CN105823890B - A kind of Subcellular Localization kit built using sorghum mosaic virus P3N PIPO - Google Patents

Abstract

The invention relates to a plant subcellular localization kit constructed by using sorghum mosaic virus P3N-PIPO, comprising four plasmodesmata localization vectors respectively labeled with green, red, yellow and cyan fluorescent proteins: SrMV-P3N-PIPO- GFP, pSrMV‑P3N‑PIPO‑RFP, pSrMV‑P3N‑PIPO‑YFP, and pSrMV‑P3N‑PIPO‑CFP; 4 control vectors without specific subcellular localization: pSAT6‑EGFP‑C1, pSAT6‑ERFP‑C1, pSAT6 ‑EYFP‑C1 and pSAT6‑ECFP‑C1; restriction enzymes Xho I, Hind III, RNase-free water and infection solution. Using this kit can quickly determine whether the target gene expression product has the characteristics of plasmodesmata localization.

Description

A subcellular localization kit constructed using sorghum mosaic virus P3N-PIPO

Technical Field The present invention relates to a kit, in particular to a subcellular localization kit constructed using Sorghum Mosaic Virus P3N-PIPO. The present invention utilizes markers with green fluorescent protein (green fluorescent protein, GFP), red fluorescent protein (red fluorescent protein, RFP), yellow fluorescent protein (yellow fluorescent protein, YFP) and cyan fluorescent protein (cyan fluorescent protein, CFP) respectively The protein SrMV-P3N-PIPO with specific plasmodesmata (PD) localization function indicates whether the gene expression protein to be verified has the property of plasmodesmata localization, and belongs to the field of biotechnology.

Background Art With the development of biotechnology, especially the development of genomics and the advancement of sequencing technology, more and more new genes have been discovered, and massive data have been formed. To clarify the function of unknown genes and explore their potential application value is the ultimate goal of genomics research. Protein is the product of gene expression. The subcellular location of protein is closely related to the structure and function of protein. Protein must be in a suitable position to perform its function. For a new gene, clarifying the subcellular location of its expression product, that is, the protein, can provide important clues for studying the function of the gene. At present, there are three main methods to determine the subcellular localization of proteins: cell fractionation, electron microscope analysis, and laser confocal method, among which laser confocal method is the most widely used. The laser confocal method uses the fluorescent signal generated by the excitation of the labeled fluorescent protein to visually observe the subcellular location of the target protein. When studying the subcellular localization of the target protein, a positive control, that is, a protein with a detectable label with a specific subcellular status, is required to support the localization of the unknown protein.

Plasmodesma (PD) is a microtubule that passes through the cell wall and connects the cytoplasm and endoplasmic reticulum of adjacent cells in plants, and is an important channel for material transport and information transmission between cells. The structure of PD is extremely complex, and it is still not completely clear, and its structural model has been in a state of supplementary update. The permeability of PD is regulated by many factors. Small molecules can freely diffuse through plasmodesmata, but the intercellular movement of macromolecules such as proteins and nucleic acids or macromolecular complexes such as virus particles and ribosomal protein complexes is restricted. Regulation of PD. For the study of plant intercellular signal transduction and material transport, although bioinformatics methods can be used to predict the subcellular location of the encoded protein of the isolated gene, the subcellular location of the encoded protein of the gene must be determined for biological analysis. Through biological experiments, it is clear whether it can be located in PD, and then whether the protein is involved in intercellular signal transduction and material transport, providing intuitive experimental evidence for the study of its function.

