Green fluorescence overexpression vector for plasma membrane localization as well as construction method and application of green fluorescence overexpression vector

Abstract

The invention belongs to the technical field of bioengineering, and discloses a plasma membrane localized green fluorescence overexpression vector as well as a construction method and application thereof. The cytoplasmic membrane localized green fluorescence overexpression vector contains an expression vector skeleton and an AtCBL1 gene connected to the expression vector skeleton, wherein the nucleotide sequence of the AtCBL1 gene is shown as SEQIDNO: 1, and the expression vector skeleton also carries a fluorescent protein gene. The construction method of the vector comprises the following steps: S1, carrying out enzyme digestion and recovery on an AtCBL1 gene sequence with a nucleotide sequence as shown in SEQIDNO: 1 obtained by amplification; s2, performing same enzyme digestion on the expression vector carrying the fluorescent protein gene, and recovering; and S3, connecting the AtCBL1 gene subjected to enzyme digestion with the expression vector to form a recombinant plasmid, converting a connection product into an escherichia coli competent cell, and extracting the recombinant plasmid from positive clone to obtain the plasma membrane localized green fluorescence overexpression vector.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a cell membrane-localized green fluorescent overexpression vector, its construction method, and its application. Background Technology

[0002] In existing technologies, expression vectors containing a green fluorescent protein tag (EGFP) initiated by a 35S promoter can introduce a target gene recombined at its multiple cloning site into transformed plant cells and integrate it into the chromosome. After integration into the plant cell chromosome, the inserted target gene can be transcribed in the cell nucleus under the action of the 35S promoter, and finally translated into the target protein; at the same time, the green fluorescent protein tag (EGFP) carried by the expression vector is also expressed.

[0003] However, there is currently no effective fluorescent labeling and localization vector for plant cell plasma membranes, which cannot meet the research needs of cell activities related to plasma membrane localization involved in signaling pathways or molecular mechanisms.

[0004] Therefore, the existing technology needs further improvement. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a green fluorescent overexpression vector for cell membrane localization, its construction method, and its applications. This expression vector can express and localize to the plant cell membrane, achieving fluorescent labeling of the plant cell membrane. It can then be applied to the dynamic observation of the plant cell membrane and the investigation of cell activities related to cell membrane localization involving signaling pathways or molecular mechanisms.

[0006] To address the above problems, this application provides the following technical solution: In a first aspect, this application provides a cell membrane-localized green fluorescent overexpression vector, comprising: an expression vector backbone and a nucleotide sequence linked to the expression vector backbone as shown in SEQ ID NO:1 (CDS sequence). AtCBL1 The expression vector backbone also carries a fluorescent protein gene.

[0007] The amino acid sequence of the AtCBL1 protein is shown in SEQ ID NO:2.

[0008] The AtCBL1 gene is recombined with an expression vector containing a green fluorescent protein tag (EGFP) initiated by the 35S promoter to form a fusion expression vector that can express the AtCBL1-EGFP protein located on the cell membrane. The protein sequence of AtCBL1 is shown in SEQ ID NO:5.

[0009] Optionally, in the cell membrane-localized green fluorescent overexpression vector, the promoter used for the fluorescent protein gene is the 35S promoter with the sequence shown in SEQ ID NO:4. This promoter is a strong constitutive promoter in plant genetic engineering, driving the gene to be continuously expressed in all tissues.

[0010] Optionally, in the cell membrane-localized green fluorescent overexpression vector, the expression vector is... pCAMBIA1300-35S-AtCBL1-EGFP .

[0011] After the AtCBL1 green fluorescent overexpression vector, located on the cell membrane, is transformed into plant cells, AtCBL1-EGFP is integrated into the chromosome, AtCBL1 protein is expressed and located on the cell membrane, and the reporter gene—green fluorescent protein—is also expressed. This allows AtCBL1 located on the cell membrane to be observed under a laser confocal microscope. This vector will be used to investigate cellular activities related to cell membrane localization involved in signaling pathways or molecular mechanisms.

[0012] Secondly, this application provides a host cell carrying the aforementioned cell membrane-localized green fluorescent overexpression vector. The host cell is *Escherichia coli* or *Agrobacterium*.

