediation material for arsenic pollution remediation in paddy fields and water environments.
Description
[ field of technology ]
The invention relates to the technical field of genetic engineering, in particular to an arsenic binding protein derived from trichoderma asperellum and having arsenite and methyl arsenite binding capacity, a gene for encoding the protein, a vector containing the gene, a recombinant engineering strain and application thereof.
[ background Art ]
Arsenic (As) is a common toxic heavy metal element in the soil environment. The source of arsenic in the soil is typically from the mining of arsenic ores, sewage irrigation, agricultural chemical fertilizer application to farms, and the like. Crop planting in severely out-of-standard arsenic polluted farmlands not only can bring hidden danger to agricultural product safety, but also can harm the physical health of people. The rice is used as an important grain crop in China, the growth environment of long-term flooding and the physiological characteristics of silicon-like and phosphorus-like are main causes of arsenic sensitivity and accumulation of the rice, and the methyl arsenic accumulated in rice grains is the main cause of the rice spike disease. At present, the environmental arsenic repair technology mainly comprises a biological repair technology, a chemical repair technology and a physical repair technology. Microbial remediation mainly relies on interaction of microorganisms and arsenic, including adsorption/resolution, precipitation/dissolution, oxidation/reduction, methylation/demethylation and other actions, so that the biological effectiveness of heavy metal pollutants is reduced, and the aim of restoring environmental arsenic pollution is fulfilled. In the early laboratory studies, a highly resistant strain of Trichoderma asperellum Trichoderma asperellum SM-12F1 was found, isolated in 2009 in the soil near the Realgar mining area of Shimen county, hunan province, with resistance to arsenate as high as 500 mg.L -1 Can mediate the oxidation of arsenite, reduction of arsenate, methylation of inorganic arsenic and demethylation of organic arsenic.
In a farmland flooding environment, arsenic exists mainly in the form of arsenite (As (III)) and reduced organic arsenic such As methyl arsenite [ MMAs (III) ], reduced dimethyl arsenics [ DMAs (III) ]. As (III) is not only absorbed by plant roots through aquaporins, but is also the primary form of metastasis in organisms; organic arsenic, especially DMAs, is absorbed by rice root systems and accumulated behind glumes, which is easy to cause rice straight spike disease, thereby causing rice yield reduction and even absolute yield. Therefore, the detoxification mechanism of the microorganism to the arsenic is known, a novel arsenic pollution restoration method and material are developed, theoretical basis and technical support are provided for restoration of the arsenic pollution in the environment, and the method has practical significance and economic effect.
[ 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 gene of the arsenic binding protein, and finds that the protein has strong binding effect on arsenite and methyl arsenite after the expression of the protein is determined through experiments, so that the arsenic content in a culture environment can be effectively reduced.
In order to achieve the above object, the present invention provides an arsenic binding protein A10, wherein the nucleic acid sequence of the encoding gene of the protein is shown as SEQ ID NO. 1; the invention provides an arsenic binding protein A10, and the nucleic acid sequence of the coding gene of the protein is shown as SEQ ID NO. 2;
the arsenic binding protein A10 of the invention is derived from Trichoderma asperellum, in particular Trichoderma asperellum Trichoderma asperellum SM-12F1 (Arsenite Resistance, accumulation, andVolatilization Properties of Trichoderma asperellum SM-12F1, penicillium janthinellum SM-12F4,and Fusarium oxysporum CZ-8F1,Xibai Zeng et al.) Trichoderma asperellum Trichoderma asperellum SM-12F1 is also described in the Chinese patent application CN 201210361866.6, written as Trichoderma asperellum Trichoderma asperellum, saved in China general microbiological culture Collection center under the preservation number CGMCC No.3186.
Based on this, the present invention also provides a recombinant vector containing the above-mentioned gene encoding arsenic-binding protein a10.
And engineering strains containing the recombinant vector, particularly preferably E.coli.
The invention also provides application of the arsenic binding protein A10 in improving resistance of a strain to arsenite and methyl arsenite, application in improving accumulation efficiency of the strain to arsenite and methyl arsenite, and application in combining arsenite and methyl arsenite in vitro.