In 2008, Chung et al. used bioinformatics methods to detect the sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV) and sugarcane streak mosaic virus (SCSMV), etc. The analysis of 48 viruses of the family Potatoviridae shows that there is a conserved structure G _1-2 A _6-7 inside the P3 gene, and the ribosome can translate a 6-7kDa gene in this conserved structure through a +2 frame shift. The protein (PIPO), which combines with the N-terminus of the P3 protein, forms a fusion protein of about 25 kDa, namely P3N-PIPO. Chung et al. cloned the P3N-PIPO coding sequence of Turnip mosaic virus (TuMV), and confirmed by experiments that TuMV-P3N-PIPO is located in plasmodesmata. Studies have shown that P3N-PIPO is likely to be the mobile protein of potato virus Y. This protein interacts with host factors to enable the virus to move between cells through plasmodesmata, and then establish a systemic infection of the host (Choi et al., 2005 ; Chung et al., 2008; Wen et al., 2010; Wei et al., 2010; Vijayapalani et al., 2012). But there is no report on the subcellular localization of SCMV-P3N-PIPO, SrMV-P3N-PIPO and SCSMV-P3N-PIPO.

SUMMARY OF THE INVENTION The object of the present invention is to provide a subcellular localization kit constructed using Sorghum Mosaic Virus P3N-PIPO, which provides an indicator for detecting whether the expression product of the target gene is localized in the plant plasmodesmata.

For realizing the purpose of the present invention, the technical scheme of the present invention is as follows.

Using fusion PCR technology, using the P3 gene coding sequence of SrMV as a template, the coding sequence of P3N-PIPO was cloned, constructed into plant expression vectors carrying different fluorescent marker protein genes, transformed into Agrobacterium, injected into N. Using corresponding laser excitation and collecting emitted light signals under a focusing microscope, clear and bright dot-like images can be observed at the plasmodesmata of N. benthamiana epithelial cells, which proves that P3N-PIPO is located in the plasmodesmata. Accordingly, we constructed a plant subcellular localization kit using P3N-PIPO of SrMV.

A subcellular localization kit constructed by using Sorghum mosaic virus P3N-PIPO of the present invention is characterized in that the kit consists of the following reagents:

(1) Green fluorescent protein-labeled PD targeting vector pSrMV-P3N-PIPO-GFP, as a positive control, 1 tube, 100 ng/μL, 50 μL/tube, cryopreserved at -20°C for later use;

(2) Red fluorescent protein-labeled PD targeting vector pSrMV-P3N-PIPO-RFP, as a positive control, 1 tube, 100 ng/μL, 50 μL/tube, stored at -20°C for later use;

(3) Yellow fluorescent protein-labeled PD targeting vector pSrMV-P3N-PIPO-YFP, as a positive control, 1 tube, 100 ng/μL, 50 μL/tube, frozen at -20°C for later use;

(4) Cyan fluorescent protein-labeled PD targeting vector pSrMV-P3N-PIPO-CFP, as a positive control, 1 tube, 100ng/μL, 50μL/tube, cryopreserved at -20°C for later use;

(5) Carrier pSAT6-EGFP-C1, 1 tube, 500ng/μL, 50μL/tube, cryopreserved at -20°C for later use;

(6) Carrier pSAT6-ERFP-C1, 1 tube, 500ng/μL, 50μL/tube, cryopreserved at -20°C for later use;

(7) Carrier pSAT6-EYFP-C1, 1 tube, 500ng/μL, 50μL/tube, cryopreserved at -20°C for later use;

(8) Carrier pSAT6-ECFP-C1, 1 tube, 500ng/μL, 50μL/tube, cryopreserved at -20°C for later use;

(9) Restriction endonuclease Xho I: 1 tube, 500 units, 100 μL/tube, frozen at -20°C for later use;

(10) Restriction endonuclease Hind III: 1 tube, 500 units, 100 μL/tube, frozen at -20°C for later use;

(11) RNase-free water: 2 bottles, 100mL/bottle, refrigerated at 4°C for later use;

(12) Infection solution: 1 tube, 50 mL/tube, frozen at -20°C for later use; the infection solution includes D-glucose, 250 mg; MES, 5 mL; Na ₃ PO ₄ ·12H ₂ O, 5 mL; Syringone, 5 μL; add ddH ₂ O to a total volume of 50 mL.

