Visualizing binary plant vector systems and uses thereof

Abstract

This invention discloses a visual plant binary vector system. The visual plant binary vector system AioFFP 2.0 includes a fluorescently labeled protein localization binary vector, a bimolecular fluorescently complementary binary vector, and an organelle marker binary vector based on the mChe signaling channel. All of these binary vectors use the pYL1300-UaUf vector as a backbone, seamlessly linked with a synthetically produced T-DNA fragment to form a circular vector. The pYL1300-UaUf vector backbone is modified with Agrobacterium-derived original replicons and selection markers to serve as a direct receptor encoding a fusion fluorescent protein expression cassette (T-DNA fragment). Experimental results of this invention show that this system can efficiently and flexibly adapt to different target genes for visual detection of protein localization and protein-protein interactions in plant cells, providing a novel vector tool for functional genomics analysis and possessing good commercial prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a visual plant binary vector system and its applications. Background Technology

[0002] Fluorescently labeled protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are currently the mainstream methods for analyzing subcellular protein localization and protein-protein interactions in living plant cells. The previous paper, "All-in-one: A Robust Fluorescent Fusion Protein Vector Toolbox for Protein Localization and BiFC Analyses in Plants," disclosed a modular and efficient integrated fluorescent fusion protein vector toolbox (AioFFP), which includes a complete set of fluorescently labeled organelle tracking vectors, fluorescent localization plasmids, and bimolecular fluorescence complementation plasmids. AioFFP utilizes Gibson assembly technology (GA) to introduce multiple unique nucleotide sequences (universally specific nucleotide sequences, UNS). It can clone a single target gene into various fluorescent fusion vectors (pUC19 or pYL322d1 backbone vectors), or insert two or more target genes into the same fluorescent fusion vector to achieve subcellular localization of the target gene and verification of protein-protein interactions. Simultaneously, it can integrate organelle-labeled genes into fluorescent fusion vectors to verify that the target protein's fluorescence targets specific subcellular compartments.

[0003] Furthermore, the article only mentions inserting a single or multiple target genes into the same fluorescent fusion transient expression vector, without mentioning inserting a single target gene into the same fluorescent fusion binary expression vector, and it does not provide the specific gene sequence of the fluorescent fusion binary expression vector. Therefore, compared to inserting multiple target genes into the same fluorescent fusion binary expression vector, the construction method for a single target gene is simpler. Successfully integrating multiple vectors into the plant genome is not only technically challenging, but also requires further validation with an empty vector control before verifying the function of the target gene. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an optimized visual plant binary carrier system.

[0005] The second objective of this invention is to provide the above-mentioned visual plant binary carrier system for easy direct application in detecting the subcellular localization and interactions of target proteins in living plants.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a visual plant binary carrier system, which includes a fluorescently labeled protein localization binary carrier, a bimolecular fluorescence complementary binary carrier, and an organelle marker binary carrier based on the mChe signaling channel. The fluorescently labeled protein positioning binary vector uses the pYL1300-UaUf vector as a backbone and is seamlessly linked with the artificially synthesized T-DNA fragment 1 to form a circular vector; the artificially synthesized T-DNA fragment is Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-Uc-restriction site 2-Ud-fluorescent protein-terminator-Ue / Uf-restriction site 3-Uf / -Uf-Ub / -Ub; Alternatively, the artificially synthesized T-DNA fragment 1 may be Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-fluorescent protein-Uc-restriction site 2-Ud-terminator-Ue / Uf-restriction site 3-Uf / -Uf-Ub / -Ub; The bimolecular fluorescent complementary binary vector consists of a pair of circular vectors with pYL1300-UaUf vectors as the backbone, seamlessly linked with artificially synthesized T-DNA fragment 2 to form a circular vector; the artificially synthesized T-DNA fragment 2 is Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-Uc-restriction site 2-Ud-truncated fluorescent protein-terminator-Ue / Uf-restriction site 3-Uf-Ub / -Ub; the truncated fluorescent proteins are Vn and Vc, constituting a set of bimolecular fluorescent complementary vectors; Alternatively, the artificially synthesized T-DNA fragment 2 may be Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-truncated fluorescent protein-Uc-restriction site 2-Ud-terminator-Ue / Uf-restriction site 3-Uf-Ub / -Ub; the truncated fluorescent proteins are Vn and Vc, constituting a set of bimolecular fluorescent complementary vectors; The organelle marker binary vector based on the mChe signaling channel is composed of a circular vector with a pYL1300-UaUf vector as the backbone and a synthetic T-DNA fragment 3 seamlessly linked together; the synthetic T-DNA fragment 3 is Ua-constitutive CaMV35S promoter-Uc-restriction site-Ud-fluorescent protein mCherry-terminator-Ub. Alternatively, the artificially synthesized T-DNA fragment 3 may be Ua-constitutive CaMV35S promoter-fluorescent protein mCherry-Uc-restriction site-Ud-terminator-Ub.