[0013] Thirdly, this application also provides a method for constructing the above-mentioned cell membrane-localized green fluorescent overexpression vector, which includes the following steps: S1. The amplified nucleotide sequence is shown in SEQ ID NO:1. AtCBL1 Gene sequence was digested with enzymes and the digested enzymes were recovered. AtCBL1 Gene; S2. The expression vector carrying the fluorescent protein gene is subjected to the same enzyme digestion, and the digestion products are recovered. S3, after enzyme digestion AtCBL1 The gene and the expression vector are ligated to form a recombinant plasmid, and the ligation product is transformed into competent E. coli cells. After extracting the plasmid to verify positive clones, the recombinant plasmid is extracted from the positive clones, which is the cell membrane-localized green fluorescent overexpression vector.

[0014] Optionally, in the method for preparing the cell membrane-localized green fluorescent overexpression vector, in step S1, the... AtCBL1 Gene sequence amplification was performed using Arabidopsis thaliana col-0 cDNA as a template, and the nucleotide sequence was amplified using primers as shown in SEQ ID NO:1. AtCBL1 Gene.

[0015] Optionally, in the method for preparing the green fluorescent overexpression vector, the primers for amplifying the AtCBL1 gene include: upstream primer F with a nucleotide sequence as shown in SEQ ID NO:2 and downstream primer R with a nucleotide sequence as shown in SEQ ID NO:3.

[0016] The nucleotide sequences of the two primers are as follows: Upstream primer: 5' ATGGGCTGCTTCCACTCAA 3' (SEQ ID NO:2) Downstream primer: 5' TGTGGCAATCTCATCGACC 3' (SEQ ID NO: 3) Fourthly, this application also provides an application of the aforementioned cell membrane-localized green fluorescent overexpression vector in the fluorescent labeling of plant cell membranes, dynamic observation of plant cell membranes, or investigation of cell membrane localization-related cellular activities involving signaling pathways or molecular mechanisms. For example, when observing the subcellular localization of unknown proteins using soybean hairy roots or tobacco leaves, the cell membrane-localized green fluorescent overexpression vector can serve as a marker for cell membrane localization, labeling the subcellular localization of unknown proteins. The invasion process of rhizobia is accompanied by the lysis and remodeling of the root hair cell membrane. During rhizobia invasion, the cell membrane-localized green fluorescent overexpression vector can be used to label the cell membrane, allowing for more direct observation of the dynamic changes in the cell membrane during rhizobia invasion.

[0017] Fifthly, this application also provides a method for preparing transgenic plants with fluorescently labeled cell membranes, comprising the following steps: The aforementioned green fluorescent overexpression vector was transformed into Agrobacterium competent cells, and then the successfully transformed recombinant Agrobacterium was transformed into plant tissues using the Agrobacterium-mediated transformation method. Transgenic plants were then obtained through further screening and culture.

[0018] By taking tissue sections from transgenic plants and observing the green fluorescent plant cell membrane under a laser confocal microscope, the activity of the plant cell membrane can be observed, and the related cellular activities involving cell membrane localization in signaling pathways or molecular mechanisms can be investigated.

[0019] Optionally, in the method for preparing the cell membrane-localized green fluorescent overexpression vector, the plant tissue is: soybean root, Arabidopsis root, Arabidopsis leaf, or tobacco leaf.

[0020] The present invention has the following beneficial effects: The green fluorescent overexpression vector provided by this invention not only has a simple construction method but also allows for precise localization to the cell membrane, achieving fluorescent labeling of plant cell membranes. Specifically, after the expression vector is transformed into plant cells, AtCBL1-EGFPWhen integrated into the chromosome, the AtCBL1 protein is expressed and located on the cytoplasmic membrane. At the same time, the reporter gene—green fluorescent protein—is also expressed and located on the cytoplasmic membrane. Therefore, transgenic plants prepared using this plasmid show fluorescently labeled cytoplasmic membranes under a laser confocal microscope. Thus, the above-mentioned vector will be used to explore cell activities related to cytoplasmic membrane localization involved in signaling pathways or molecular mechanisms, contributing to the study of cytoplasmic membrane-related signaling pathways or molecular mechanisms. Attached Figure Description