The construction method of the green fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-GFP: use XhoI and Hind III to carry out double digestion of the vector pSAT6-EGFP-C1 (GenBank: AY818374), and then use T4DNA ligase to digest the enzyme The cut product was ligated with the insert fragment S, and transformed into competent Escherichia coli (Escherichia coli) DH5α, and positive clones were picked for verification;

The construction method of the red fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-RFP: use XhoI and Hind III to carry out double digestion of the vector pSAT6-ERFP-C1 (GenBank: DQ005474), and then use T4DNA ligase to digest the enzyme The cut product was ligated with the insert fragment S, and transformed into competent Escherichia coli (Escherichia coli) DH5α, and positive clones were picked for verification;

The construction method of the yellow fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-YFP: use XhoI and Hind III to carry out double digestion of the vector pSAT6-EYFP-C1 (GenBank: AY818380), and then use T4 DNA ligase to digest the enzyme The cut product was ligated with the insert fragment S, and transformed into competent Escherichia coli (Escherichia coli) DH5α, and positive clones were picked for verification;

The construction method of the cyan fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-CFP: use XhoI and Hind III to carry out double enzyme digestion on the vector pSAT6-ECFP-C1 (GenBank: AY818374), and then use T4 DNA ligase to digest the enzyme The cut product was ligated with the insert fragment S, and transformed into competent Escherichia coli (Escherichia coli) DH5α, and positive clones were picked for verification.

The nucleotide sequence of the insertion fragment S is the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing.

Advantages and beneficial effects of the present invention: using the kit of the present invention, the target gene can be quickly constructed into a fluorescently labeled vector, and whether its expression product has the characteristic of plasmodesmata localization is clarified. This kit provides 4 kinds of fluorescent labeling vectors, which provide users with more choices and meet the needs of studying whether different gene expression products are co-localized in plasmodesmata. Using this kit can quickly complete the research on the subcellular localization of the expression product of the target gene.

Description of drawings:

Fig. 1 is the plasmid map of the vector pSrMV-P3N-PIPO-GFP labeled with green fluorescent protein;

Fig. 2 is the plasmid map of the vector pSrMV-P3N-PIPO-RFP marked with red fluorescent protein;

Fig. 3 is the plasmid map of the vector pSrMV-P3N-PIPO-YFP marked with yellow fluorescent protein;

Fig. 4 is the plasmid map of the vector pSrMV-P3N-PIPO-CFP marked with cyan fluorescent protein;

Figure 5 is the plasmid map of the Arabidopsis thaliana AtREM1.3 subcellular localization vector pAtREM1.3-RFP labeled with red fluorescence;

Figure 6 is a merged picture of the subcellular localization maps of pAtREM1.3-RFP and pSrMV-P3N-PIPO-GFP;

Figure 7 is the subcellular localization of GFP vector pSAT6-EGFP-C1;

Figure 8 shows the subcellular localization of the RFP vector pSAT6-ERFP-C1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to further clarify the present invention rather than limit the present invention, the following examples will be used for illustration. The experimental methods described in the following examples are conventional methods unless otherwise specified. The reagents and biological materials can be obtained from commercial sources unless otherwise specified.

Example 1: Cloning of SrMV-P3N-PIPO coding sequence

The present invention clones the P3 protein coding sequence of Sorghum mosaic virus (SrMV) by using PCR technology; takes the P3 gene as a template, utilizes fusion PCR technology to clone the coding sequence of P3N-PIPO of SrMV, and converts the The sequences were linked into expression vectors with different fluorescent protein markers, and four subcellular expression vectors capable of specifically targeting plasmodesmata were obtained, namely the green fluorescent protein-labeled PD localization vector pSrMV-P3NPIPO-GFP, red fluorescent protein The labeled PD targeting vector pSrMV-P3NPIPO-RFP, the yellow fluorescent protein labeled PD targeting vector pSrMV-P3NPIPO-YFP and the cyan fluorescent protein labeled PD targeting vector pSrMV-P3NPIPO-CFP. The carrier construction method is as follows:

