Yeast expression vector system for bimolecular fluorescence complementation experiment and application
By using a specific expression vector system in yeast, the problem of instability of protein expression in BiFC experiments in plant cells was solved, and stable protein expression and efficient protein interaction detection were achieved.
Patent Information
- Application Number
- CN202510108066.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-05-06
AI Technical Summary
In plant cells, especially mature tissues, gene conversion efficiency is low, resulting in unstable or insufficient expression levels of fusion proteins in BiFC experiments, affecting the intensity of the signal and the reliability of the results.
A yeast expression vector system is provided, including expression vector 1 and expression vector 2, vector 1 contains the encoding gene of the C-terminal residue of the fluorescent protein and the ADH1 promoter, and vector 2 contains the encoding gene of the N-terminal residue of the fluorescent protein and the ADH1 promoter. The protein is expressed in yeast through these vector systems and is used for bimolecular fluorescent complementary experiments.
This yeast expression vector system achieves stable protein expression in yeast, improves the signal strength and reliability of BiFC experiments, and is suitable for high-throughput screening and protein interaction verification.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a yeast expression vector system and application thereof for a bimolecular fluorescence complementation experiment. Background Art
[0002] Bimolecular Fluorescent Complementation (BiFC) technology is based on the principle of protein fragment complementation and is used to study protein-protein interactions. Plant cells have strong cell walls, which makes plasmid transfer and fusion protein expression more difficult; at the same time, the presence of cell walls may interfere with the detection and positioning of fluorescent signals. In plants, especially mature tissues, gene transformation efficiency is low, resulting in unstable or insufficient expression levels of fusion proteins in BiFC experiments, affecting signal intensity and reliability of results. The Agrobacterium infiltration method commonly used for transient expression is limited in efficiency and takes a long time to express proteins. Yeast bimolecular fluorescence complementation technology combines the advantages of yeast genetics and live cell fluorescence imaging, providing an economical, efficient and stable method for protein interaction research, especially for high-throughput screening and interaction verification. Its advantages make it an important tool in the study of protein function and interaction networks. Summary of the invention
[0003] The purpose of the present invention is to provide a vector system for expressing proteins in yeast and for bimolecular fluorescence complementation experiments and its application. The technical problems to be solved are not limited to the technical themes described, and those skilled in the art can clearly understand other technical themes not mentioned in this article through the following description.
[0004] To achieve the above-mentioned object, the present invention first provides a yeast expression vector system, which comprises an expression vector 1 and an expression vector 2, wherein the expression vector 1 contains a gene encoding a C-terminal residue of a fluorescent protein and a promoter operably connected thereto, and the expression vector 2 contains a gene encoding a N-terminal residue of a fluorescent protein and a promoter operably connected thereto, and the promoter may be a yeast ADH1 promoter.
[0005] The fluorescent protein N-terminal residue and the fluorescent protein C-terminal residue can form a complete fluorescent protein.
[0006] Furthermore, the nucleotide sequence of the promoter may be as shown in SEQ ID NO:1.
[0007] Furthermore, the expression vector 1 may be a recombinant expression vector obtained by cloning the promoter and the gene encoding the C-terminal residue of the fluorescent protein described in this article into the pNH603 vector; the expression vector 2 may be a recombinant expression vector obtained by cloning the promoter and the gene encoding the N-terminal residue of the fluorescent protein described in this article into the pNH605 vector.
[0008] The fluorescent protein includes yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP) and blue fluorescent protein (BFP) but is not limited thereto.
[0009] Furthermore, the fluorescent protein may be yellow fluorescent protein (YFP).
[0010] Furthermore, the nucleotide sequence of the gene encoding the C-terminal residue of the fluorescent protein may be as shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the N-terminal residue of the fluorescent protein may be as shown in SEQ ID NO:3.
[0011] Furthermore, the nucleotide sequence of the expression vector 1 may be as shown in SEQ ID NO:10, and the nucleotide sequence of the expression vector 2 may be as shown in SEQ ID NO:11.
[0012] The present invention also provides an application of any yeast expression vector system described herein, wherein the application can be used to detect protein interaction in a bimolecular fluorescence complementation experiment.
