CN116380857A - Cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology and application thereof - Google Patents
Cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology and application thereof Download PDFInfo
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a cadmium ion biosensor based on a fluorescence lifetime resonance energy transfer technology and application thereof. Specifically, the invention takes metallothionein type 2metallothionein as an identification probe, the type 2metallothionein can specifically identify and combine cadmium ions and generate conformational change, so that the fluorescence life of green fluorescent protein is reduced, and based on the fact, a biosensor for detecting cadmium ions based on the fluorescence life is established and is used for detecting the content of cadmium ions in plants. The method has the advantages of high sensitivity, good stability, high resolution, simple and convenient operation and the like, and has important significance for breeding low-cadmium plant varieties.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a cadmium ion biosensor based on a fluorescence lifetime resonance energy transfer technology and application thereof.
Background
FRET biosensors have been widely used to determine intracellular ion content. However, previously developed biosensors have many drawbacks, mainly in terms of: (1) The dynamic sensitization range of the conventional cyan and yellow fluorescent protein pairs is low, and the transient and weak biochemical reactions in cells are difficult to monitor. (2) The phototoxicity is high, and the experimental result can be greatly deviated when living cells are detected. (3) Measurement methods based on fluorescence intensity, such as techniques based on fluorescence intensity of a donor (acceptor photobleaching, FRET AB) or acceptor (sensitized emission, FRET SE), are affected not only by the concentration of the subject under investigation, but also by illumination intensity, photobleaching, and matrix absorption and shadow effects, etc., resulting in deviations in experimental results. Therefore, there is a need to develop highly sensitive sensors that can be used as more accurate measurement tools.
Fluorescence lifetime resonance energy transfer FLIM-FRET is Fluorescence Lifetime Imaging (FLIM) with
Resonance Energy Transfer (FRET) combined techniques, because fluorescence lifetime is independent of dye concentration, illumination intensity, and absorption and scattering of fluorescent signals in the sample, do not affect intermolecular interactions; on the other hand, fluorescence lifetime is significantly affected by the molecular microenvironment, which allows FLIM to sensitively measure molecular microenvironment parameters.
There is currently no cadmium ion biosensor based on FLIM-FRET.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a cadmium ion biosensor based on a fluorescence lifetime resonance energy transfer technology and application thereof. The present inventors have made extensive efforts to screen a fusion protein capable of recognizing and binding cadmium ions and undergoing conformational changes, resulting in a change in fluorescence lifetime, thereby completing the present invention.
It is therefore a first object of the present invention to provide a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology. The sensor comprises green fluorescent protein, red fluorescent protein, metallothionein 2 combined with cadmium ions, a connecting arm 1 and a connecting arm 2; the amino acid sequence of the metallothionein type 2metallothionein is shown in SEQ ID NO. 1; the amino acid sequence of the connecting arm 1 is shown as SEQ ID NO. 2; the amino acid sequence of the connecting arm 2 is shown as SEQ ID NO. 3.
Preferably, the green fluorescent protein is mNanGreen; the red fluorescent protein is mCherry.
Preferably, the connecting arm 1 connects the C-terminal of the green fluorescent protein with the N-terminal of the metallothionein type 2 metallothionein; the connecting arm 2 connects the C-terminal of metallothionein type 2metallothionein with the N-terminal of red fluorescent protein.
A second object of the present invention is to provide an application of the above-mentioned cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology in detecting cadmium ion content.
Preferably, the application of the cadmium ion biosensor based on the fluorescence lifetime resonance energy transfer technology in detecting the cadmium ion content in plants is provided.
The third object of the invention is to provide the application of the cadmium ion biosensor based on the fluorescence lifetime resonance energy transfer technology in the breeding of plant varieties with low cadmium content.
A fourth object of the present invention is to provide a method for detecting cadmium ions by using a cadmium ion biosensor based on a fluorescence lifetime resonance energy transfer technology, comprising the following steps: in the presence of cadmium ions, the metallothionein type 2metallothionein of the biosensor is combined with the cadmium ions to generate protein conformational change, so that the distance between the green fluorescent protein and the red fluorescent protein is close, and the concentration of the cadmium ions is measured by measuring the fluorescence lifetime change of the green fluorescent protein.
The fifth object of the present invention is to provide a method for preparing the above-mentioned cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, which is prepared by DNA recombination technology.