(1) Obtaining of SrMV-P3 coding sequence: Design specific PCR primers SrMV-P3-F (upstream primer) and SrMV-P3-R (downstream primer) for the coding sequence of SrMV-P3, and use the cDNA of SrMV as a template to carry out PCR amplification, the PCR product is separated and recovered by agarose gel electrophoresis, and the coding sequence of SrMV-P3 is obtained. The nucleotide sequence of the upstream primer SrMV-P3-F is the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing; the nucleotide sequence of the downstream primer SrMV-P3-R is the SEQ ID NO in the sequence listing : the nucleotide sequence shown in 2; the nucleotide sequence of the SrMV-P3 coding sequence is the nucleotide sequence shown in SEQID NO: 3 in the sequence listing;

(2) Obtaining of SrMV-P3N coding sequence: Design specific PCR primers, SrMV-P3N-F (upstream primer) and SrMV-P3N-R (downstream primer), introduce restriction site in upstream primer SrMV-P3N-F Xho I. Using the SrMV-P3 coding sequence as a template, use primers SrMV-P3N-F and SrMV-P3N-R for PCR amplification. The reaction system is 25 μL: 10×PCR Buffer 2.5 μL, dNTPs 2.0 μL, upstream primer SrMV-P3N-F (10 μmol/L) 1.0 μL, downstream primer SrMV-P3N-R (10 μmol/L) 1.0 μL, Taq enzyme (5U/μL) 0.125 μL, ddH ₂ O 17.375 μL, template (cDNA) 1.0 μL, total volume 25 μL. PCR reaction program: 94°C pre-denaturation for 4min; then run 35 cycles, 94°C for 30s, 50°C for 30s, 72°C for 1min; finally 72°C for 10min. After the PCR reaction, the PCR product was separated by agarose gel electrophoresis, recovered and concentrated to 400 ng/μL, and the coding sequence of SrMV-P3N was obtained. The nucleotide sequence of the upstream primer SrMV-P3N-F is the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing; the nucleotide sequence of the downstream primer SrMV-P3N-R is the SEQ ID NO in the sequence listing : the nucleotide sequence shown in 5; the nucleotide sequence of the SrMV-P3N coding sequence is the nucleotide sequence shown in SEQ ID NO: 6 in the sequence listing;

(3) Obtaining of the SrMV-PIPO coding sequence: design specific PCR primers, SrMV-PIPO-F (upstream primer) and SrMV-PIPO-R (downstream primer), and introduce restriction sites into the downstream primer SrMV-P3N-R Hind III. Using the SrMV-P3 coding sequence as a template, use primers SrMV-PIPO-F and SrMV-PIPO-R for PCR amplification. The reaction system is 25 μL: 10×PCR Buffer 2.5 μL, dNTPs 2.0 μL, upstream primer SrMV-PIPO-F (10 μmol/L) 1.0 μL, downstream primer SrMV-PIPO-R (10 μmol/L) 1.0 μL, Taq enzyme (5U/μL) 0.125 μL, ddH ₂ O 17.375 μL, template (cDNA) 1.0 μL, total volume 25 μL. PCR reaction program: 94°C pre-denaturation for 4min; then run 35 cycles, 94°C for 30s, 50°C for 30s, 72°C for 1min; finally 72°C for 10min. After the PCR reaction, the PCR product was separated by agarose gel electrophoresis, recovered and concentrated to 400 ng/μL, and the coding sequence of SrMV-PIPO was obtained. The nucleotide sequence of the upstream primer SrMV-PIPO-F is the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing; the nucleotide sequence of the downstream primer SrMV-PIPO-R is SEQ ID NO in the sequence listing: The nucleotide sequence shown in 8; the nucleotide sequence of the SrMV-PIPO coding sequence is the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing;