The invention discovers the protein A10 with strong binding capacity to arsenite and methyl arsenite for the first time, constructs a recombinant vector and engineering bacteria, and verifies that the engineering bacteria have strong resistance to arsenite (60 mu M) and methyl arsenite (3 mu M). The invention is characterized in that
The combination efficiency of the engineering bacteria on arsenite and reducing monomethyl arsenic is up to 92.24% and 76.69%, and the combination efficiency of the target protein A10 on arsenite and reducing monomethyl arsenic is up to 96.70% and 83.33%, so that arsenic in a culture environment can be effectively removed.
The protein A10 with strong binding capacity to arsenite and methyl arsenite provides a novel bioremediation material for arsenic pollution remediation in paddy fields and water environments.
Compared with the prior art, the binding protein is discovered in a fungus body for the first time, and has high binding capacity for reduced inorganic arsenic and organic arsenic. For example, compared with the technical proposal disclosed in the Chinese patent application No. CN 111996207A, the recombinant bacterium disclosed by the invention not only relates to the removal of arsenic in one form, but also has the removal rate obviously higher than that of the prior art.
[ description of the drawings ]
FIG. 1 is an electrophoresis diagram of arsenic binding protein A10 purification;
FIG. 2 is a graph showing the effect of arsenic binding gene a10 on resistance to arsenite in E.coli;
FIG. 3 is a graph showing the effect of arsenic binding gene a10 on E.coli on methyl arsenite resistance;
FIG. 4 is the accumulation of arsenite by recombinant strain BL21 (pET 30a-a 10);
FIG. 5 shows the accumulation of methyl arsenite by recombinant strain BL21 (pET 30a-a 10);
FIG. 6 is a graph showing the binding efficiency of arsenic binding protein A10 to arsenite;
FIG. 7 shows the binding efficiency of arsenic binding protein A10 to methyl arsenite.
[ detailed description ] of the invention
The following examples serve to illustrate the technical solution of the invention without limiting it.
In the invention, unless otherwise specified, "%" for specifying the concentration is mass percent ": "all are mass ratios.
The invention relates to the following culture media and chemical reagents:
PGP liquid medium: 100g of potato (cut into 1cm potato pieces, boiled for about 30 min), 10g of glucose and 2.5g of peptone, the volume of the potato is 1000ml after boiling, and the potato is sterilized at 121 ℃ for 20min.
LB medium: yeast extract 5g, tryptone 10g, sodium chloride 10g, constant volume to 1L (ph=7), sterilized at 121 ℃ for 20min.
ST10 -1 Culture medium: 0.05g of yeast powder, 0.5g of peptone, 5g of glucose, and fixing the volume to 1L,
sterilizing at 121deg.C for 20min.
ST medium: 0.5g of yeast phenol, 5g of peptone, 5g of glucose, and sterilizing at 121 ℃ for 20min.
Sodium arsenite (As (III)) NaAsO 2 The method comprises the steps of carrying out a first treatment on the surface of the Sodium methyl arsenite (MMA (III)) was reduced by sodium methyl arsenite, reduction system: 0.2mM methyl arsenate MMA (V), 27mM NaS 2 O 3 ，66 mM NaS 2 O 5 ，82mM H 2 S0 4 ，pH＝6。
Antibiotics: kanamycin sulfate (Kana); induction of expression: isopropyl- β -D-thiogalactoside (IPTG).
The invention relates to the following primers:
TABLE 1 primer information
EXAMPLE 1 construction of arsenic-binding Gene vector, expression Strain and purification of arsenic-binding protein
1.1 DNA extraction of Trichoderma asperellum Trichoderma asperellum SM-12F1
Trichoderma asperellum was taken in Trichoderma asperellum SM-12F1, fungal DNA was extracted according to the DNA kit (Quick-DNATM Fungal/Bacterial Midiprep Kit, zymo Research, orange, calif., USA) extraction procedure, the target DNA bands were detected by 10% agarose gel electrophoresis, and the concentration of purified DNA was confirmed by Nanodrop detection.