[0013] The present invention also provides a method for preparing a yeast expression vector system, which may include the following steps:
[0014] A1) cloning the gene encoding the C-terminal residue of yellow fluorescent protein and the promoter driving its expression into the pNH603 vector to obtain expression vector 1;
[0015] A2) cloning the gene encoding the N-terminal residue of yellow fluorescent protein and the promoter driving its expression into the pNH605 vector to obtain expression vector 2;
[0016] The promoter may be the promoter described herein (yeast ADH1 promoter, such as the promoter shown in SEQ ID NO: 1).
[0017] The promoter and the coding gene driven by it can be cloned into the vector successively, or first connected in series and then cloned into the vector.
[0018] In the above method, the nucleotide sequence of the gene encoding the C-terminal residue of the yellow fluorescent protein may be shown as SEQ ID NO:2; the nucleotide sequence of the gene encoding the N-terminal residue of the yellow fluorescent protein may be shown as SEQ ID NO:3.
[0019] Furthermore, the method for preparing the yeast expression vector system may include the following steps:
[0020] (1) cloning the yeast ADH1 promoter (e.g., the DNA molecule shown in SEQ ID NO: 1) into the pNH603 vector between the PspOMI and XhoI restriction sites to obtain the intermediate vector pNH603-ADH1pr;
[0021] (2) cloning the gene encoding the C-terminal residue of yellow fluorescent protein (e.g., the DNA molecule shown in SEQ ID NO: 2) into the intermediate vector pNH603-ADH1pr between the BamHI and NotI restriction sites to obtain the pNH603-ADH1pr-CYFP vector, which is the expression vector 1;
[0022] (3) cloning the yeast ADH1 promoter (e.g., the DNA molecule shown in SEQ ID NO: 1) into the pNH605 vector between the PspOMI and XhoI restriction sites to obtain the intermediate vector pNH605-ADH1pr;
[0023] (4) cloning the gene encoding the N-terminal residue of yellow fluorescent protein (e.g., the DNA molecule shown in SEQ ID NO: 3) into the intermediate vector pNH605-ADH1pr between the BamHI and NotI restriction sites to obtain the pNH605-ADH1pr-NYFP vector, which is the expression vector 2;
[0024] The expression vector 1 and the expression vector 2 constitute the yeast expression vector system of the present invention.
[0025] Furthermore, the gene encoding the C-terminal residue of the yellow fluorescent protein, the gene encoding the N-terminal residue of the yellow fluorescent protein, and the promoter can be cloned into a corresponding vector by homologous recombination.
[0026] Furthermore, the homologous recombination method can be achieved by adding vector homology arms to both ends of the DNA molecule to be cloned into the vector.
[0027] The present invention also provides a method for detecting protein interaction based on a bimolecular fluorescence complementation experiment, the method comprising detecting a pair of proteins to be detected (A and B) using any of the yeast expression vector systems described herein.
[0028] Furthermore, the method may include the following steps:
[0029] (1) cloning the coding gene of the protein A to be tested into the upstream or downstream of the coding gene of the C-terminal residue of the yellow fluorescent protein in the expression vector 1 (such as the pNH603-ADH1pr-CYFP vector), and fusion expressing with it to obtain a yeast expression vector in which the protein A to be tested is fused with the C-terminal residue of the yellow fluorescent protein;
[0030] (2) cloning the coding gene of the protein B to be tested into the upstream or downstream of the coding gene of the N-terminal residue of the yellow fluorescent protein in the expression vector 2 (such as the pNH605-ADH1pr-NYFP vector), and fusion expressing with it to obtain a yeast expression vector in which the protein B to be tested is fused with the N-terminal residue of the yellow fluorescent protein;
[0031] (3) co-transforming the two yeast expression vectors obtained in steps (1) and (2) into yeast;
[0032] (4) Detect whether fluorescence appears.
[0033] Furthermore, the appearance of fluorescence indicates that the test protein A and the test protein B interact with each other.
[0034] Furthermore, between step (2) and step (3), a step of linearizing the two yeast expression vectors obtained in steps (1) and (2) may be included.
[0035] Furthermore, linearization can be performed using PmeI endonuclease.
[0036] Furthermore, the linearized enzyme digestion system can be: 1 μg of vector (plasmid), 1 μL of endonuclease 10×Buffer, 0.5 μL of restriction endonuclease PmeI (10U / μL), and double distilled water to make up to 10 μL.
[0037] Furthermore, in step (1), the coding gene of the protein A to be tested can be cloned upstream of the coding gene of the C-terminal residue of the yellow fluorescent protein in the expression vector 1 after removing the stop codon, and expressed in fusion with the gene.