(4) Obtaining the coding sequence of SrMV-P3N-PIPO: the PCR products of P3N and PIPO with a concentration of 400ng/μL were mixed according to 1:1 as a template, and primers SrMV-P3N-F and SrMV-PIPO-R were used. The coding sequence of SrMV-P3NPIPO was cloned by fusion PCR method. The PCR reaction system was 25 μL: 10×PCR Buffer 2.5 μL, dNTPs 2.0 μL, upstream primer SrMV-P3N-F (10 μmol/L) 1.0 μL, downstream primer SrMV-PIPO-R (10 μmol/L) 1.0 μL, Taq enzyme ( 5U/μL) 0.125 μL, ddH ₂ O 12.375 μL, template (cDNA) 6.0 μL, total volume 25 μL. PCR reaction program: 94°C pre-denaturation for 4 minutes; then run 35 cycles, 94°C for 30s, 55°C for 30s, 72°C for 1min; finally 72°C for 10min. After the PCR reaction, the PCR product was separated and recovered by agarose gel electrophoresis to obtain the coding sequence of SrMV-P3N-PIPO. The nucleotide sequence of the SrMV-P3N-PIPO coding sequence is the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing;

(5) Obtaining the insert S: using Xho I and Hind III to double-enzyme digest the SrMV-P3N-PIPO coding sequence, separate and recover the digested products by agarose gel electrophoresis, and obtain the insert S; The nucleotide sequence of the insert fragment S is the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing;

(6) Construction of the green fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-GFP: Carry out double digestion of the vector pSAT6-EGFP-C1 (GenBank: AY818374) with Xho I and Hind III, and pass the digested product through agar Sugar gel electrophoresis for separation and recovery. Then use T4 DNA ligase to ligate the digested product with the insert fragment S, and transform the ligated product into competent Escherichia coli (Escherichia coli) DH5α, pick positive clones for verification, and obtain the green fluorescent protein-labeled PD positioning vector pSrMV-P3N -PIPO-GFP, the plasmid map of which is shown in Figure 1;

(7) Construction of the red fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-RFP: Carry out double digestion of the vector pSAT6-ERFP-C1 (GenBank: DQ005474) with Xho I and Hind III, and pass the digested product through agar Sugar gel electrophoresis for separation and recovery. Then use T4 DNA ligase to ligate the digested product with the insert fragment S, and transform the ligated product into competent Escherichia coli (Escherichia coli) DH5α, pick positive clones for verification, and obtain the red fluorescent protein-labeled PD positioning vector pSrMV-P3N - PIPO-RFP, the plasmid map of which is shown in Figure 2;

(8) Construction of yellow fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-YFP: Carry out double enzyme digestion on the vector pSAT6-EYFP-C1 (GenBank: AY818380) with Xho I and Hind III, and pass the digested product through agar Sugar gel electrophoresis for separation and recovery. Then use T4 DNA ligase to ligate the digested product with the insert fragment S, and transform the ligated product into competent Escherichia coli (Escherichia coli) DH5α, pick positive clones for verification, and obtain the yellow fluorescent protein-labeled PD positioning vector pSrMV-P3N -PIPO-YFP, the plasmid map of which is shown in Figure 3;

(9) Construction of the cyan fluorescent protein-labeled PD positioning vector pSrMV-P3N-PIPO-CFP: Carry out double digestion of the vector pSAT6-ECFP-C1 (GenBank: AY818374) with Xho I and Hind III, and pass the digested product through agar Sugar gel electrophoresis for separation and recovery. Then use T4 DNA ligase to ligate the digested product with the insert fragment S, and transform the ligated product into competent Escherichia coli (Escherichia coli) DH5α, pick positive clones for verification, and obtain the cyan fluorescent protein-labeled PD positioning vector pSrMV-P3N - PIPO-CFP, the plasmid map of which is shown in FIG. 4 .

