A cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology and its application

Abstract

This invention discloses a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology and its applications. Specifically, the invention uses type 2 metallothionein as a recognition probe. Type 2 metallothionein can specifically recognize and bind to cadmium ions, undergoing conformational changes that cause a decrease in the fluorescence lifetime of green fluorescent protein. Based on this, a biosensor for detecting cadmium ions based on fluorescence lifetime is developed for measuring cadmium ion content in plants. This method has the advantages of high sensitivity, good stability, high resolution, and simple operation, and is of great significance for the breeding of low-cadmium plant varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology and applications thereof. Background Art

[0002] FRET biosensors have been widely used to measure intracellular ion content. However, previously developed biosensors have many drawbacks, mainly manifested in the following aspects: (1) The dynamic sensitivity range of the commonly used cyan and yellow fluorescent protein pairs is low, making it difficult to monitor transient and weak biochemical reactions in cells. (2) The phototoxicity is high, which can lead to large deviations in experimental results when detecting living cells. (3) Measurement methods based on fluorescence intensity, such as techniques based on donor (acceptor photobleaching, FRETAB) or acceptor (sensitized emission, FRET SE) fluorescence intensity, are affected not only by the concentration of the research object, but also by illumination intensity, photobleaching, matrix absorption, and shadow effects, leading to deviations in experimental results. Therefore, there is a need to develop highly sensitive sensors that can be used as more accurate measurement tools.

[0003] Fluorescence Lifetime Resonance Energy Transfer (FLIM-FRET) is a technique that combines fluorescence lifetime imaging (FLIM) with The technology combined with resonance energy transfer (FRET) has no effect on intermolecular interactions because the fluorescence lifetime is independent of the dye concentration, light intensity, and the absorption and scattering of the fluorescence signal in the sample; on the other hand, the fluorescence lifetime is significantly affected by the molecular microenvironment, which makes FLIM a sensitive measurement of molecular microenvironment parameters.

[0004] There is currently no FLIM-FRET-based cadmium ion biosensor. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention aims to provide a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer (FLRET) technology and its applications. After extensive research, the inventors identified a fusion protein that can recognize and bind to cadmium ions, undergoing conformational changes that result in changes in fluorescence lifetime, thus completing the present invention.

[0006] Therefore, a first object of the present invention is to provide a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology. The sensor comprises green fluorescent protein, red fluorescent protein, a cadmium ion-binding metallothionein type 2, and linkers 1 and 2. The amino acid sequence of the metallothionein type 2 is shown in SEQ ID NO. 1; the amino acid sequence of linker 1 is shown in SEQ ID NO. 2; and the amino acid sequence of linker 2 is shown in SEQ ID NO. 3.

[0007] Preferably, the green fluorescent protein is mNeonGreen; and the red fluorescent protein is mCherry.

[0008] Preferably, the linker arm 1 connects the C-terminus of green fluorescent protein and the N-terminus of metallothionein type 2 metallothionein; the linker arm 2 connects the C-terminus of metallothionein type 2 metallothionein and the N-terminus of red fluorescent protein.

[0009] The second object of the present invention is to provide the application of the above-mentioned cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology in detecting cadmium ion content.

[0010] Preferably, the application of the above-mentioned cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology in detecting the cadmium ion content in plants is provided.

[0011] The third object of the present invention is to provide the application of the above-mentioned cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology in the breeding of low-cadmium plant varieties.

[0012] A fourth object of the present invention is to provide a method for detecting cadmium ions using a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, comprising the following steps: in the presence of cadmium ions, the metallothionein type 2 metallothionein of the above-mentioned biosensor undergoes protein conformational changes after binding to the cadmium ions, thereby shortening the distance between the green fluorescent protein and the red fluorescent protein, and measuring the concentration of the cadmium ions by measuring the change in the fluorescence lifetime of the green fluorescent protein.

[0013] 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.