The sixth object of the present invention is to provide an application of metallothionein type 2, metallothionein, connecting arm 1 or connecting arm 2 in preparing a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, wherein the amino acid sequence of the metallothionein type 2 is shown as SEQ ID No. 1; the amino acid sequence of the connecting arm 1 is shown as SEQ ID NO. 2; the amino acid sequence of the connecting arm 2 is shown as SEQ ID NO. 3.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers for the first time that the fluorescence lifetime change of the FLIM-FRET biosensor fused with metallothionein type 2metallothionein is in direct proportion to the concentration of cadmium ions, and can be applied to the detection of the content of cadmium ions. Specifically, the invention takes metallothionein type 2metallothionein as an identification probe, the type 2metallothionein can specifically identify and combine cadmium ions and generate conformational change, so that the fluorescence life of green fluorescent protein is reduced, and based on the fact, a biosensor for detecting cadmium ions based on the fluorescence life is established and is used for detecting the content of cadmium ions in plants. The method has the advantages of high sensitivity, good stability, high resolution, simple and convenient operation and the like, and has important significance for breeding low-cadmium plant varieties.
Drawings
FIG. 1 is a schematic diagram showing structural changes, changes in FRET efficiency, and changes in fluorescence lifetime of green fluorescent protein in the presence or absence of cadmium ions for FLIM-FRET biosensors.
FIG. 2 is a predicted three-dimensional structure of a recombinant protein.
FIG. 3 shows fluorescence lifetime change after binding cadmium ions for a biosensor.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used in the present invention and the laboratory procedures described below is well known and commonly employed in the art.
The definition of the main terms used for describing the present invention is as follows:
the term "FLIM" (fluorescence lifetime) as used herein refers to the average length of time a fluorophore retains its excited state before the emitted fluorescent photons return to the ground state. Depending on the molecular composition of the fluorophore and the nano-environment.
The term "FLIM-FRET" (fluorescence lifetime resonance energy transfer) as used herein refers to non-radiative energy transfer between two fluorophores having different emission wavelengths, wherein the excitation energy of a fluorescence donor in an excited state is transferred to a fluorescence acceptor, whereby a change in the fluorescence lifetime of the fluorescence donor is observed.
The term "green fluorescent protein" as used herein refers to a fluorophore as a donor in the FLIM-FRET phenomenon, and the term "red fluorescent protein" refers to a fluorophore as an acceptor in the FLIM-FRET phenomenon.
The term "linker arm" as used herein refers to a polypeptide that is used to link between a metallothionein and a fluorescent protein.
In the present invention, the fusion protein constituting the FLIM-FRET biosensor includes metallothionein and a linker arm containing green fluorescent protein and red fluorescent protein, and binding cadmium ions. Wherein the green fluorescent protein and the red fluorescent protein can be fused to both ends of the metallothionein through a linker arm. In view of the extinction coefficient, quantum efficiency, light stability, and convenience of green and red fluorescent proteins used in FLIM-FRET biosensors, fluorescent proteins mneon green (green fluorescent protein) and mCherry (red fluorescent protein) are preferably used.
The method for detecting and measuring the concentration of cadmium ions according to the present invention adopts "FLIM-FRET", the optical property of fluorescence, and the principle thereof is shown in FIG. 1. In general, the fluorescence process is understood as the energy transition in a molecule from the electron ground state to its excited state. The absorbed energy is stored by fluorescent molecules for a short period of time before it can be emitted as fluorescence, the time the molecules are in an excited state being called fluorescence lifetime. FRET is another process of relaxation from an excited state. By FRET excitation, energy is transferred to the acceptor molecule in a non-radiative manner, and then the acceptor molecule is relaxed in a fluorescent manner. Since donor fluorescence and energy transfer are competing processes, the rate of consumption of the excited state increases in the presence of FRET. Thus, FRET shortens the donor fluorescence lifetime. FRET efficiency can be calculated from the ratio of the lifetime τqueue of the FRET donor to the lifetime τ of the non-FRET donor:
using the above FLIM-FRET principle, the present inventors fabricated a FLIM-FRET cadmium ion biosensor by fusing fluorescent proteins mneon green (green fluorescent protein) and mCherry (red fluorescent protein) as a fluorescent donor and a fluorescent acceptor, respectively, to both ends of metallothionein.