Example 2: Subcellular localization of Arabidopsis AtREM1.3

1. Cloning of Arabidopsis AtREM1.3 gene

A Remorin gene was cloned from Arabidopsis and cloned into the cloning vector pMD19-T. Phylogenetic tree analysis showed that the gene belonged to type 3 of subgroup 1 of Remorin gene family, named AtREM1.3. Literature shows that Remorin can be located in plasma membrane and plasmodesmata, but bioinformatics analysis shows that Remorin has no transmembrane domain and signal peptide. In order to verify that AtREM1.3 can be localized in plasmodesmata, this kit was used to study its subcellular localization. The nucleotide sequence of the AtREM1.3 coding sequence is the nucleotide sequence shown in SEQ ID NO: 12 in the sequence listing;

2. Construction of subcellular localization vector pAtREM1.3-RFP

(1) Design specific PCR primers, AtREM1.3-F (upstream primer) and AtREM1.3-R (downstream primer), introduce restriction site Xho I in the upstream primer AtREM1.3-F, and introduce restriction site Xho I in the downstream primer AtREM1. A restriction enzyme cutting site Hind III was introduced into 3-R. Using the pair of primers, PCR amplification was performed using the cloning vector of the AtREM1.3 gene as a template. The PCR reaction system was 25 μL: 10×PCR Buffer 2.5 μL, dNTPs 2.0 μL, upstream primer AtREM1.3-F (10 μmol/L) 1.0 μL, downstream primer AtREM1.3-R (10 μmol/L) 1.0 μL, Taq enzyme ( 5U/μL) 0.125 μL, ddH ₂ O 17.375 μL, template (cDNA) 1.0 μL, total volume 25 μL. PCR reaction program: 94°C pre-denaturation for 4min; then run 35 cycles, 94°C for 30s, 50°C for 30s, 72°C for 1min; finally 72°C for 10min. After the PCR reaction, use Xho I and Hind III to carry out double enzyme digestion on the PCR product, separate and recover the enzyme digestion product by agarose gel electrophoresis, and obtain the insert fragment InsTarget; the nucleus of the upstream primer AtREM1.3-F The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 13 in the sequence listing; the nucleotide sequence of the downstream primer AtREM1.3-R is the nucleotide sequence shown in SEQ ID NO: 14 in the sequence listing; insert The nucleotide sequence of the fragment InsTarget is the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing;

(2) Take the RFP control vector, use Xho I and Hind III to carry out double enzyme digestion on the vector pSAT6-ERFP-C1, and separate and recover the digestion products by agarose gel electrophoresis. Then use T4 DNA ligase to connect the digested product with the insert fragment InsTarget, and transform the ligated product into competent Escherichia coli (Escherichia coli) DH5α, pick positive clones for verification, and obtain the red fluorescent protein-labeled PD positioning vector pAtREM1.3 -RFP, its plasmid map is as shown in Figure 5;

3. Subcellular localization experiment

(1) Using the liquid nitrogen freeze-thaw method, the subcellular localization vector pAtREM1.3-RFP and the GFP-labeled PD localization vector pSrMV-P3N-PIPO-GFP were transferred into competent Agrobacterium strain EHA105; cultured in 5 mL LB The transformed Agrobacterium was cultured in medium (containing Rif, 34 μg/mL; Kan, 50 μg/mL), and cultured overnight at 28 °C and 200 rpm;

(2) Centrifuge at 5,000 rpm for 5 minutes at room temperature, discard the supernatant, and add 1 mL of infection solution to resuspend the bacteria;

(3) Repeat step (2) to wash away the antibiotics contained in the culture medium;

(4) Add 1 mL of infection solution, measure the OD ₆₀₀ of the bacterial solution, and adjust the OD ₆₀₀ value to 0.1;

