A heavy metal cadmium-specific binding protein gene BjHMA4R and encoding protein and application thereof

A protein-binding and heavy metal-binding technology, applied in the fields of molecular biology and genetic engineering technology research, can solve problems such as limiting the application of microbial methods, improve cadmium tolerance and enrichment ability, strong salt and alkali resistance, and broad market application foreground effect

Inactive Publication Date: 2019-01-15
YULIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there is currently no strain that has strong salt and alkali resistance, adsorption and flocculation capabilities, and strong cadmium tolerance. application in

Method used

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  • A heavy metal cadmium-specific binding protein gene BjHMA4R and encoding protein and application thereof
  • A heavy metal cadmium-specific binding protein gene BjHMA4R and encoding protein and application thereof
  • A heavy metal cadmium-specific binding protein gene BjHMA4R and encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: BjHMA4R gene fragment cloning and sequence analysis

[0030] The HMA4 gene BjHMA4 (NCBI accession number JQ673430) obtained in the early stage was used as a template to amplify the target gene BjHMA4R, and the sequence of the upstream primer used was BjHMA4R-F-KpnI: 5`-GGGGTACCATGAAGAAACCAAGTAGT-3`, the downstream The primer sequence of SEQ ID NO: 4 is BjHMA4R-R-XhoI:5'-CCGCTCGAGTCAAAGCAGTCCCCACATG-3'. The target gene BjHMA4R was amplified using Pyrobest DNA Polymerase PCR Enzyme (TakaraBio); PCR reaction conditions: 95°C for 1min; 94°C for 30s, 55°C for 30s, 72°C for 40s, 30 cycles; 72°C for 5min; the reaction system (10 μL) was 1μL cDNA, 1μL 10×Advantage 2 PCR Buffer, 0.5μL 50×dNTP Mix (10mM each), 0.2μL Forward Primer (10μM), 0.2μL Reverse Primer (10μM), 0.2μL PyrobestDNA Polymerase Mix, 6.9μL PCR-Grade water; after PCR, take 5 μL for agarose gel electrophoresis to detect the specificity and size of the amplified product.

[0031] The PCR product obtaine...

Embodiment 2

[0032] Example 2: Yeast overexpression vector construction, transformation and screening of transformants

[0033] The Escherichia coli plasmid pMD-18T-BjHMA4R inserted into BjHMA4R and the plasmid of yeast expression vector pYES2 were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted Integrity and concentration of the plasmid; use restriction endonucleases KpnI (TaKaRa) and XhoI (TaKaRa) to double-enzyme digest the plasmids pMD-18T-BjHMA4R and pYES2 respectively (100μL system), the reaction system and operation process are as follows: 20 μL pMD-18T-BjHMA4R and pYES2 plasmids, add 10 μL 10×K buffer, 4 μL KpnI, 6 μL XhoI, 60 μL ddH in sequence 2 O, after mixing, centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then perform gel recovery on the BjHMA4R fragment and the large f...

Embodiment 3

[0038] Example 3: Escherichia coli overexpression vector construction, transformation and screening of transformants

[0039] Use the recovered BjHMA4R fragment and pEASY-Blunt E1 expression vector (full-style gold biology) to connect when constructing the yeast expression vector above. After mixing, centrifuge for a short time, and then react overnight in a water bath at 16°C. Then, the ligation product was transformed into Escherichia coli DH5α by heat shock transformation method, and positive clones were screened with solid medium containing 50 mg / L kanamycin (Km). Select a single colony and shake the bacteria, and use the bacterial liquid as a template to perform PCR with the specific primers for amplifying BjHMA4R, and select the clone that successfully connects BjHMA4R and pYES2. If the detected strain is positive, add glycerol and store at -80°C for future use.

[0040] The pEASY-Blunt E1-BjHMA4R plasmid in the above Escherichia coli was extracted and purified, and the...

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Abstract

The invention provides a heavy metal cadmium-specific binding protein gene BjHMA4R, a protein encoded by the gene BjHMA4R and an application thereof. The nucleotide sequence of the gene BjHMA4R is shown in SEQ ID NO: 1 and encodes a protein having the amino acid sequence shown in SEQ ID NO: 2. The C-terminal (carboxyl-terminal) binding domain small fragment BjHMA4R of the heavy metal transporter BjHMA4 is found in an Indian mustard plant for super-enriching heavy metals, can specifically bind to Cd<2+> at low concentrations, and the experiment show that the gene can improve the cadmium tolerance and enrichment ability of microorganisms. The recombinant gene is recombined into the microorganisms with strong salt tolerance, alkali tolerance, adsorption and flocculation ability, so that the microorganisms not only have strong salt tolerance, alkali tolerance, adsorption and flocculation ability, but also have strong cadmium tolerance and enrichment ability, so as to promote the application of microbial process in the treatment of cadmium-containing wastewater.

Description

technical field [0001] The invention belongs to the research fields of molecular biology and genetic engineering technology, and relates to a heavy metal cadmium-specific binding protein gene BjHMA4R, its encoded protein and its application. Background technique [0002] Cadmium is a highly toxic heavy metal, and most of its compounds are toxic substances, so it is considered a dangerous environmental pollutant. A very small amount of cadmium can cause harm to the human body. It is enriched through the food chain, has the characteristics of stability, accumulation and is not easy to eliminate, and can cause chronic poisoning to the human body. It mainly accumulates in the liver, kidney and bones, causing kidney and other organs to develop disease, and even cause death. Cadmium is widely used in electroplating, automobile and aviation, pigments, paints, printing and other industries. Cadmium-containing wastewater discharged from factories is the main source of cadmium pollut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/14C12N15/70C12N15/80
CPCC12N9/14C12N15/70C12N15/80C12Y306/03003
Inventor 王建武相微微代惠萍段义忠梁爽亢福仁
Owner YULIN UNIV
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