[0038] Furthermore, in step (2), the coding gene of the protein B to be tested can be cloned upstream of the coding gene of the N-terminal residue of the yellow fluorescent protein in the expression vector 2 after removing the stop codon, and expressed in fusion with the gene.
[0039] Furthermore, in step (1), the coding gene of the protein A to be tested can be cloned into the expression vector 1 (pNH603-ADH1pr-CYFP vector) between the XhoI and XmaI restriction sites after removing the stop codon.
[0040] Furthermore, in step (2), the coding gene of the protein B to be tested can be cloned into the expression vector 2 (pNH605-ADH1pr-NYFP vector) between the XhoI and XmaI restriction sites after removing the stop codon.
[0041] The yeast expression vector system of the present invention can be a yeast expression vector combination or a yeast expression vector composition.
[0042] The term "operably linked" in the present invention generally refers to the physical and / or functional connection of a DNA segment to another DNA segment in a manner that allows the segment to function in its intended manner. DNA encoding a gene product is operably linked to a regulatory element, which can directly or indirectly regulate the transcription of the DNA. For example, when a promoter is operably linked to DNA encoding a gene product, it can initiate (drive) DNA transcription.
[0043] The term "fusion expression" in the present invention generally refers to splicing two or more protein or polypeptide coding genes together and expressing them as a new open reading frame.
[0044] Bimolecular fluorescence complementation (BiFC) is a technique for studying protein interactions. In this experiment, a fluorescent protein is divided into two fragments (N-fragment and C-fragment) without fluorescent activity, and fused with two target proteins (A and B) respectively. When the target proteins A and B interact with each other, the two fluorescent protein fragments will be close to each other in space, and then reassembled into a complete fluorescent protein with fluorescent activity. Under the excitation light of a specific wavelength, the complete fluorescent protein will emit a fluorescent signal, thereby indicating the interaction between A and B. On the contrary, if there is no interaction between A and B, the fragments of the fluorescent protein cannot be renatured, so no fluorescent signal is generated. The present invention transforms yeast expression vectors pNH603 and pNH605, and finally obtains a fluorescent protein C-terminal residue expression vector pNH603-ADH1pr-CYFP driven by a yeast ADH1 promoter and a fluorescent protein N-terminal residue expression vector pNH605-ADH1pr-NYFP driven by a yeast ADH1 promoter. The target proteins A and B are constructed into pNH603-ADH1pr-CYFP and pNH605-ADH1pr-NYFP, respectively, and finally the pNH603-ADH1pr-A-CYFP and pNH605-ADH1pr-B-NYFP vectors are obtained. After linearization, the pNH603-ADH1pr-A-CYFP and pNH605-ADH1pr-B-NYFP vectors are co-transformed into the recipient yeast strain W303a, and the interaction between the target proteins A and B is determined by observing the fluorescence signal. The experimental results show that the yeast expression vector system (pNH603-ADH1pr-CYFP vector and pNH605-ADH1pr-NYFP vector) designed and constructed by the present invention can be well applied to the protein interaction detection of the bimolecular fluorescence complementation experiment (BiFC). The present invention provides a vector tool for realizing efficient detection of interacting gene pairs, and utilizes the characteristics of fast yeast cell reproduction, good cell repeatability, high conversion efficiency, and low cost, so that the candidate proteins can be verified for interaction in the cell body. By adopting the technical solution provided by the present invention, the interaction verification of the proteins to be tested in vivo can be completed efficiently. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 This is the pNH603 vector map.
[0046] Figure 2 This is the pNH605 vector map.
[0047] Figure 3 This is the pNH603-ADH1pr vector map.
[0048] Figure 4 This is the map of the pNH603-ADH1pr-CYFP vector.
[0049] Figure 5 This is the pNH605-ADH1pr vector map.
[0050] Figure 6 This is the map of the pNH605-ADH1pr-NYFP vector.
[0051] Figure 7 This is a diagram showing the interaction between Arabidopsis HSFB1 and RPL11. Figure 7 A in the middle is a yeast two-hybrid experiment to verify the interaction between Arabidopsis HSFB1 and RPL11. SD / -Leu-Trp represents yeast culture medium lacking leucine and tryptophan, and SD / -Leu-Trp-His represents yeast culture medium lacking leucine, tryptophan, and histidine; Figure 7 Middle B is a luciferase protein complementation assay (LCA) that proves that HSFB1 interacts with RPL11 in Nicotiana benthamiana leaves; Figure 7 C shows the bimolecular fluorescence complementation (BiFC) assay that demonstrates the interaction between HSFB1 and RPL11 in Nicotiana benthamiana leaves. Scale bar, 50 μm.