(5) Take 750 μL of bacterial liquid in a 2 mL sterile centrifuge tube, mix well, and place it for 3-5 hours; mix the bacterial liquid containing the subcellular localization carrier pAtREM1.3-GFP and the bacterial liquid containing the YFP-labeled carrier in equal proportions, Conduct infection experiments;

(6) Get healthy Nicotiana benthamiana plant (before infecting, light treatment tobacco 1h), select two large blades, inject bacterial solution on the back side of tobacco blade (between two leaf veins) and make a mark;

(7) Culture the infected tobacco under normal conditions. After 2 days, 1-2 cm ² of infected leaves were taken, with the back facing up, and observed with a laser confocal microscope. When collecting fluorescent signals, collect different fluorescent signals for the same cell. The pAtREM1.3-RFP to be detected is labeled with red fluorescent protein. When the red fluorescent protein signal is collected, a bright spot will appear at the position indicated by the arrow, indicating that AtREM1.3 is expressed on the cell membrane, but it cannot be determined that it is located in PD; this reagent The PD positioning vector pSrMV-P3N-PIPO-GFP provided in the box is marked with green fluorescent protein. When the green fluorescent signal is collected, bright spots will also appear, indicating that the location of the bright spots is plasmodesmata; when the two pictures are merged, red Accurate superimposition of the bright spot and the green bright spot is displayed as a yellow bright spot, indicating that AtREM1.3 is located in the plasmodesmata, as shown in FIG. 6 .

(8) According to the above procedures and methods, the GFP vector pSAT6-EGFP-C1 and the RFP vector pSAT6-ERFP-C1 were expressed in the epidermal cells of Nicotiana benthamiana leaves, and the fluorescent signals were detected under a confocal laser microscope. The green fluorescent signal is in a diffuse state (Figure 7), and the red fluorescent signal is in a diffuse state (Figure 8), indicating that there is no specific subcellular localization of green fluorescent protein and red fluorescent protein, and the subcellular localization of the target protein is not caused by green fluorescent protein or red fluorescent protein , thereby demonstrating that AtREM1.3 can localize to plasmodesmata.

The agarose gel electrophoresis refers to the method of agarose gel electrophoresis in the first section of Chapter 6 of "Molecular Cloning Experiment Guide" (second edition); the connection product is transformed into competent Escherichia coli DH5α, transformed Methods refer to the method for preparing and transforming competent Escherichia coli with calcium chloride in the fifth section of the first chapter of "Molecular Cloning Experiment Guide" (Second Edition); The identification method of the bacterial colony containing the recombinant plasmid in the sixth section of the first chapter of the "Guide" (Second Edition); the method of extracting plasmid DNA, refer to the instruction manual of the plasmid extraction kit; For the instructions of the enzyme; for the recovery method, refer to the instructions of the gel recovery kit; for the ligation method with T4-DNA ligase, refer to the T4-DNA ligase operation instructions.

Claims (2)

1. A kind of 1. preparation method of the PD positioning carriers of fluorescent protein labeling, it is characterised in that：

   (1) for coded sequence design the specific PCR primers SrMV-P3-F and SrMV-P3-R of SrMV-P3, with the cDNA of SrMV For template, PCR amplification is carried out, PCR product is separated and recovered from by agarose gel electrophoresis, obtains the volume of SrMV-P3 Code sequence；The nucleotides sequence of the SrMV-P3-F is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 1；SrMV- The nucleotides sequence of P3-R is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 2；The nucleotide of SrMV-P3 coded sequences Sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 3；

   (2) specific PCR primers SrMV-P3N-F and SrMV-P3N-R are designed, restriction enzyme site Xho I are introduced in SrMV-P3N-F； Using SrMV-P3 coded sequences as template, PCR amplification is carried out using primer SrMV-P3N-F and SrMV-P3N-R, PCR reactions terminate Afterwards, PCR product is separated by agarose gel electrophoresis, recycles and be concentrated into 400ng/ μ L, obtain the volume of SrMV-P3N Code sequence；The nucleotides sequence of the SrMV-P3N-F is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 4；SrMV- The nucleotides sequence of P3N-R is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 5；The nucleosides of SrMV-P3N coded sequences Acid sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 6；