Genomic DNA of insert size 20kb SMRT bell was constructed using SMRT bell template preparation kit (SMRTbell Template Prep Kits, pacific Biosciences). The DNA samples were sheared to fragments of less than 400bp using the Illumina TruSeq Nano DNA Library Prep Kits kit. The 20kb and 400bp libraries were sequenced using the PacBIO RS II platform and Illumina Hiseq 2000, respectively.
And then carrying out gene prediction and annotation on the assembled genome of the trichoderma spinosum SM-12F 1. The coding sequence CDS was predicted using Augustus v3.3.2 and GeneMark-ES v 4.46. The obtained genes were submitted to databases such as Cluster of Orthologous Groups of proteins (COG), gene on log (GO), kyoto Encyclopediaof Genes and Genomes (KEGG) using a BLAST, diamond, HMMER sequence alignment tool. The results are annotated to 1 dioxygenase a10 associated with protein binding.
1.2 construction of the amplification and expression Strain of the arsenic binding protein encoding Gene
1.2.1 PCR amplification, cleavage and recovery of the arsenic-binding protein-encoding Gene
The target gene is obtained by using the inverted trichoderma spinosum SM-12F1 DNA gene as a template and using a primer for amplifying the arsenic binding protein encoding gene.
(1) Amplification system of target gene a 10:
(2) Target gene amplification procedure:
95℃ 5min；
95 ℃ 30sec,65 ℃ 30sec, -1 ℃/cycle,72 ℃ 1min,14 cycles;
95℃30sec,62℃30sec,72℃1min,72℃10min,16℃10min, 32 cycles.
The amplified PCR system is subjected to 1% agarose gel electrophoresis, and the target fragment is obtained through gel cutting and recovery.
(3) EcoR I and Hind III were double digested, the digestion system was as follows:
the water bath kettle is opened in advance, the temperature is set to be 37 ℃, and the enzyme digestion system is immersed and reacts for 12-16 hours. Then, 5. Mu.L was taken for 1% agarose gel detection and recovered using a common DNA product purification kit.
1.2.2 cleavage and recovery of the vector
For plasmid pET-30a (+) double digestion was likewise carried out using EcoR I and Hind III double digestion, the digestion system being as follows:
the enzyme cutting condition and the recovery process are the same as before.
1.2.3 ligation of the Gene of interest with vector
The DNA concentration of the target gene and the vector was determined by using a microplate quantification method, and the ligation system was as follows:
the above system was thoroughly mixed and then placed in a metal bath at 16 ℃ for reaction overnight.
1.2.4 construction and Positive identification of recombinant expression Strain
The recombinant plasmid is introduced into competent cells of escherichia coli TOP10, and after culturing, bacterial liquid is used as a PCR template for identifying positive clones, and T7ter are used as primers for amplification. The PCR products were detected by 1% agarose gel electrophoresis, and the size of the target band was determined.
The amplification system is as follows:
the PCR amplification procedure was as follows:
95℃ 5min；
95℃30sec,55℃30sec,72℃1min 30sec,29 cycles; 72℃for 10min and 16℃for 10min.
The TOP10 strain with the same sequencing result was cultured, and then the plasmid was extracted and introduced into BL21 (DE 3) for expression. The PCR products were detected by 1% agarose gel electrophoresis using T7 and T7ter as primers, and the target band sizes were sequenced.
Transformants sequenced correctly were designated BL21 (pET 30a-a 10).
1.2.5 expression and purification of arsenite and methyl arsenite binding proteins
Recombinant expression strain BL21 (pET 30a-a 10) was cultured in LB medium to OD 600 After 0.6 to 0.8 nm, 0.4mM IPTG (isopropyl-. Beta. -D-thiogalactoside) was added and incubated overnight at 16℃and 200 rpm. After the culture, the cells were collected by centrifugation at 8000rpm for 10min at 4℃and resuspended in 20mmol/L Tris-HCl buffer, and disrupted by an ultrasonic disruption apparatus, and the disrupted supernatant was purified by nickel ion affinity chromatography to As-collectin A10, and the purification effect was examined by SDS-PAGE protein electrophoresis.