[0007] Existing technologies disclose a modular, integrated fluorescent fusion protein (AioFFP 1.0) transient expression vector toolkit, which can facilitate research on protein localization and bimolecular fluorescence complementarity (BiFC) experiments in plants. However, the AioFFP 1.0 system is suitable for protoplast transfection, but its effectiveness is limited in Agrobacterium-mediated transient expression or stable genetic transformation studies because the system vector does not carry the Agrobacterium replication origin. This invention introduces a plant binary vector, incorporating Agrobacterium tumefaciens replication origin and adaptor selection markers, enabling the direct cloning of target genes into the binary vector pYL1300-UaUf. This new system is named AioFFP 2.0. AioFFP 2.0 not only complements the former but also represents a comprehensive upgrade addressing the operational limitations of the original system. By circumventing the complex cloning process of the original system, it promotes large-scale, high-throughput functional genomics research on protein (co)localization and BiFC analysis, with significant application value, especially for Agrobacterium tumefaciens-mediated transient and stable transformation in plants.

[0008] Furthermore, the visualized plant binary carrier system consists of a fluorescently labeled protein localization binary carrier and an organelle marker binary carrier based on the mChe signaling channel, used for subcellular localization studies; or it consists of a bimolecular fluorescent complementary binary carrier and an organelle marker binary carrier based on the mChe signaling channel, used for protein interaction and its role in the cell.

[0009] Furthermore, the fluorescent protein is selected from one of StayGold, eGFP, mCherry, Venus, and eCFP. StayGold, derived from the jellyfish (Cytaeis uchidae), encodes a fluorescent protein that exhibits higher photostability and faster maturation compared to eGFP and its derivatives.

[0010] Furthermore, the restriction enzyme site is selected from... Asc I. Sma Ⅰ or Sbf I.

[0011] Preferably, the nucleotide sequence of the artificially synthesized T-DNA fragment 1 in the fluorescently labeled protein positioning binary vector can be selected from any one of SEQ ID NO.1 to 8.

[0012] Furthermore, the nucleotide sequence of the artificially synthesized T-DNA fragment 2 in the bimolecular fluorescent complementary binary vector can be selected from any one of SEQ ID NO. 9 to 12.

[0013] Furthermore, the enzyme cleavage sites in the organelle marker binary vector of the mChe signaling channel can be inserted into gene fragments targeting the cell nucleus, endoplasmic reticulum, plasmids, vacuoles, cytoplasm, mitochondria, plasma membrane, Golgi apparatus, peroxisomes, plasmodesmata, or cytoskeleton.

[0014] Preferably, the nucleotide sequence of the artificially synthesized T-DNA fragment 3 in the organelle marker binary vector of the mChe signaling channel can be selected from any one of SEQ ID NO.13 to 24.

[0015] This invention utilizes chimeric primers (containing Uc and Ud sequences) to amplify the open reading frame (ORF) of the target gene. Through the Uc / Ud site, the target gene is cloned into the pYL1300-UaUf binary expression vector via single-tube Gibson assembly to construct an N-terminal or C-terminal fluorescent fusion protein. Furthermore, multiple fluorescent fusion protein expression units can be integrated into the pYL1300-UaUf binary expression vector using Ua / Ub, Ue / Uf, etc., to improve co-expression efficiency. Results show that the protein localization and bimolecular fluorescence complementary binary vector provided by this invention can be used for protein subcellular localization and protein-protein interaction studies. Therefore, this invention successfully constructs a modular fluorescent fusion protein vector (AioFFP 2.0) suitable for plant protein colocalization and protein interaction analysis.

[0016] In terms of construction efficiency, this vector relies on standardized and modular assembly logic, which greatly shortens the vector construction cycle and significantly improves the success rate of vector construction. At the same time, this vector is highly versatile and can be flexibly adapted to different target genes for basic scientific research gene function verification, which greatly reduces the difficulty of technical operation and labor costs, and has broad prospects for promotion and excellent commercial transformation value.

