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Protein A10 with arsenite and methyl arsenite combining capacity, engineering strain containing protein gene and application

A methylarsenite, protein-binding technology, applied in the restoration of polluted soil, microorganism-based methods, bacteria, etc., to achieve high binding capacity and high removal rate

Active Publication Date: 2022-06-10
INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Planting crops in arsenic-contaminated farmland that seriously exceeds the standard will not only bring hidden dangers to the safety of agricultural products, but also endanger human health

Method used

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  • Protein A10 with arsenite and methyl arsenite combining capacity, engineering strain containing protein gene and application
  • Protein A10 with arsenite and methyl arsenite combining capacity, engineering strain containing protein gene and application
  • Protein A10 with arsenite and methyl arsenite combining capacity, engineering strain containing protein gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of arsenic-binding gene vector, expression strain and purification of arsenic-binding protein

[0035] 1.1 DNA Extraction of Trichoderma asperellum SM-12F1

[0036] Take Trichoderma asperellum SM-12F1 from Trichoderma asperellum SM-12F1, extract fungal DNA according to the DNA kit (Quick-DNATM Fungal / Bacterial Midiprep Kit, Zymo Research, Orange, CA, USA) extraction steps, and detect the target by 10% agarose gel electrophoresis DNA bands, using Nanodrop detection to confirm the concentration of purified DNA.

[0037] SMRTbell template prep kits (SMRTbell Template Prep Kits, Pacific Biosciences) were used to construct genomic DNA with an insert size of 20 kb SMRTbell. DNA samples were sheared into fragments smaller than 400bp using the Illumina TruSeq NanoDNA Library Prep Kits kit. The 20kb and 400bp libraries were sequenced using the PacBio RS II platform and Illumina Hiseq 2000, respectively.

[0038]Then, gene prediction and annotation were ...

Embodiment 2

[0073] Embodiment 2 resistance experiment

[0074] 2.1 Arsenic-binding genes enhance the resistance of Escherichia coli to arsenite

[0075] The recombinant expression strain BL21 (pET30a-a10) that embodiment 1 obtains is in LB medium (50mg L - 1 After culturing overnight in Kana), inoculate 1% of the inoculum into LB medium and grow to OD value of 0.6-0.8, add 0.4mM IPTG, and cultivate at 16°C for 18 hours. After centrifuging to remove the supernatant, elute the bacteria with an equal volume of ST medium, add an equal volume of ST medium to resuspend, insert 2% of the inoculum into 30ml ST medium, and add antibiotics and 0μM , 10μM, 20μM, 40μM, 60μM As(III), take 1ml of the bacterial solution at 0, 6, 10, 14, 22, and 24h, and measure the OD value; at the same time, set the empty load strain BL21 (pET30a) as the control, each group of experiments Set up three replicates, culture at 30°C, 200rpm. The bacteria liquid was measured with a UV spectrophotometer at a wavelength o...

Embodiment 3

[0081] Example 3 Binding efficiency of recombinant expression strains to arsenite and methyl arsenite

[0082] 3.1 Absorption efficiency of arsenite by recombinant expression strain BL21 (pET30a-a10)

[0083] Express the strain BL21 (pET30a-a10) in LB medium (50 mg·L -1 After culturing overnight in Kana), inoculate 1% inoculum into LB medium and grow to OD 600 When the value is 0.6-0.8, add 0.4mM IPTG and incubate at 25°C for 6 hours. After centrifugation to remove the supernatant, use an equal volume of ST10 -1 After the cells were eluted from the culture medium, an equal volume of ST10 was added -1 Medium resuspended (50 mg·L -1 Kana), and 50 μM As(III) was added, and samples were taken after culturing for 1 h.

[0084] At the same time, an empty strain (pET30a) was set as a control, and three replicates were set for each group of experiments. The arsenic form of the supernatant filter membrane was measured by HPLC-ICP-MS, and the result was extracellular arsenic; the ...

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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 disclosed by the invention can be used for improving the resistance of a strain to arsenite and methyl arsenite, improving the accumulation efficiency of the strain to the arsenite and the methyl arsenite and combining the arsenite and the methyl arsenite in vitro. The engineering bacterium has strong resistance to arsenite and methyl arsenite, the binding efficiency of the target protein A10 to arsenite and reducing monomethyl arsenic is as high as 92.24% and 76.69%, the binding efficiency of the target protein A10 to arsenite and reducing monomethyl 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 novel bioremediation material for arsenic pollution remediation in paddy fields and water environments.

Description

【Technical field】 [0001] The invention relates to the technical field of genetic engineering, in particular to an arsenic-binding protein derived from Trichoderma aculeatus and capable of binding arsenite and methyl arsenite, and also relates to a gene encoding the protein, containing the Gene carrier, recombinant engineering strain and its application. 【Background technique】 [0002] Arsenic (As) is a common toxic heavy metal element in the soil environment. The sources of arsenic in soil usually come from the mining of arsenic mines, sewage irrigation, and pesticides and fertilizers applied on farmland. Planting crops in arsenic-contaminated farmland that seriously exceeds the standard will not only bring hidden dangers to the safety of agricultural products, but also endanger human health. Rice is an important food crop in my country. The long-term flooded growth environment and the physiological characteristics of silicon and phosphorus are the main reasons for the sen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/70C12N1/21C02F3/34B09C1/10C12R1/19C02F101/20
CPCC07K14/37C12N15/70C02F3/34B09C1/10C02F2101/103C02F2101/20Y02P10/20
Inventor 苏世鸣李丽娟曾希柏张洋张楠
Owner INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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