[0014] A sixth object of the present invention is to provide a use of metallothionein type 2 metallothionein, linker 1, or linker 2 in the preparation of a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, wherein the amino acid sequence of the metallothionein type 2 metallothionein is shown in SEQ ID NO.1; the amino acid sequence of the linker 1 is shown in SEQ ID NO.2; and the amino acid sequence of the linker 2 is shown in SEQ ID NO.3.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This study first discovered that the fluorescence lifetime of a FLIM-FRET biosensor fused to the metallothionein type 2 metallothionein changes in direct proportion to cadmium ion concentration, making it suitable for detecting cadmium ion content. Specifically, the present invention uses the metallothionein type 2 metallothionein as a recognition probe. Type 2 metallothionein can specifically recognize and bind to cadmium ions, undergoing conformational changes that cause a decrease in the fluorescence lifetime of green fluorescent protein. Based on this, a biosensor for detecting cadmium ions based on fluorescence lifetime was established for detecting cadmium ion content in plants. This method has the advantages of high sensitivity, good stability, high resolution, and simple operation, and is of great significance for the selection and breeding of low-cadmium plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram showing the structural changes of the FLIM-FRET biosensor, the changes in FRET efficiency, and the changes in the fluorescence lifetime of green fluorescent protein in the presence or absence of cadmium ions.

[0018] Figure 2 The predicted three-dimensional structure of the recombinant protein.

[0019] Figure 3 This is the change in fluorescence lifetime after the biosensor binds to cadmium ions. DETAILED DESCRIPTION

[0020] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as conventionally understood by those of ordinary skill in the art to which the present invention pertains. Generally, the specific nomenclature used in the present invention and the experimental methods described below are well known and commonly used in the art.

[0022] The definitions of the main terms used to describe the present invention are as follows:

[0023] The term "FLIM" (fluorescence lifetime) as used herein refers to the average length of time a fluorophore remains in its excited state before emitting a fluorescent photon and returning to the ground state. This depends on the molecular composition and nanoenvironment of the fluorophore.

[0024] The term "FLIM-FRET" (Fluorescence Lifetime Resonance Energy Transfer) used in the present invention refers to the non-radiative energy transfer between two fluorophores with different emission wavelengths, wherein the excitation energy of the excited fluorescent donor is transferred to the fluorescent acceptor, thereby observing the change in the fluorescence lifetime of the fluorescent donor.

[0025] As used herein, the term "green fluorescent protein" refers to a fluorophore that acts as a donor in the FLIM-FRET phenomenon, and the term "red fluorescent protein" refers to a fluorophore that acts as an acceptor in the FLIM-FRET phenomenon.

[0026] The term "linker" used in the present invention refers to a polypeptide used to connect metallothionein and fluorescent protein.

[0027] In the present invention, the fusion protein constituting the FLIM-FRET biosensor includes a fusion protein containing green fluorescent protein and red fluorescent protein, a metallothionein that binds cadmium ions, and a linker. The green fluorescent protein and red fluorescent protein can be fused to both ends of the metallothionein via the linker. Given the extinction coefficient, quantum efficiency, photostability, and convenience of the green and red fluorescent proteins used in FLIM-FRET biosensors, the fluorescent proteins mNeonGreen (green fluorescent protein) and mCherry (red fluorescent protein) are preferred.

[0028] 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 its principle is as follows Figure 1 As shown. Generally, the fluorescence process is understood as an energy transition from the electronic ground state to its excited state in a molecule. The absorbed energy is stored by the fluorescent molecule for a short period of time before it can be emitted as fluorescence, and the time the molecule is in the excited state is called the fluorescence lifetime. FRET is another process of relaxation from the excited state. Through FRET excitation, energy is transferred to the acceptor molecule in a non-radiative manner, and the acceptor molecule then relaxes in a fluorescent manner. Since donor fluorescence and energy transfer are competing processes, the consumption rate of the excited state increases in the presence of FRET. Therefore, FRET shortens the donor fluorescence lifetime. The FRET efficiency can be calculated as the ratio of the lifetime τquench of the donor undergoing FRET to the lifetime τ when FRET does not occur:

[0029]

[0030] Using the above FLIM-FRET principle, the present inventors fabricated a FLIM-FRET cadmium ion biosensor by fusing the fluorescent proteins mNeonGreen (green fluorescent protein) and mCherry (red fluorescent protein), which serve as a fluorescence donor and a fluorescence acceptor, respectively, to the two ends of metallothionein.

[0031] To identify protein domains capable of binding to cadmium ions and undergoing conformational changes, the inventors screened metallothioneins from various species and fused these metallothioneins to FLIM-FRET biosensors. Upon exogenous addition of cadmium ions, the researchers evaluated changes in the fluorescence lifetime of the FLIM-FRET biosensor. After extensive research, they discovered that the fluorescence lifetime of the FLIM-FRET biosensor fused to type 2 metallothionein changes in direct proportion to the cadmium ion concentration.