[0017] The present invention also provides the application of the above-mentioned visual plant binary carrier system in detecting the subcellular localization and interaction of target proteins in living plants.

[0018] Furthermore, when tobacco mesophyll cells are co-transformed with a binary vector containing a bimolecular fluorescent complementary vector fused with the target protein encoding gene and a binary vector containing an organelle marker of the mChe signaling channel, two non-fluorescent expression cassettes are observed to be brought closer together and produce fluorescence using a laser confocal / fluorescence microscope. At the same time, the organelle marker of the mChe signaling channel produces red fluorescence that specifically indicates cell localization, thus verifying the interacting genes of the target protein and their position in the cell.

[0019] Furthermore, when tobacco mesophyll cells are co-transformed with a fluorescent marker protein localization binary vector that integrates the target protein encoding gene and an organelle marker binary vector that integrates the mChe signaling channel, the fluorescence of the target protein can be observed by laser confocal / fluorescence microscopy. At the same time, the organelle marker of the mChe signaling channel produces red fluorescence that can specifically indicate cell localization, thus verifying the localization of the target protein in the cell.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a visual plant binary vector system. The visual plant binary vector system AioFFP2.0 includes a fluorescently labeled protein localization binary vector, a bimolecular fluorescently complementary binary vector, and an organelle marker binary vector based on the mChe signaling channel. All of these binary vectors use the pYL1300-UaUf vector as a backbone, seamlessly linked with a synthetically produced T-DNA fragment to form a circular vector. The pYL1300-UaUf vector backbone is modified with Agrobacterium-derived original replicons and selection markers to serve as a direct receptor encoding a fusion fluorescent protein expression cassette (T-DNA fragment). Experimental results of this invention show that this system can efficiently and flexibly adapt to different target genes for visual detection of protein localization and protein-protein interactions in plant cells, providing a novel vector tool for functional genomics analysis and possessing good commercial prospects. Attached Figure Description

[0021] Figure 1 A binary carrier map designed and constructed for the AioFFP2.0 system. Among them, Figure 1 AD stands for StayGold fluorescent protein carrier; EF stands for mChe fluorescent protein carrier; GH stands for eCFP fluorescent protein carrier; and IL stands for BiFC fluorescent protein carrier.

[0022] Figure 2 This is a plasmid map of 12 mChe signaling channel organelles (markers) in the AioFFP2.0 system. Among them, Figure 2 All ALs in the images are organelle carriers, all emit red light, and all belong to the mChe laser signaling pathway.

[0023] Figure 3 This is a protein colocalization and bimolecular fluorescence complementation diagram of the AioFFP2.0 binary vector system. Figure 3 In the diagram, A represents a schematic of the vector combination; B represents organelle markers; C represents BiFC verification; and D represents subcellular co-localization verification.

[0024] Figure 4 The fluorescence intensity of StayGold and eGFP proteins in the FTPL vector binary vector is quantified.

[0025] Figures 5-28 Nucleotide sequences of artificially synthesized T-DNA fragments in binary carriers for localizing fluorescent labeled proteins, bimolecular fluorescent complementary binary carriers, and organelle marker binary carriers for the mChe signaling channel. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] Example 1: Design and construction of a binary carrier for the AioFFP2.0 system The AioFFP 2.0 system consists of a series of modular binary vectors containing multiple expression cassettes. These cassettes are based on a modified pYL1300-UaUf vector backbone and are driven by a constitutive CaMV35S promoter to express different fluorescent protein genes. The AioFFP 2.0 system comprises three parts: a fluorescently labeled protein localization (FTPL) binary vector, a bimolecular fluorescence complementarity (BiFC) binary vector, and an organelle marker (OM) binary vector based on the mChe signaling channel.

[0029] 1. Construction of FTPL and BiFC binary expression vectors Vectors were constructed via gene synthesis, Q-PCR (Chen et al., 2013), and GA-mediated multi-type plasmid modification (Zhu et al., 2014), and validated by Sanger sequencing. The cauliflower mosaic virus 35S promoter (P35s or CaMV35S), cauliflower mosaic virus 35S terminator (T35s), and cauliflower base synthase terminator (Tnos) were used as regulatory elements for gene expression. A pair of universally specific nucleotide sequences (UNSs), namely Uc and Ud, were added to the 5' or 3' end of the fluorescent protein gene to maintain the efficiency and high throughput of Gibson cloning. Multiple pairs of UNSs (Ua and Ub; Ua and Ue; Ue and Uf; and Uf and Ub) were retained, which can be used to construct multiple localization expression cassettes or multiple fluorescent protein expression cassettes within the T-DNA region for transient, independent, and co-expression to detect the stability, localization, and photostability of the target protein. StayGold, eGFP, mChe, eCFP, Vn, and Vc are used as fluorescent tags. Detailed vector construction procedures can be found in the reference "All-in-one: A Robust Fluorescent Fusion Protein Vector Toolbox for Protein Localization and BiFC Analyses in Plants".