To find a protein domain that binds to cadmium ions and undergoes conformational changes, the inventors screened metallothionein from different species and fused the above metallothionein to a FLIM-FRET biosensor, with the addition of cadmium ions exogenously, assessed changes in fluorescence lifetime of the FLIM-FRET biosensor. Through extensive efforts, it was found that the fluorescence lifetime change of the FLIM-FRET biosensor fused with metallothionein type 2metallothionein was proportional to the cadmium ion concentration.
Specifically, in one embodiment of the invention, the concentration of cadmium ions is analyzed according to the change in fluorescence lifetime of the green fluorescent protein in the FLIM-FRET biosensor. 100. Mu. Mol of cadmium ions are added to plant protoplasts transformed with FLIM-FRET biosensors, and the fluorescence lifetime of the green fluorescent protein is reduced from 2.66ns before cadmium ions are not added to 2.12ns after cadmium ions are added. The FLIM-FRET cadmium ion biosensor can sensitively detect the concentration change of cadmium ions in cells.
Example 1: preparation of FLIM-FRET biosensor
1. Construction of expression vector for preparing fusion protein for FLIM-FRET biosensor
First, in order to provide a biosensor containing a protein represented by the following formula 1, an expression vector MT-FLIM was constructed in the following manner.
Wherein MT is metallothionein selected from type 2metallothionein (shown as SEQ ID NO. 1); l1 and L2 are linker peptides composed of polypeptides, L1 (shown as SEQ ID NO. 2) connects the C-terminal end of the green fluorescent protein mNannGreen with the N-terminal end of MT, and L2 (shown as SEQ ID NO. 3) connects the C-terminal end of MT with the N-terminal end of the red fluorescent protein mCherry.
The amino acid sequence of metallothionein type 2Metallothionein (MT) is shown in SEQ ID NO.1, and specifically comprises: MSSCCAGKCGCGDGCKCGSSCTGCKKYPDLGYSGEGTSGETMIIGFAPEKNYFEGSEMSV GAENDGCQCGANCTCNPCNCK.
The amino acid sequence of the connecting arm 1 (L1) is shown as SEQ ID NO.2, and specifically comprises the following steps: EAAARGTAPG.
The amino acid sequence of the connecting arm 2 (L2) is shown as SEQ ID NO.3, and specifically comprises the following steps: EAAARGGEAAAR.
According to the sequence, a total gene synthesis mode is adopted to construct a cadmium ion sensor-expression vector.
SEQ ID NO.4:5’-TCTGATTAACAGGGATCCgccaccatggtgagcaagggcg-3’
SEQ ID NO.5:5’-cttgcggctgcttccccgggtgcggtacctctagcagcagcttcCTTGTACAGCTCGTC-3’
SEQ ID NO.6:
5’-CACCCGGGGAAGCAGCCGCAAGAGGAGGCGAGGCTGCCGCAAGGatggtgagcaagggc-3’
SEQ ID NO.7:5’-ccaaatgtttgaacgatctgcagTTACTTGTACAGCTCGTC-3’
SEQ ID NO.8:
5’-GAAGCTGCTGCTAGAGGTACCGCACCCGGGGAAGCAGCCGCAAGAGGAGGCGAGG CTGCCGCAAGG-3’
First, the coding sequences of the fluorescent proteins mNanngreen (SEQ ID NO.4 and SEQ ID NO. 5) and mCherry (SEQ ID NO.6 and SEQ ID NO. 7) were amplified by PCR using the pDRF1-GW mNanngreen-T2A-mCherry plasmid (professor of Bas Teusink, amsterdam university; addgene goods No. 125698) as a template, and the coding sequences (SEQ ID NO.5 and SEQ ID NO. 6) containing a linker for binding the protein to the cleavage site were directly synthesized by Beijing family Biotech Co., ltd.) for embedding into the fluorescent protein amplification primer sequences (SEQ ID NO.5 and SEQ ID NO. 6). The amplified fragment was introduced into BamHI/PstI double digested pUC119-eGFP-HA vector (given to the university of Zhongshan Showcase professor) by multi-fragment homologous recombination. The ClonExpress MultiS One Step Cloning Kit kit (C113-01) for the multiple fragment homologous recombination experiments was purchased from Nannoovozan Biotechnology Co., ltd, and the experiments were carried out by referring to the instructions. Finally, pUC119-mNG-mCherry vector was constructed. Then, MT sequence SEQ ID NO.1 was synthesized through total gene ligation to pUC119-mNG-mCherry vector through smaI restriction enzyme site, thereby constructing vector MT-FLIM.