[0021] Figure 1 for AtCBL1 Electrophoresis diagram of gene amplification; Figure 2 Electrophoresis results of colony PCR for Agrobacterium transformed with recombinant expression vector; Figure 3 Under a laser confocal microscope AtCBL1 The cell membranes of genetically modified soybeans are labeled with green fluorescent markers. Figure 4 The fluorescence in a single EGFP transgenic soybean under a laser confocal microscope was localized in the cell membrane, cytoplasm, and nucleus. Figure 5 Under a laser confocal microscope AtROP2 The cell membranes of genetically modified soybeans are labeled with green fluorescent markers. Figure 6 Under a laser confocal microscope AtGEF10 The cell membranes of genetically modified soybeans are labeled with green fluorescent markers. The above Figures 3-6 The three images, from left to right, are: a green fluorescence image obtained by laser confocal scanning, a bright-field image, and a superimposed image of green fluorescence and bright-field scanning; the scale bar for each image is 10 μm. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0023] Reagent kits used in the experiment 1. Ultrapure RNA Extraction Kit (DNase І) Purchased from CWBio, catalog number: CW0597S (50 preps), contains the following ingredients:

[0024] 2. Reverse transcription kit: The 5X All-in-One RTMasterMix (with AccuRT Genomic DNA Removal Kit), purchased from ABM, catalog number G492, was used. The included reagents are as follows:

[0025] 3. DNA Gel Rapid Purification Kit: The EasyPure Quick Gel Extraction Kit, purchased from TRANS (catalog number EG101-02), was used. The reagents included are as follows:

[0026] 4. Plasmid miniprep kit: The EasyPure Plasmid MiniPrep Kit was purchased from TRANS (catalog number EM101-02). The reagents included are as follows:

[0027] Example 1 AtCBL1 Acquisition of genes In this embodiment, specific primers were used to amplify Arabidopsis thaliana col-0 cDNA using PCR as a template. AtCBL1 Gene. AtCBL1 The gene is an upstream regulatory gene that overexpresses a potassium channel protein gene (e.g., AKT1). This expressed protein is located in the plant cell membrane. It can guide the fluorescent labeling gene to enter the plant cell along with the recombinant expression vector, and express and locate it in the plant cell membrane, thereby achieving fluorescent labeling of the plant cell membrane.

[0028] 1. Extraction of RNA from Arabidopsis thaliana 1) Sprinkle Arabidopsis thaliana col-0 on 1 / 2 MS solid medium, vernalize for 2-3 days, grow in a light incubator for 4 days, collect the material, flash freeze in liquid nitrogen, and store in a -80°C freezer.

[0029] 2) After thoroughly grinding 30-50 mg of tissue in liquid nitrogen, add 1 ml of TRlzon Reagent.

[0030] 3) After adding TRlzon Reagent to the sample, repeatedly pipette and agitate several times to ensure complete lysis of the sample. Incubate at room temperature for 5 minutes to allow the protein-nucleic acid complex to be completely separated.

[0031] 4) Add 200 μL of TRlzon Pal to every 1 ml of TRlzon Reagent. TM Cover the tube, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes.

[0032] 5) Centrifuge at 4℃ and 12000 rpm for 10 minutes. At this time, the sample is divided into three layers: red organic phase, middle layer and upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0033] 6) Add an equal volume of 70% ethanol (prepared with RNase-free water) to the obtained aqueous solution and mix by inverting.

[0034] 7) Add all the solution obtained in the previous step to the adsorption column (Spin Columns RM) that has been loaded into the collection tube. If the solution cannot be added all at once, it can be added in several batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0035] 8) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 20s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0036] 9) Preparation of DNase I mixture: Take 52 μL of RNase-Free Water, add 8 μL of 10×Reaction Buffer and 20 μL of DNase I (1 U / μL), mix well to prepare a reaction solution with a final volume of 80 μL.

[0037] 10) Add 80 μL of DNase I mixture directly to the adsorption column and incubate at 20-30℃ for 15 minutes.

[0038] 11) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube.

[0039] 12) Add 500 μL of Buffer RW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Repeat step 12). 13) Centrifuge at 12000 rpm for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column at room temperature for several minutes to dry completely. Note: The purpose of this step is to remove the residual ethanol in the adsorption column. Residual ethanol will affect subsequent enzymatic reactions (enzyme digestion, PCR, etc.).