[0030] The binary carrier map designed and constructed using the AioFFP2.0 system is as follows: Figure 1 As shown, for the FTPL binary vector, four fluorescent proteins (StayGold, eGFP, eCFP, and mChe) provide multiple options for plant co-localization studies, all of which can be easily detected by Agrobacterium-mediated co-transfection technology. The nucleotide sequences of the insert fragments containing fluorescent proteins in this FTPL binary vector are referenced in SEQ ID NO. 1–8. Figures 5-12 For the BiFC binary vector, Vn and Vc provide options for studying the interaction and action site of the target gene in plant cells. The nucleotide sequence of the fluorescent protein insert fragment contained in the BiFC binary vector is referenced in SEQ ID NO. 9–12. Figures 13-16 ).

[0031] 2. Construction of binary vectors for organelle markers To complement the aforementioned FTPL and BiFC binary vectors, an organelle marker (OM) binary vector based on the mChe signaling pathway was also constructed. The mChe-containing expression cassette was subcloned into the pYL1300-UaUf vector backbone, thereby targeting each mChe fluorescence to different subcellular compartments.

[0032] To construct a series of organelle marker binary vectors, DNA sequences encoding signal peptides or proteins located in each subcellular compartment were obtained by synthesis or amplification from plant cDNA and cloned into the 5' or 3' end of mChe. For NLS-mChe (nuclear localization signal peptide), ER-mChe (endoplasmic reticulum localization signal peptide KTl1 1-25aa), Pt-mChe (PsrbcS truncated sequence PsrbcS 1-57aa), Tp-mChe (AtCBL2 truncated sequence AtCBL2 1-28aa), CF-mChe (AtTZF1 full-length coding sequence), Mito-mChe (OsCOX11 full-length coding sequence), and Pd-mChe (AtPDCB1 full-length coding sequence), the signal peptides are fused to the 5' end of mChe, respectively. For mChe-PM (full-length OsRac3 sequence), mChe-Golgi (full-length AtRER1B sequence), mChe-Px (peroxisome localization signal peptide ihhprelsrl), and mCh-Cs1 (cytoskeleton signal peptide AtFIM1 364-688), the signal peptides are fused to the 5' end of mChe, respectively. aa) and mCh-Cs2 (cytoskeleton signal peptide AtADF1), the signal peptides are fused to the 3' end of mChe, and the constructed vector sequences are referenced in SEQ ID NO.13~24 ( Figures 17-28 ).

[0033] The AioFFP 2.0 system contains various marker (OM) binary vector maps, such as... Figure 2 As shown, these binary carriers target different organelles and accumulate in the cytoplasm and nucleus (mChe), respectively: nucleus (NLS-mChe), endoplasmic reticulum (ER-mChe), plastids (Pt-mChe), vacuoles (Tp-mChe), cytoplasm (CF-mChe), mitochondria (Mito-mChe), plasma membrane (PM-mChe), Golgi apparatus (Golgi-mChe), peroxisomes (Px-mChe), plasmodesmata (Pd-mChe), or cytoskeleton (CS1-mChe and CS2-mChe).

[0034] In summary, a set of carrier system tools based on the same carrier skeleton is provided. The modules of this system are fully functional, mutually compatible, and support random combination and use to form a complete toolbox.

[0035] Example 2: Verification of the localization of various marker (OM) carriers in the AioFFP 2.0 system I. Experimental Methods 1. Agrobacterium-mediated infiltration of Fumigia benthamiana Seeds of *Nicotiana benthamiana* germinated and grew in peat soil at 25°C under a 14-hour light / 10-hour dark cycle. Various marker (OM) binary plasmids from the AioFFP 2.0 system constructed in Example 1 were electroporated into *Agrobacterium tumefaciens* strain GV3101, and then transiently transformed into leaves of 4-week-old *Nicotiana benthamiana* plants via syringe-mediated infiltration (Blatt and Grefen, 2014). The inoculated leaves were observed after incubation for approximately 24-48 hours.