The predicted three-dimensional structure of the recombinant protein expressed by the recombinant plasmid MT-FLIM is shown in figure 2.
2. Preparation of FLIM-FRET biosensor
The constructed MT-FLIM vector was transformed into E.coli, which was then inoculated into LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, water) containing 50. Mu.g/mL ampicillin and cultured with shaking at 37℃for 12 hours.
After completion of the cultivation, the cultivated bacteria were recovered at a speed of 5000rpm using a centrifuge, and plasmid extraction was performed. After plasmid extraction, the plasmid was concentrated to a concentration of 2000. Mu.g/. Mu.L using a vacuum concentrator and stored at-20℃for further use.
Example 2: fluorescence lifetime analysis of FLIM-FRET biosensor
1. Transformed plant protoplasts
Protoplasts of arabidopsis thaliana were extracted using an eductive enzyme and a pectic enzyme. 10. Mu.g MT-FLIM plasmid was added to the centrifuge tube, 100. Mu.L of protoplast was added, and after mixing, 100. Mu.L of 40% PEG solution (40% PEG, 100mM CaCl) was added 2 250mM mannitol, water as solvent), and immediately gently mixed. After 10 minutes, the reaction was terminated by adding 2 times the total volume of the W5 solution, and the protoplasts were collected by centrifugation at 800rpm for 2 minutes, and then 500. Mu.L of the W5 solution was added and allowed to standThe shaker was gently shaken at 200rpm and incubated with light for 16 hours.
2. FLIM-FRET biosensor for measuring cadmium ion concentration
The cultured protoplast is divided into a plurality of parts, cadmium ions with different concentration gradients are respectively added, 20 mu M saponin is added, and the fluorescence lifetime change of the green fluorescent protein is immediately measured by using a fluorescence lifetime microscope. And calculating the cadmium ion concentration of the plant cells to be detected according to the change relation of the concentration and the fluorescence lifetime. In this example, the fluorescence lifetime of the green fluorescent protein after 5 minutes was 2.66,2.59,2.56,2.40,2.12ns, respectively, using 0,1, 10, 50, 100. Mu.M cadmium ion and 20. Mu.M saponin (FIG. 3).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (9)
1. The cadmium ion biosensor based on the fluorescence lifetime resonance energy transfer technology is characterized by comprising green fluorescent protein, red fluorescent protein, and metallothionein 2 and a connecting arm 1 and a connecting arm 2 combined with cadmium ions; the amino acid sequence of the metallothionein type 2metallothionein is shown in SEQ ID NO. 1; the amino acid sequence of the connecting arm 1 is shown as SEQ ID NO. 2; the amino acid sequence of the connecting arm 2 is shown as SEQ ID NO. 3.
2. The cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology of claim 1, wherein the green fluorescent protein is mneon green; the red fluorescent protein is mCherry.
3. The cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to claim 1, wherein the connecting arm 1 connects the C-terminal of green fluorescent protein with the N-terminal of metallothionein type 2 metallothionein; the connecting arm 2 connects the C-terminal of metallothionein type 2metallothionein with the N-terminal of red fluorescent protein.
4. Use of a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technique according to any one of claims 1-3 for detecting cadmium ion content.
5. The method according to claim 4, wherein the method is used for detecting the content of cadmium ions in plants.
6. Use of a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to any one of claims 1-3 in the breeding of cadmium low-content plant varieties.
7. A method for detecting cadmium ions by a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, which is characterized by comprising the following steps: in the presence of cadmium ions, the metallothionein type 2metallothionein of the biosensor of claim 1 undergoes a protein conformational change after binding with cadmium ions, and further the distance between the green fluorescent protein and the red fluorescent protein is approached, and the concentration of cadmium ions is measured by measuring the fluorescence lifetime change of the green fluorescent protein.
8. A method for preparing the cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to claim 1, which is characterized by being prepared by a DNA recombination technology.
9. The application of metallothionein type 2, connecting arm 1 or connecting arm 2 in the preparation of a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology is disclosed, wherein the amino acid sequence of the metallothionein type 2 is shown as SEQ ID NO. 1; the amino acid sequence of the connecting arm 1 is shown as SEQ ID NO. 2; the amino acid sequence of the connecting arm 2 is shown as SEQ ID NO. 3.
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