[0052] Figure 8 This is the result diagram of the interaction between wheat TaHSFB1 and TaMYB4. Figure 8 A in the middle is a yeast two-hybrid experiment to verify the interaction between wheat TaHSFB1 and TaMYB4. SD / -Leu-Trp represents the yeast culture medium lacking leucine and tryptophan, and SD / -Leu-Trp-His-Ade represents the yeast culture medium lacking leucine, tryptophan, histidine and adenine; Figure 8 Middle B is a luciferase protein complementation experiment (LCA) demonstrating that TaHSFB1 interacts with TaMYB4 in Nicotiana benthamiana leaves; Figure 8 Middle C is a bimolecular fluorescence complementation experiment (BiFC) demonstrating that TaHSFB1 and TaMYB4 interact in wheat leaf protoplasts.
[0053] Fig. 9 This is the map of the pNH603-ADH1pr-AtHSFB1-CYFP vector.
[0054] Fig.10 This is the map of the pNH605-ADH1pr-AteIF3G1-NYFP vector.
[0055] Fig.11 This is the map of the pNH605-ADH1pr-AtRPL11-NYFP vector.
[0056] Fig.12This is the map of the pNH603-ADH1pr-TaHSFB1-CYFP vector.
[0057] Fig.13 This is the map of the pNH605-ADH1pr-TaWRKY70-NYFP vector.
[0058] Fig.14 This is the map of the pNH605-ADH1pr-TaMYB4-NYFP vector.
[0059] Fig.15 The protein interaction results of pNH603-ADH1pr-CYFP vector and pNH605-ADH1pr-NYFP vector were verified in bimolecular fluorescence complementation (BiFC) experiment. DETAILED DESCRIPTION
[0060] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0061] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0062] The pNH603 vector map in the following examples is as follows Figure 1 As shown; pNH605 vector map as Figure 2 The pNH603 vector and the pNH605 vector are described in the following literature: Fordyce PM, Pincus D, Kimmig P, Nelson CS, El-Samad H, Walter P, DeRisi JL. Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses. Proc Natl Acad Sci US A. 2012, 109(45): E3084-E3093. The public can obtain them from the applicant. The biological materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
[0063] The pCYFP vector and pNYFP vector in the following embodiments are recorded in the following literature: Gao J, Zhang R, Zheng L, Song L, Ji M, Li S, Wang J, Yang J, Kang G, Zhang P, Shi Y, Jiao Y, Pincus D, Zheng X. Blue light receptor CRY1 regulates HSFA1d nuclear localization topromote plant thermotolerance. Cell Rep 2023, 42(9): 113117. The public can obtain it from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
[0064] Example 1. Construction of pNH603-ADH1pr-CYFP vector
[0065] Using the genomic DNA of wild-type Saccharomyces cerevisiae strain W303a as a template, the ADH1 promoter sequence (SEQ ID NO: 1) was PCR amplified to obtain an amplified product, which contained the ADH1 promoter (SEQ ID NO: 1) and had a homology arm and restriction enzyme sites of the pNH603 vector at both ends of the ADH1 promoter. The amplified product was ligated between the PspOMI and XhoI restriction enzyme sites of the pNH603 vector to obtain the pNH603-ADH1pr vector (the map of the intermediate vector is shown in Figure 3 shown).
[0066] The primer sequences used to amplify the ADH1 promoter are as follows:
[0067] Upstream primer pNH603-ADH1pr-F: 5′-TCGGCTTTGGGTACCgggcccATCCTTTTGTTGTTTCCGGG-3′;
[0068] Downstream primer pNH603-ADH1pr-R: 5′-CACCTGCCTTGCTCCctcgagAGTTGATTGTATGCTTGGTA-3′.
[0069] The bold sequence is the homologous sequence of the pNH603 vector after restriction endonuclease digestion; the lowercase sequence is the restriction enzyme site sequence.
[0070] The C-terminal residue of the fluorescent protein YFP (CYFP fragment, sequence SEQ ID NO: 2) was amplified from the pCYFP vector to obtain a CYFP fragment amplification product, which contained the CYFP fragment (SEQ ID NO: 2) and had a homology arm and restriction enzyme cutting site of the pNH603-ADH1pr vector at both ends of the CYFP fragment. The amplification product was ligated between the BamHI and NotI restriction enzyme cutting sites of the pNH603-ADH1pr vector to obtain the pNH603-ADH1pr-CYFP vector.