[0036] 2. Confocal laser scanning microscope Cells expressing fluorescently labeled fusion proteins were observed using a confocal laser scanning microscope (Carl Zeiss LSCM 710, Oberkochen, Germany). Excitation / emission wavelengths: eCFP 405 nm / 445-485 nm, eGFP 488 nm / 490-560 nm, Venus 514 nm / 520-570 nm, mChe 543 nm / 580-660 nm. Images were acquired and processed using Zen software (Carl Zeiss).

[0037] II. Experimental Results The results are as follows Figure 3 As shown in Figure B, red fluorescence was detected in all types of marker (OM) binary vectors in the plant cells and organelles at the expected locations. This indicates that the AioFFP 2.0 system can achieve good expression in plant cells with relatively complete location coverage in terms of transient expression.

[0038] Example 3: Validation of FTPL and BiFC vectors in the AioFFP 2.0 system I. Experimental Methods 1. Plasmid construction for testing BiFC vectors To evaluate the practicality of AioFFP 2.0 for dual fluorescence complementation (BiFC), two known nuclear localization interacting proteins from rice (Oryza sativa), the 18 kDa Sin3A-associated protein (OsSAP18) and histone deacetylase 701 (OsHDA710), were used. BiFC binary vectors pYL1300 / OsSAP18-Vn and pYL1300 / OsHDA710-Vc were constructed to generate OsSAP18 fused to the N-terminal half of the Venus protein and OsHDA710 fused to the C-terminal half of the Venus protein, respectively. These two plasmids were then co-transfected with the nuclear localization marker vector pYL1300 / NLS-mChe from the organelle marker binary vector.

[0039] 2. Plasmid construction for OsCBSX subcellular localization analysis To evaluate AioFFP 2.0 for subcellular localization (FTPL), coding sequences of members of the OsCBSX gene family were amplified from rice cDNA, flanked by Uc and Ud adapters, and cloned into Sma I-digested pYL1300 / N-eGFP and pYL1300 / N-StayGold via Uc and Ud sequences. These were then co-transfected with the nuclear localization marker vector pYL1300 / NLS-mChe in the organelle marker binary vector.

[0040] II. Experimental Results The detection results of BiFC vector are as follows: Figure 3 As shown in Figure C, a green signal was detected in the cell nucleus via Venus signaling, indicating that the Venus protein was fused and reconstituted in this compartment. This demonstrates that the AioFFP 2.0 system provides a simple and rapid protocol for the detection of bimolecular fluorescence complementarity (BiFC).

[0041] FTPL vector detection results are as follows Figure 3 D and Figure 4 As shown, when OsCBSX4 protein is fused with eGFP or StayGold, it is localized in the nucleus, cytoplasm, and mitochondria. However, the fluorescence signal is significantly enhanced when fused with StayGold, further validating the superior fluorescence properties of StayGold protein compared to eGFP protein. These results indicate that the AioFFP 2.0 FTPL vector is more efficient and convenient in the subcellular (co)localization module, accurately localizing target proteins to their corresponding subcellular compartments with better results.

[0042] Therefore, the AioFFP 2.0 system provided by this invention is not only suitable for transient detection, but also eliminates the need for intermediate cloning vectors. The same target gene can be more easily subcloned into the AioFFP 2.0 binary vector, which can then be directly used for Agrobacterium transformation. In practical use, this vector exhibits significant advantages such as ease of operation, high construction efficiency, strong stability, and wide adaptability, effectively overcoming the industry pain points of cumbersome, lengthy, high-failure-rate, and costly traditional vector construction processes; and providing important technical support for large-scale genetic transformation experiments. Furthermore, StayGold has been found to be a more ideal labeling tool in plant cell imaging research. The AioFFP 2.0 system also supports protein subcellular localization and protein-protein interaction studies, providing an efficient, rapid, simple, and reliable technical platform for in-depth exploration of protein function.