As shown in FIG. 1, the expression of the arsenic binding protein in Escherichia coli shows that the broken supernatant has a specific protein band at 19.2 kDa, which indicates that the arsenic binding gene realizes soluble expression in Escherichia coli and the expression is better.
Example 2 resistance experiment
2.1 arsenic-binding Gene increases resistance of E.coli to arsenite
The recombinant expression strain BL21 (pET 30a-a 10) obtained in example 1 was cultured in LB medium (50 mg.L) - 1 Kana) was cultured overnight, and inoculated into LB medium at an inoculum size of 1% for growthWhen the OD value is 0.6-0.8, 0.4mM IPTG is added and the mixture is incubated 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, re-suspending, inoculating into 30ml of ST culture medium at 2% inoculation amount, adding antibiotics, and adding 0 μM,10 μM,20 μM,40 μM, and 60 μM As (III), taking 1ml bacterial liquid at 0, 6, 10, 14, 22, and 24 hr, and measuring OD value; at the same time, empty strain BL21 (pET 30 a) was used as a control, and three replicates were set for each experiment, and cultured at 30℃and 200 rpm. The growth of the cells was measured at a wavelength of 600nm with an ultraviolet spectrophotometer.
As shown in FIG. 2, after expressing arsenic binding gene a10 in arsenic-sensitive E.coli, the resistance of recombinant expression strain pET30a-a10 to arsenite can reach 60. Mu.M, and the growth condition of recombinant strain pET30a-a10 in 60. Mu.M As (III) is superior to that of empty strain pET30a grown under 10. Mu.M As (III), which indicates that the arsenic binding gene a10 can significantly improve the resistance of E.coli to arsenite.
2.2 arsenic-binding Gene increases E.coli resistance to methyl arsenite
The expression strain BL21 (pET 30a-a 10) obtained in example 1 was cultured in LB medium (mg.L) -1 Kana) was cultured overnight, and inoculated in 1% of the inoculum size into LB medium to grow to OD 600 At a value of 0.6-0.8, 0.4mM IPTG was added and incubated 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, re-suspending, inoculating into 30ml ST culture medium at 2% inoculation amount, and adding 50mg.L -1 1ml of each of Kana, 0. Mu.M, 1. Mu.M, 3. Mu.M, and 9. Mu.M MMA (III) was collected at 0, 2, 4, 6, 12, 14, and 24 hours, and the OD was measured by a spectrophotometer.
As shown in FIG. 3, after expressing arsenic-binding gene a10 in arsenic-sensitive E.coli, the resistance of expression strain pET30a-a10 to methyl arsenite was as high as 3. Mu.M in MMA (III) resistance experiments; whereas the resistance of the empty strain pET30a is only 1 mu M, which is significantly improved by about 3 times. After expressing arsenic binding gene a10 in arsenic-sensitive E.coli, the resistance of the expression strain methyl arsenite was significantly improved.
Taken together, the results in example 2 show that the expression strain BL21 (pET 30a-a 10) has both high resistance to arsenite and methyl arsenite, and is the gene that was first found to be resistant to two reducing arsenic species in eukaryotic microorganisms.
EXAMPLE 3 binding efficiency of recombinant expression Strain to arsenite and methyl arsenite
3.1 absorption efficiency of recombinant expression Strain BL21 (pET 30a-a 10) on arsenite
Expression strain BL21 (pET 30a-a 10) was cultured in LB medium (50mg.L) -1 Kana) was cultured overnight, and inoculated in 1% of the inoculum size into LB medium to grow to OD 600 At a value of 0.6-0.8, 0.4mM IPTG was added and incubated at 25℃for 6 hours. Centrifuging to remove supernatant, and concentrating with equal volume of ST10 -1 After the cells were eluted in the medium, an equal volume of ST10 was added -1 Culture medium resuspension (50 mg.L) -1 Kana) and 50 μm As (III) was added and sampled after 1h of incubation.