   (3) acquisition of SrMV-PIPO coded sequences：Specific PCR primers SrMV-PIPO-F and SrMV-PIPO-R are designed, Restriction enzyme site Hind III are introduced in SrMV-P3N-R；Using SrMV-P3 coded sequences as template, primer SrMV-PIPO-F is used PCR amplification is carried out with SrMV-PIPO-R, PCR is separated by agarose gel electrophoresis after reaction, by PCR product, is returned Receive and be concentrated into 400ng/ μ L, obtain the coded sequence of SrMV-PIPO；The nucleotides sequence of the SrMV-PIPO-F is classified as sequence SEQ ID NO in table：Nucleotide sequence shown in 7；The nucleotides sequence of SrMV-PIPO-R is classified as SEQ ID NO in sequence table：8 Shown nucleotide sequence；The nucleotides sequence of SrMV-PIPO coded sequences is classified as SEQ ID NO in sequence table：Nucleosides shown in 9 Acid sequence；

   (4) acquisition of SrMV-P3N-PIPO coded sequences：Be by concentration the P3N and PIPO of 400ng/ μ L PCR product according to 1 ︰ 1 mixing is used as template, and using primer SrMV-P3N-F and SrMV-PIPO-R, SrMV- is cloned using fusion DNA vaccine method P3NPIPO coded sequences；PCR is separated and recovered from by agarose gel electrophoresis after reaction, by PCR product, is obtained The coded sequence of SrMV-P3N-PIPO；The nucleotides sequence of the SrMV-P3N-PIPO coded sequences is classified as SEQ ID in sequence table NO：Nucleotide sequence shown in 10；

   (5) acquisition of Insert Fragment S：Double digestion is carried out to SrMV-P3N-PIPO coded sequences using Xho I and Hind III, Digestion products are separated and recovered from by agarose gel electrophoresis, obtain Insert Fragment S；The nucleosides of the Insert Fragment S Acid sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 11；

   (6) acquisition of the PD positioning carriers of fluorescent protein labeling：Using Xho I and Hind III respectively to carrier pSAT6- EGFP-C1, pSAT6-ERFP-C1, pSAT6-EYFP-C1 and pSAT6-ECFP-C1 carry out double digestion, and digestion products are passed through fine jade Sepharose electrophoresis is separated and recovered from；Then digestion products are connected with Insert Fragment S with T4DNA ligases, and will be even Thing of practicing midwifery is converted into competence bacillus coli DH 5 alpha, and picking positive colony verification, the fluorescent protein labeling PD obtained respectively determines Position carrier pSrMV-P3N-PIPO-GFP, pSrMV-P3N-PIPO-RFP, pSrMV-P3N-PIPO-YFP, pSrMV-P3N- PIPO-CFP；The nucleotides sequence of the Insert Fragment S is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 11.
2. A kind of 2. subcellular fraction of the PD positioning carriers comprising the fluorescent protein labeling being prepared using claim 1 the method Location reagent box, it is characterised in that the kit is made of following reagent：

   (1) the PD positioning carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (2) the PD positioning carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (3) the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein mark, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (4) the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein mark, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (5) carrier pSAT6-EGFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (6) carrier pSAT6-ERFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (7) carrier pSAT6-EYFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (8) carrier pSAT6-ECFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；

   (9) restriction enzyme Xho I：1 pipe, 500units, 100 μ L/ pipe；

   (10) restriction enzyme Hind III：1 pipe, 500units, 100 μ L/ pipe；

   (11) without RNase water：2 bottles, 100mL/ bottles；

   (12) liquid is infected：1 pipe, 50mL/ pipes, -20 DEG C of freezen protectives are spare.