[0071] The primer sequences used to amplify the CYFP fragment are as follows:
[0072] Upstream primer pNH603-ADH1pr-CYFP-F: 5′-TTCCTGCAGCCCGGGggatccGACAAGCAGAAGAACGGCAT-3′;
[0073] Downstream primer pNH603-ADH1pr-CYFP-R: 5′-CTCCACCGCGGTGgcggccgcTTACTTGTACAGCTCGTCCA-3′.
[0074] The bold sequence is the homologous sequence of the pNH603-ADH1pr vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0075] The pNH603-ADH1pr-CYFP vector constructed above is a recombinant expression vector obtained by cloning the ADH1 promoter sequence (SEQ ID NO: 1) and the CYFP fragment (SEQ ID NO: 2) into the pNH603 vector. The ADH1 promoter is placed at the N-terminus of the CYFP fragment to drive the expression of the CYFP fragment. The map of the pNH603-ADH1pr-CYFP vector is shown in FIG. Figure 4 The nucleotide sequence of the pNH603-ADH1pr-CYFP vector is SEQ ID NO:10.
[0076] Example 2, pNH605-ADH1pr-NYFP vector construction
[0077] Using the wild-type Saccharomyces cerevisiae strain W303a genomic DNA as a template, the ADH1 promoter sequence (SEQ ID NO: 1) was PCR amplified to obtain an amplified product, which contained the ADH1 promoter (SEQ ID NO: 1) and had a homology arm and restriction enzyme sites of the pNH605 vector at both ends of the ADH1 promoter. The amplified product was ligated between the PspOMI and XhoI restriction enzyme sites of the pNH605 vector to obtain the pNH605-ADH1pr vector (the map of the intermediate vector is shown in Figure 5 shown).
[0078] The primer sequences used to amplify the ADH1 promoter are as follows:
[0079] Upstream primer pNH605-ADH1pr-F: 5′-GAAACGAACGGTACCgggcccATCCTTTTGTTGTTTCCGGG-3′;
[0080] Downstream primer pNH605-ADH1pr-R: 5′-CACCTGCCTTGCTCCctcgagAGTTGATTGTATGCTTGGTA-3′.
[0081] The bold sequence is the homologous sequence of the pNH605 vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme site sequence.
[0082] The N-terminal residue of the fluorescent protein YFP (NYFP fragment, sequence SEQ ID NO: 3) was amplified from the pNYFP vector to obtain a NYFP fragment amplification product, which contained the NYFP fragment (SEQ ID NO: 3) and had a homology arm and restriction enzyme cutting site of the pNH605-ADH1pr vector at both ends of the NYFP fragment. The amplification product was ligated between the BamHI and NotI restriction enzyme cutting sites of the pNH605-ADH1pr vector to obtain the pNH605-ADH1pr-NYFP vector.
[0083] The primer sequences used to amplify the NYFP fragment are as follows:
[0084] Upstream primer pNH605-ADH1pr-NYFP-F: 5′-TTCCTGCAGCCCGGGggatccATGGTGAGCAAGGGCGAGGA-3′;
[0085] Downstream primer pNH605-ADH1pr-NYFP-R: 5′-AGCTCCACCGCGGTGgcggccgcTTAGGCCATGATATAGACGT-3′.
[0086] The bold sequence is the homologous sequence of the pNH605-ADH1pr vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0087] The pNH605-ADH1pr-NYFP vector constructed above is a recombinant expression vector obtained by cloning the ADH1 promoter sequence (SEQ ID NO: 1) and the NYFP fragment (SEQ ID NO: 3) into the pNH605 vector. The ADH1 promoter is placed at the N-terminus of the NYFP fragment to drive the expression of the NYFP fragment. The map of the pNH605-ADH1pr-NYFP vector is shown in FIG. Figure 6 The nucleotide sequence of pNH605-ADH1pr-NYFP vector is SEQ ID NO:11.