The empty strain (pET 30 a) was set up simultaneously as a control, and three replicates were set up for each set of experiments. The supernatant filtering membrane is subjected to HPLC-ICP-MS to measure arsenic form, and the result is extracellular arsenic; the bacterial pellet was eluted with PBS and lyophilized, and arsenic was extracted with water to determine the morphology, which resulted in intracellular arsenic. The method comprises weighing lyophilized Escherichia coli 0.01g, adding into a lysis tube containing 0.5g glass beads (0.01-0.1 mm), adding 600 μl of ultrapure water, and standing in Fastprep-24 (MB biomedicals) for 6.5 m.s -1 Crushing for 60s; centrifuge at 12000rpm for 1Min, aspirate supernatant, add to a new tube containing glass strain and continue disruption. Until the supernatant was clear and transparent. Filtering the supernatant with 0.22 μm filter membrane, adding 10% H 2 O 2 The arsenic morphology was measured after 30min of standing.
As shown in FIG. 4, the extracellular arsenic concentration (arsenic concentration in the medium) of the empty strain (pET 30 a) was 3125.51. Mu.g.kg after 1 hour of stress of the expression strain BL21 in 50. Mu.M As (III) medium -1 While the extracellular arsenic concentration of the expression strain pET30a-a10 is as low as 291 mug.kg -1 92.24% of arsenite in the culture medium is removed; at the same time, in the control strain of 0.01g, the same was foundThe phenomenon of (2): the intracellular accumulation of arsenic in recombinant expression strain pET30a-a10 reaches 1993.93 mug.kg -1 334.25. Mu.g.kg, far higher than the empty strain (pET 30 a) -1 . In summary, the expression strain containing the arsenic binding gene a10 can significantly remove arsenite in the stress environment by accumulating arsenite from outside to inside within 1 hour, and the removal rate is as high as 92.24%.
3.2 absorption efficiency of recombinant expression Strain on methyl arsenite
Expression strain BL21 (pET 30a-a 10) was cultured in LB medium (50mg.L) -1 Kana) was cultured overnight, and inoculated in 1% of the inoculum size into LB medium to grow to OD 600 At a value of 0.6-0.8, 0.4mM IPTG was added and incubated at 25℃for 6 hours. Centrifuging to remove supernatant, and concentrating with equal volume of ST10 -1 After the cells were eluted in the medium, an equal volume of ST10 was added -1 The medium was resuspended, 2. Mu.M MMA (III) was added separately and the culture was sampled after 1 h. The empty strain BL21 (pET-30 a) was also set as a control, and three replicates were set for each set of experiments. Intracellular arsenic and extracellular arsenic are determined as described in example 3, step 3.1.
As shown in FIG. 5, the extracellular organic arsenic concentration of recombinant expression strain pET30a-a10 was reduced to 29.76. Mu.g.kg after 1 hour of stress in 2. Mu.M MMA (III) medium -1 Far below 94.52. Mu.g.kg of the empty strain -1 The method comprises the steps of carrying out a first treatment on the surface of the Meanwhile, 0.01g of expression strain pET30a-a10 has intracellular arsenic content of 329 mu g.kg -1 Is far higher than 180.02 mug.kg of empty vector 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 is removed, and the removal rate is as high as 79.69%.
In summary, the results in example 3 show that the recombinant expression strain pET30a-a10 has high accumulation of arsenite and also has high accumulation of methyl arsenite, and the gene a10 has the capability of accumulating various reduced arsenic, so that the recombinant engineering strain containing the gene is a biological material suitable for repairing various arsenic pollution.
Example 4 in vitro Activity of protein A10 of the order of America on arsenite and methyl arsenite
4.1 in vitro Activity test of protein A10 of interest against arsenite
The 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 protein addition. After standing at 30 ℃ for 30Min, a part of arsenic is directly filtered to measure the arsenic form, and the part of arsenic is arsenic content in an enzyme activity reaction system before oxidation; another portion was added with 10% by volume of H 2 O 2 Terminating the reaction, centrifuging to extract supernatant, and measuring arsenic form by HPLC-ICP-MS, wherein part of arsenic is arsenic content released by enzyme activity system due to protein denaturation after oxidation.