[0032] Specifically, in one embodiment of the present invention, cadmium ion concentration is analyzed based on changes in the fluorescence lifetime of green fluorescent protein in a FLIM-FRET biosensor. When 100 μmol of cadmium ions were added to plant protoplasts transformed with the FLIM-FRET biosensor, the fluorescence lifetime of green fluorescent protein decreased from 2.66 nanoseconds before cadmium addition to 2.12 nanoseconds after addition. The FLIM-FRET cadmium ion biosensor can sensitively detect changes in intracellular cadmium ion concentration.

[0033] Example 1: Preparation of FLIM-FRET biosensor

[0034] 1. Construction of expression vector for preparing fusion protein for FLIM-FRET biosensor

[0035] First, in order to provide a biosensor containing a protein represented by the following formula 1, an expression vector MT-FLIM was constructed as follows.

[0036]

[0037] Wherein, MT is a metallothionein selected from type 2 metallothionein (as shown in SEQ ID NO.1); L1 and L2 are linker peptides composed of polypeptides, L1 (as shown in SEQ ID NO.2) connects the C-terminus of the green fluorescent protein mNeonGreen and the N-terminus of MT, and L2 (as shown in SEQ ID NO.3) connects the C-terminus of MT and the N-terminus of the red fluorescent protein mCherry.

[0038] The amino acid sequence of metallothionein type 2 (MT) is shown in SEQ ID NO. 1, specifically: MSSCCAGKCGCGDGCKCGSSCTGCKKYPDLGYSGEGTSGETMIIGFAPEKNYFEGSEMSVGAENDGCQCGANCTCNPCNCK.

[0039] The amino acid sequence of linker arm 1 (L1) is shown in SEQ ID NO. 2, specifically: EAAARGTAPG.

[0040] The amino acid sequence of linker arm 2 (L2) is shown in SEQ ID NO. 3, specifically: EAAARGGEAAAR.

[0041] According to the sequence, a cadmium ion sensor-expression vector was constructed by whole gene synthesis.

[0042] SEQ ID NO.4:5'-TCTGATTAACAGGGATCCgccaccatggtgagcaagggcg-3'

[0043] SEQ ID NO.5:5'-cttgcggctgcttccccgggtgcggtacctctagcagcagcttcCTTGTACAGCTCGTC-3'

[0044] SEQ ID NO.6:

[0045] 5'-CACCCGGGGAAGCAGCCGCAAGAGGAGGCGAGGCTGCCGCAAGGatggtgagcaagggc-3'

[0046] SEQ ID NO.7:5'-ccaaatgtttgaacgatctgcagTTACTTGTACAGCTCGTC-3'

[0047] SEQ ID NO.8:

[0048] 5'-GAAGCTGCTGCTAGAGGTACCGCACCCGGGGAAGCAGCCGCAAGAGGAGGCGAGGCTGCCGCAAGG-3'

[0049] First, the coding sequences for the fluorescent proteins mNeongreen (SEQ ID NOs. 4 and 5) and mCherry (SEQ ID NOs. 6 and 7) were amplified by PCR using the pDRF1-GW mNeongreen-T2A-mCherry plasmid (a gift from Professor Bas Teusink at the University of Amsterdam, the Netherlands; Addgene catalog number 125698) as a template. A linker coding sequence (SEQ ID NO. 8) for binding protein ligation into restriction sites was synthesized directly by Beijing Qingke Biotechnology Co., Ltd. and incorporated into the fluorescent protein amplification primers (SEQ ID NOs. 5 and 6). The amplified fragments were then introduced into the BamHI / PstI double-digested pUC119-eGFP-HA vector (a gift from Professor Xiao Shi at Sun Yat-sen University) by multi-fragment homologous recombination. The ClonExpress MultiS One-Step Cloning Kit (C113-01) was purchased from Nanjing Novozymes Biotech Co., Ltd. and performed according to the manufacturer's instructions. Finally, the pUC119-mNG-mCherry vector was constructed. The MT sequence SEQ ID NO. 1 was then fully synthesized and ligated into the pUC119-mNG-mCherry vector via the smaI restriction enzyme site to construct the MT-FLIM vector.