[0040] 14) Place the adsorption column in a new RNase-free centrifuge tube, add 40 μL RNase-Free Water to the middle of the adsorption column, incubate at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect the RNA solution, and store the RNA at -70℃ to prevent degradation.

[0041] 2. The obtained RNA was reverse transcribed into cDNA. 1) Thaw the RNA template prepared in the previous step and 5×All-In-One RT MasterMix in an ice box. Gently shake up and down to mix the solution.

[0042] 2) Set up the reaction system in the PCR tubes according to the following conditions, and perform the entire process on ice:

[0043] 3) Thoroughly mix all reaction components and briefly centrifuge. Place the mixture in a PCR instrument and react according to the following procedure: 25℃ for 10 min 42℃ for 50 min 85℃ for 5 min An ice bath stops the reaction.

[0044] 3. AtCBL1 Gene amplification Using the above cDNA as a template, the following methods were employed. AtCBL1 Gene-specific primers are used to amplify the gene. The primer sequences are as follows: Upstream primer: 5' ATGGGCTGCTTCCACTCAA 3' Downstream primer: 5' TGTGGCAATCTCATCGACC 3' The PCR reaction system is as follows:

[0045] The PCR reaction procedure is as follows: (1 cycle) 98℃ for 1 min (35 cycles) 98℃ 10 sec 62℃ for 5 seconds 72℃ 7 sec (1 cycle) 72℃ for 1 min (1 cycle) Hold at 4℃.

[0046] After the PCR reaction, the amplification products were subjected to agarose gel electrophoresis. The electrophoresis results are shown in the figure. Figure 1 The results show that the amplified target gene... AtCBL1 The length is 642bp, which meets the expected length.

[0047] Example 2: Construction of a cell membrane-localized green fluorescent overexpression vector 1. Enzyme digestion of the target vector In this embodiment, the target vector uses the plant expression plasmid pCAMBIA1300-35S-EGFP. In this plasmid, the EGFP gene uses the 35S promoter, which is a strong constitutive promoter in plant genetic engineering, driving the gene to be continuously expressed in all tissues.

[0048] The enzyme digestion system is as follows: pCAMBIA1300-35S-EGFP 8.7 μL 10× Flycut Buffer 5 μL BamHⅠ1 μL XbaⅠ1 μL ddH2O 34.3 μL After the enzyme digestion reaction was completed, the digestion products were recovered by agarose gel electrophoresis.

[0049] 2. Recovery of target gene and target vector 1) Extract the target DNA band (the digested vector / ) from the agarose gel. AtCBL1 (Gene), place it in a clean centrifuge tube and weigh it. If the gel weighs 100 mg, it can be considered as 100 μL.

[0050] 2) Add 3 times the volume of GSB solution and melt the gel in a 55°C water bath for 6-10 minutes, mixing intermittently to ensure complete melting. Once the gel is completely melted, observe the color of the solution. If the color is purple, add an appropriate amount of 3M sodium acetate (pH 5.2) to adjust the color to match the GSB color (yellow). (To increase DNA recovery, add 1 volume of the melted gel solution; for example, if the gel weight is 100 mg, add 100 μL of isopropanol). 3) Once the gel has melted and reached room temperature (the DNA binding ability of the centrifuge column is weak at high temperatures), add it to the centrifuge column and let it stand for a few minutes. Centrifuge at 10,000×g for 1 minute and discard the eluent. Pass the entire column once, then pass it multiple times, adding the remaining eluent as needed.

[0051] 4) Add 650 μL of WB solution, centrifuge at 10,000×g for 1 minute, and discard the effluent. 5) Centrifuge at 10,000×g for 1-2 minutes to completely remove residual WB.

[0052] 6) Attach the centrifuge column to a clean centrifuge tube, open the cap and let it stand for 1 minute to allow the residual ethanol to evaporate completely. Add 40 μL of EB or deionized water (pH>7.0) to the center of the column and let it stand at room temperature for 1 minute. 7) Centrifuge at 10,000×g for 1 minute to elute the DNA, and store the eluted DNA at -20℃. 3. One-step connection Add each component according to the following ligation system, place in a PCR instrument, and incubate at 50°C for 15 min.