As shown in FIG. 6, the concentration of inorganic arsenic in the reaction system after 30Min of reaction with arsenite was 123.54. Mu.g.kg -1 Is far lower than 3447 mug.kg of a blank CT reaction system -1 About 96.70% of the arsenite was incorporated. To add H 2 O 2 After that, the protein structure was destroyed to release arsenic, and at this time, the arsenic concentration in the reaction system A10 was 3124. Mu.g.kg -1 The binding ability of the target protein to arsenic is shown.
4.1 in vitro Activity assay of protein A10 of interest against methyl arsenite.
The 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 protein addition. Termination reactions and sample treatments to be tested were as in example 4.1.
As shown in FIG. 7, the purified target protein A10 was reacted with methyl arsenite for 30Min, and the concentration of organic arsenic was 7.52. Mu.g.kg in the reaction system of the target protein A10 -1 Arsenic concentration of a blank CT reaction system is 142.56 mug.kg -1 About 83.33% of methyl arsenite is bound by the protein; when the protein structure is destroyed by adding oxidant, a large amount of organic arsenic is released, and the arsenic concentration in the A10 reaction system is 132.57 mug.kg -1 No significant difference exists between the blank group before and after oxidation, and the concentration is 143.6 mug.kg -1 . The protein A10 has obvious binding effect on methyl arsenite.
In conclusion, the target protein A10 has remarkable binding effect on arsenite and methyl arsenite, and the binding rate is 96.70% and 83.33% respectively.
According to the invention, from trichoderma asperellum Trichoderma asperellum SM-12F1, the arsenic binding protein A10 is obtained, and experiments prove that when the gene of the arsenic-sensitive escherichia coli BL21 is expressed, 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, so that the arsenic binding protein has obvious application prospect and environmental protection significance.
Sequence listing
<110> national academy of agricultural sciences agricultural environment and sustainable development institute
<120> protein A10 having binding ability to arsenite and methyl arsenite, engineering strain containing the protein gene and use thereof
<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 (3)
1. The application of the arsenic binding protein A10 in improving the resistance of strains to arsenite and methyl arsenite is provided, the nucleic acid sequence of the coding gene of the protein is shown as SEQ ID NO.1, the amino acid sequence of the coding gene is shown as SEQ ID NO.2, and the arsenic binding protein A10 is derived from trichoderma asperellum.
2. The application of the arsenic binding protein A10 in improving the absorption efficiency of strains on arsenite and methyl arsenite is provided, the nucleic acid sequence of the coding gene of the protein is shown as SEQ ID NO.1, the amino acid sequence of the coding gene is shown as SEQ ID NO.2, and the arsenic binding protein A10 is derived from trichoderma asperellum.
3. The application of the arsenic binding protein A10 in-vitro binding arsenite and methyl arsenite is characterized in that the nucleic acid sequence of a coding gene of the protein is shown as SEQ ID NO.1, the amino acid sequence of the coding gene is shown as SEQ ID NO.2, and the arsenic binding protein A10 is derived from trichoderma asperellum.
Priority Applications (1)
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| WO2022023583A1 (en) * | 2020-07-31 | 2022-02-03 | Biotalys NV | Expression host |
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| JP2003265186A (en) * | 2002-03-19 | 2003-09-24 | Japan Science & Technology Corp | Gene involved in arsenic tolerance |
| CN102876586A (en) * | 2012-09-26 | 2013-01-16 | 中国农业科学院农业环境与可持续发展研究所 | Method for screening trichoderma asperellum and application thereof |
| CN108559710A (en) * | 2018-01-03 | 2018-09-21 | 中国农业科学院农业环境与可持续发展研究所 | A kind of reduction crop absorbs seed coat agent and preparation method thereof to arsenic |
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