[0050] The predicted three-dimensional structure of the recombinant protein expressed by the recombinant plasmid MT-FLIM is shown in Figure 2 .

[0051] 2. Preparation of FLIM-FRET Biosensor

[0052] The constructed MT-FLIM vector was transformed into Escherichia coli, and then Escherichia coli was inoculated into LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, solvent: water) containing 50 μg / mL ampicillin and cultured with shaking at 37°C for 12 hours.

[0053] After the culture is complete, the cultured bacteria are recovered using a centrifuge at 5000 rpm and plasmids are extracted. After plasmid extraction, the plasmid is concentrated to a concentration of 2000 μg / μL using a vacuum concentrator and stored at -20°C until use.

[0054] Example 2: Fluorescence Lifetime Analysis of FLIM-FRET Biosensor

[0055] 1. Transformation of plant protoplasts

[0056] Arabidopsis protoplasts were extracted using macerate and pectinase. 10 μg of MT-FLIM plasmid was added to a centrifuge tube, followed by 100 μL of protoplasts. After mixing, 100 μL of 40% PEG solution (40% PEG, 100 mM CaCl2, 250 mM mannitol, in water) was added and gently mixed. After 10 minutes, the reaction was terminated by adding twice the total volume of W5 solution. The protoplasts were collected by low-speed centrifugation at 800 rpm for 2 minutes, and 500 μL of W5 solution was added. The tube was gently shaken at 200 rpm and incubated under light for 16 hours.

[0057] 2. FLIM-FRET biosensor for measuring cadmium ion concentration

[0058] The cultured protoplasts were divided into several portions, and different concentration gradients of cadmium ions were added to each portion. Then, 20 μM saponin was added. The fluorescence lifetime change of green fluorescent protein was immediately measured using a fluorescence lifetime microscope. The cadmium ion concentration of the plant cells to be tested can be calculated based on the relationship between the change in concentration and fluorescence lifetime. In this example, 0, 1, 10, 50, and 100 μM cadmium ions and 20 μM saponin were used. After 5 minutes, the fluorescence lifetime of green fluorescent protein changed from 2.66, 2.59, 2.56, 2.40, and 2.12 ns, respectively. Figure 3 ).

[0059] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, characterized in that: The sensor includes green fluorescent protein, red fluorescent protein, metallothionein type 2 that binds cadmium ions, and connecting arm 1 and connecting arm 2; the amino acid sequence of the metallothionein type 2 is shown in SEQ ID NO.1; the amino acid sequence of the connecting arm 1 is shown in SEQ ID NO.2; and the amino acid sequence of the connecting arm 2 is shown in SEQ ID NO.

3.

2. The cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to claim 1, characterized in that: The green fluorescent protein is mNeonGreen; the red fluorescent protein is mCherry.

3. The cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to claim 1, characterized in that: The connecting arm 1 connects the C-terminus of green fluorescent protein and the N-terminus of metallothionein type 2 metallothionein; the connecting arm 2 connects the C-terminus of metallothionein type 2 metallothionein and the N-terminus of red fluorescent protein.

4. Use of the cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to any one of claims 1 to 3 in detecting cadmium ion content.

5. The use according to claim 4, characterized in that It is used in detecting the cadmium ion content in plants.

6. Use of the cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology according to any one of claims 1 to 3 in the breeding of plant varieties with low cadmium content.

7. A method for detecting cadmium ions using a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, characterized in that: The following steps are involved: In the presence of cadmium ions, the metallothionein type 2 metallothionein of the biosensor according to claim 1 undergoes a protein conformational change after binding to the cadmium ions, thereby shortening the distance between the green fluorescent protein and the red fluorescent protein. The concentration of the cadmium ions is measured by measuring the change in the fluorescence lifetime 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, characterized in that: Prepared by DNA recombinant technology.

9. Use of a metallothionein type 2 metallothionein, linker 1, or linker 2 in the preparation of a cadmium ion biosensor based on fluorescence lifetime resonance energy transfer technology, wherein the amino acid sequence of the metallothionein type 2 metallothionein is shown in SEQ ID NO. 1; the amino acid sequence of linker 1 is shown in SEQ ID NO. 2; and the amino acid sequence of linker 2 is shown in SEQ ID NO. 3.

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