[0053] The connection system is as follows:

[0054] 4. Transformation of E. coli competent cells (1) All the ligation products were transformed into 10 μL of competent Escherichia coli (DH5α), placed on ice for 20 min, heat-shocked at 42℃ for 45 s, cooled on ice for 2 min, and then added to 250 μL of LB liquid medium. The mixture was then recovered in a shaker at 37℃ for 30 min.

[0055] (2) Centrifuge at 12000×g for 2 min, remove part of the supernatant, resuspend the bacteria by pipetting, and spread them all on LB solid medium containing kanamycin.

[0056] 5. Sequencing and amplification Two single colonies were picked up with a pipette tip and streaked onto LB solid medium containing antibiotics. The remaining bacteria from the pipette tip were then inoculated into 700 μL of LB medium containing the corresponding antibiotic and incubated at 37°C. Single colonies were sequenced using upstream primers. After sequencing, the sequences were compared, and the streaks corresponding to single colonies with completely identical alignment results were selected and inoculated into 6 ml of LB medium and incubated overnight at 37°C. Plasmids were extracted the next day.

[0057] 6. Extract plasmids (1) Take the overnight cultured bacterial solution, centrifuge at 10,000×g for 1 minute, and remove the supernatant (try to remove as much as possible). Collect the culture by centrifugation in 3 separate times. (2) Add 250 μL of colorless solution RB (containing RNase A), shake to suspend the bacterial precipitate, and leave no small bacterial clumps. (3) Add 250 μL of blue solution LB and gently mix by turning it upside down 4-6 times to fully lyse the cells and form a clear blue solution. The color changes from semi-clear to clear blue, indicating complete lysis (not more than 5 minutes). (4) Add 340 μL of yellow solution NB and gently mix 5-6 times (the color changes completely from blue to yellow, indicating that the mixture is uniform and neutralized completely) until a firm yellow agglomerate is formed. Let it stand at room temperature for 2 minutes. (5) Centrifuge at 12,000×g for 5 minutes, carefully aspirate the supernatant and add it to the centrifuge column. Centrifuge at 12,000×g for 1 minute and discard the eluent. (6) Add 650 μL of solution WB, centrifuge at 12,000×g for 1 minute, and discard the effluent. (7) Centrifuge at 12,000×g for 1-2 minutes to completely remove residual WB. (8) Place the centrifuge column in a clean centrifuge tube, add 40 μL of EB or deionized water (pH>7.0) to the center of the column, and let it stand at room temperature for 1 minute. (Preheating the EB or deionized water in a water bath at 60-70℃ will improve the effect). (9) Centrifuge at 10,000×g for 1 minute to elute the DNA. Store the eluted DNA at -20℃.

[0058] The final extracted recombinant plasmid is the cell membrane-localized green fluorescent overexpression vector. It is subsequently used to transform Agrobacterium and further used to prepare transgenic plants with cell membrane labeling.

[0059] Example 3: Preparation and application of transgenic soybeans with fluorescently labeled cell membranes. 3.1 Experimental Methods 3.1.1 Transformation of Agrobacterium competent cells (1) Take 5 μL of Agrobacterium K599 competent cells, thaw them on ice, add 1 μL of plasmid extracted in Example 2, gently mix, place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and then cool on ice to room temperature.

[0060] (2) Add 700 μL of LB medium to the competent cells and shake at 28°C for 2 hours.

[0061] (3) Centrifuge at 12000×g for 2 min, discard the supernatant, resuspend, spread, and culture to obtain a single colony.

[0062] 3.1.2 Colony PCR Identification Four single colonies were picked from the selection plate using a pipette tip and streaked onto solid YEP medium containing str / kan. The remaining bacteria were then placed into the prepared PCR system, with the plasmid serving as a positive control and no template added as a negative control.