Claims

1. A visual plant binary carrier system, characterized in that, The visualized plant binary carrier system includes a fluorescently labeled protein localization binary carrier, a bimolecular fluorescent complementary binary carrier, and an organelle marker binary carrier based on the mChe signaling channel. The fluorescently labeled protein positioning binary vector uses the pYL1300-UaUf vector as a backbone and is seamlessly linked with the artificially synthesized T-DNA fragment 1 to form a circular vector; the artificially synthesized T-DNA fragment is Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-Uc-restriction site 2-Ud-fluorescent protein-terminator-Ue / Uf-restriction site 3-Uf / -Uf-Ub / -Ub; Alternatively, the artificially synthesized T-DNA fragment 1 may be Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-fluorescent protein-Uc-restriction site 2-Ud-terminator-Ue / Uf-restriction site 3-Uf / -Uf-Ub / -Ub; The bimolecular fluorescent complementary binary vector consists of a pair of circular vectors with pYL1300-UaUf vectors as the backbone, seamlessly linked with artificially synthesized T-DNA fragment 2 to form a circular vector; the artificially synthesized T-DNA fragment 2 is Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-Uc-restriction site 2-Ud-truncated fluorescent protein-terminator-Ue / Uf-restriction site 3-Uf-Ub / -Ub; the truncated fluorescent proteins are Vn and Vc, constituting a set of bimolecular fluorescent complementary vectors; Alternatively, the artificially synthesized T-DNA fragment 2 may be Ua-restriction site 1-Ub / Ue-constitutive CaMV35S promoter-truncated fluorescent protein-Uc-restriction site 2-Ud-terminator-Ue / Uf-restriction site 3-Uf-Ub / -Ub; the truncated fluorescent proteins are Vn and Vc, constituting a set of bimolecular fluorescent complementary vectors; The organelle marker binary vector based on the mChe signaling channel is composed of a circular vector with a pYL1300-UaUf vector as the backbone and a synthetic T-DNA fragment 3 seamlessly linked together; the synthetic T-DNA fragment 3 is Ua-constitutive CaMV35S promoter-Uc-restriction site-Ud-fluorescent protein mCherry-terminator-Ub. Alternatively, the artificially synthesized T-DNA fragment 3 may be Ua-constitutive CaMV35S promoter-fluorescent protein mCherry-Uc-restriction site-Ud-terminator-Ub.

2. The visual plant binary carrier system according to claim 1, characterized in that, The fluorescent protein is selected from one of StayGold, eGFP, mCherry, Venus, and eCFP.

3. The visual plant binary carrier system according to claim 1, characterized in that, The enzyme cleavage site is selected from Asc I. Sma Ⅰ or Sbf I.

4. The visualization plant binary carrier system according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the artificially synthesized T-DNA fragment 1 in the fluorescently labeled protein localization binary vector can be selected from any one of SEQ ID NO.1 to 8.

5. The visualization plant binary carrier system according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the artificially synthesized T-DNA fragment 2 in the bimolecular fluorescent complementary binary vector can be selected from any one of SEQ ID NO. 9 to 12.

6. The visual plant binary carrier system according to claim 1, characterized in that, The enzyme cleavage sites in the organelle marker binary vector of the mChe signaling channel can be inserted into gene fragments targeting the cell nucleus, endoplasmic reticulum, plasmids, vacuoles, cytoplasm, mitochondria, plasma membrane, Golgi apparatus, peroxisomes, plasmodesmata, or cytoskeleton.

7. The visual plant binary carrier system according to claim 6, characterized in that, The nucleotide sequence of the artificially synthesized T-DNA fragment 3 in the organelle marker binary vector of the mChe signaling channel can be selected from any one of SEQ ID NO.13 to 24.

8. The application of the visualization plant binary carrier system according to any one of claims 1 to 7 in detecting the subcellular localization and interaction of target proteins in living plants.

9. The application according to claim 8, characterized in that, When tobacco mesophyll cells are co-transformed with a binary vector containing a bimolecular fluorescent complementary binary vector fused with the target protein encoding gene and an organelle marker binary vector containing the mChe signaling channel, two non-fluorescent expression cassettes are observed to be brought closer together and produce fluorescence using a laser confocal / fluorescence microscope. At the same time, the organelle marker of the mChe signaling channel produces red fluorescence that specifically indicates cell localization, thus verifying the interacting genes of the target protein and their position in the cell.

10. The application according to claim 8, characterized in that, When tobacco mesophyll cells are co-transformed with a fluorescent marker protein localization binary vector that fuses the target protein encoding gene and an organelle marker binary vector for the mChe signaling channel, the fluorescence of the target protein can be observed by laser confocal / fluorescence microscopy. At the same time, the organelle marker for the mChe signaling channel produces red fluorescence that specifically indicates cell localization, thus verifying the localization of the target protein in the cell.

Images