[0063] The primer sequences are as follows: Upstream primer: 5' ATGGGCTGCTTCCACTCAA 3' (SEQ ID NO: 2) Downstream primer: 5' TGTGGCAATCTCATCGACC 3' (SEQ ID NO: 3) The PCR reaction system is as follows: Easy Taq Mix 5 μL Primer F0.2 μL Primer R 0.2 μL ddH2O 3.6 μL The PCR reaction procedure is as follows: (1 cycle) 94℃ for 10 min (35 cycles) 94℃ 30 sec 58℃ for 30 seconds 72℃ for 35 seconds (1 cycle) 72℃ for 5 min (1 cycle) 4℃ Hold Experimental Results and Analysis: Colony PCR results are attached. Figure 2 Based on the length of the amplification results, positive colonies are screened and stored at -80°C.

[0064] 3.1.3 Transformation of recombinant expression vector into soybean hairy roots (1) Soybean cultivation (Williams 82) (2) The Agrobacterium tumefaciens culture stored at -80°C was activated on solid YEP medium containing str / kan to make it active and grow single colonies.

[0065] (3) After about 5-7 days, the hypocotyl of soybean elongates and the cotyledons open slightly. At this time, the activated Agrobacterium (2) is inoculated into 4 ml of YEP medium (50 ml conical flask), the corresponding antibiotic is added, and it is placed at 28℃ and cultured at 200 rpm for 12 hours.

[0066] (4) Add 400 μL of Agrobacterium tumefaciens prepared in step (3) to 100 ml YEP liquid culture medium and incubate at 28℃ and 200 rpm until the bacterial solution OD600=1.

[0067] (5) The Agrobacterium tumefaciens activated in step (2) was streaked onto YEP solid medium without antibiotics for culture.

[0068] (6) Prepare 1 / 10 MS medium: Add 0.043 g imported MS, 2 g sucrose and 0.39 g MES to 100 ml water and adjust the pH to 5.4. After sterilizing 1 / 10 MS, add AS (acetylsuccinone, the final concentration of AS is 100 μg / ml, that is, 100 μL of 0.1 g / ml AS is added to 100 ml of medium).

[0069] (7) Centrifuge the bacterial culture prepared in step (4) at 5000 rpm for 10 minutes, discard the supernatant, and resuspend it with an equal volume of 1 / 10 MS with added AS for later use.

[0070] (8) Cuttings: Wet the vermiculite with sterilized water, select soybeans with open cotyledons and unopened true leaves, cut at a 45° angle 0.7 cm-1 cm away from the cotyledon node, cover the wound with the Agrobacterium tumefaciens prepared in step (5) and carefully bury it in the moist vermiculite, then pour 5 ml of the suspension obtained in step (7) into each soybean.

[0071] (9) When the soybeans have grown for 17 days, wash off the vermiculite on the soybean roots. The hairy roots that grow from the wounds are the transgenic roots. Cut off the transgenic roots and place them on a glass slide for observation under a laser confocal microscope.

[0072] 3.1.4 Setting up the control group: The expression vector constructed in this patent pCAMBIA1300-35S-AtCBL1-EGFP The AtCBL1 gene, which is located in the cell membrane, was replaced with other protein-coding genes in Arabidopsis thaliana that are located in the cell membrane, including... AtROP2 (The sequence is shown in SEQ ID NO:6) and AtGEF10 (Sequence shown in SEQ ID NO:7) to evaluate the cell membrane fluorescent labeling of the subsequently constructed recombinant soybean hairy roots.

[0073] Specifically, expression vectors are constructed according to the aforementioned methods. pCAMBIA1300-35S-AtROP2-EGFP、 pCAMBIA1300-35S-AtGEF10-EGFP, The two recombinant expression vectors were then transformed into soybean hairy roots, and transgenic roots were obtained using the method described above. These roots were then observed under a laser confocal microscope.

[0074] 3.2 Experimental Results like Figure 3 As shown, it can be clearly seen under a laser confocal microscope. AtCBL1 genetically modified Soybean plant cell plasma membranes were labeled with green fluorescence, and the plasma membrane of each cell could be clearly observed. Correspondingly, pCAMBIA1300-35S-EGFP was localized in the plasma membrane, cytoplasm, and nucleus (e.g., ...). Figure 4 (As shown).

[0075] like Figure 5 and Figure 6 In the control group shown, it was observed under a laser confocal microscope that... AtROP2 Genetically modified soybean cell membrane and AtGEF10 The boundaries of the cell membranes in genetically modified soybeans are indistinct, and fluorescence is even localized within the cytoplasm. Clearly, AtROP2 and AtGEF10 Not suitable as a marker for plasma membrane localization. The above results indicate that not all proteins that are localized to the cell membrane are suitable as markers for plasma membrane localization.

[0076] Therefore, the green fluorescent overexpression vector for cell membrane localization provided in this application can be used for precise fluorescent labeling of plant cell membranes, and can be widely applied in dynamic observation or investigation of cell membrane localization related to signaling pathways or molecular mechanisms. For example, when observing the subcellular localization of unknown proteins using soybean hairy roots or tobacco leaves, the green fluorescent overexpression vector for cell membrane localization can serve as a marker for cell membrane localization to label the subcellular localization of unknown proteins. The invasion process of rhizobia is accompanied by the lysis and remodeling of the root hair cell membrane. During the rhizobia invasion process, the green fluorescent overexpression vector for cell membrane localization can be used to label the cell membrane, allowing for more direct observation of the dynamic changes in the cell membrane during rhizobia invasion.

[0077] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A cytoplasmic membrane-localized green fluorescent overexpression vector, characterized by, comprises: an expression vector backbone and a nucleotide sequence as shown in SEQ ID NO: 1 linked to the expression vector backbone AtCBL1 gene, the expression vector backbone further carrying a fluorescent protein gene.

2. The cytoplasmic membrane-localized green fluorescent overexpression vector of claim 1, characterized by, The promoter adopted by the fluorescent protein gene is a 35s promoter with a sequence as shown in SEQ ID NO:

4.

3. The cytoplasmic membrane-localized green fluorescent overexpression vector of claim 1, wherein, The expression vector is pCAMBIA1300-35S-AtCBL1-EGFP .

4. A host cell, characterized in that, The cytomembrane-located green fluorescent overexpression vector as claimed in claim 1.

5. The method for constructing a cytoplasmic membrane-localized green fluorescent overexpression vector according to any one of claims 1 to 3, wherein The method comprises the following steps: S1, the amplified nucleotide sequence is shown as SEQ ID NO: 1 AtCBL1 gene sequence is cut by enzyme and the cut gene is recovered AtCBL1 gene; S2, the expression vector carrying the fluorescent protein gene is subjected to the same enzyme digestion, and the enzyme digestion product is recovered; S3, the enzyme cut AtCBL1 The gene and the expression vector are connected to form a recombinant plasmid, and the connection product is transformed into an E. coli competent cell. After extracting the plasmid to verify a positive clone, the recombinant plasmid is extracted from the positive clone, which is a green fluorescent overexpression vector for cytomembrane localization.

6. The method for preparing the cell membrane-localized green fluorescent overexpression vector according to claim 5, characterized in that, In the S1 step, the AtCBL1 The gene sequence is amplified using the cDNA of Arabidopsis thaliana col-0 as a template to amplify the nucleotide sequence of the gene shown in SEQ ID NO:

1. AtCBL1 gene.

7. The method for preparing the cell membrane-localized green fluorescent overexpression vector according to claim 6, characterized in that, The primers for amplifying the AtCBL1 gene comprise: an upstream primer F with a nucleotide sequence as shown in SEQ ID NO: 2 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO:

3.

8. The cytomembrane-located green fluorescent overexpression vector as claimed in claim 1 is applied to plant cytomembrane fluorescent labeling and dynamic observation or exploration of signal pathways or cytomembrane-located related cell activities of molecular mechanisms.

9. A method for the production of a transgenic plant with a fluorescent marker in the cytoplasmic membrane, characterized in that, The method comprises the following steps: The green fluorescent overexpression vector as claimed in claim 1 is transformed into Agrobacterium competent cells, and the successfully transformed recombinant Agrobacterium is further transformed into plant tissues based on the Agrobacterium-mediated method, and the transgenic plants are obtained through further screening and culture.

10. The method of claim 1, wherein the method is for preparing a cytoplasmic membrane-localized green fluorescent overexpression vector, and the vector comprises: a) a green fluorescent protein gene; b) a cytoplasmic membrane-localized signal sequence; c) a promoter; and d) a terminator. The plant tissues are soybean roots, Arabidopsis roots, Arabidopsis leaves or tobacco leaves.

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