[0088] Example 3: Application of pNH603-ADH1pr-CYFP vector and pNH605-ADH1pr-NYFP vector
[0089] This example verifies the application of the yeast expression vector system (pNH603-ADH1pr-CYFP vector and pNH605-ADH1pr-NYFP vector) in the bimolecular fluorescence complementation experiment (BiFC), as follows:
[0090] 1. Construction of target vector
[0091] In order to verify the effectiveness of the yeast expression vector system, the system was used to verify the proteins that have been identified to interact. The protein constructed into the pNH603-ADH1pr-CYFP vector is protein A, and the protein constructed into the pNH605-ADH1pr-NYFP vector is protein B. Among them: (1) A protein AtHSFB1 interacts with B protein AteIF3G1 (Zhang Qianlong. Molecular mechanism of transcription factor HSFB1 balancing plant growth and drought resistance [D]. Henan Agricultural University, 2024. DOI: 10.27117 / d.cnki.ghenu.2024.000431); (2) A protein AtHSFB1 interacts with B protein AtRPL11 ( Figure 7 ); (3) A protein TaHSFB1 interacts with B protein TaWRKY70 (Zhang Qianlong. Molecular mechanism of transcription factor HSFB1 balancing plant growth and drought resistance [D]. Henan Agricultural University, 2024. DOI: 10.27117 / d.cnki.ghenu.2024.000431); (4) A protein TaHSFB1 interacts with B protein TaMYB4 ( Figure 8 ).
[0092] The coding sequence (CDS) of the gene encoding the AtHSFB1 protein (AtHSFB1 gene) is shown in SEQ ID NO:4.
[0093] The coding sequence (CDS) of the gene encoding the AtEIF3G1 protein (AtEIF3G1 gene) is shown in SEQ ID NO:5.
[0094] The coding sequence (CDS) of the gene encoding the AtRPL11 protein (AtRPL11 gene) is shown in SEQ ID NO:6.
[0095] The coding sequence (CDS) of the gene encoding the TaHSFB1 protein (TaHSFB1 gene) is shown in SEQ ID NO:7.
[0096] The coding sequence (CDS) of the gene encoding TaWRKY70 protein (TaWRKY70 gene) is shown in SEQ ID NO:8.
[0097] The coding sequence (CDS) of the gene encoding TaMYB4 protein (TaMYB4 gene) is shown in SEQ ID NO:9.
[0098] 1-1. Construction of yeast expression vector for fusion of AtHSFB1 (A protein) and CYFP protein
[0099] Arabidopsis thaliana (Columnbia, Col-0) RNA was extracted and reverse transcribed into cDNA. The CDS sequences of AtHSFB1, AtEIF3G1 and AtRPL11 were amplified using Arabidopsis thaliana cDNA as template.
[0100] After removing the stop codon, the AtHSFB1 gene (SEQ ID NO: 4) was cloned into the pNH603-ADH1pr-CYFP vector between the XhoI and XmaI restriction sites by homologous recombination to obtain the yeast expression vector pNH603-ADH1pr-AtHSFB1-CYFP (vector map as shown in Fig. 9 The primer sequences used to amplify the CDS of the AtHSFB1 gene are as follows:
[0101] Upstream primer pNH603-ADH1pr-AtHSFB1-CYFP-F: 5′-AGCATACAATCAACTctcgagATGACGGCTGTGACGGCGGC-3′;
[0102] Downstream primer pNH603-ADH1pr-AtHSFB1-CYFP-R: 5′-CTGCTTGTCGGATCCcccgggGTTGCAGACTTTGCTGCTTT-3′.
[0103] The bold sequence is the homologous sequence of the pNH603-ADH1pr-CYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0104] 1-2. Construction of yeast expression vector for AtEIF3G1 (B protein) and NYFP protein fusion
[0105] After the stop codon was removed from the AtEIF3G1 gene (SEQ ID NO: 5), the AtEIF3G1 gene (SEQ ID NO: 5) was cloned into the pNH605-ADH1pr-NYFP vector between the XhoI and XmaI restriction sites by homologous recombination to obtain the yeast expression vector pNH605-ADH1pr-AteIF3G1-NYFP containing the fusion protein of AtEIF3G1 and NYFP (see vector map for details). Fig.10 The primer sequences used to amplify the CDS of the AtEIF3G1 gene are as follows:
[0106] Upstream primer pNH605-ADH1pr-AteIF3G1-NYFP-F: 5′-AGCATACAATCAACTctcgagATGACGATCGATTCGCAGCA-3′;
[0107] Downstream primer pNH605-ADH1pr-AteIF3G1-NYFP-R: 5′-GCTCACCATGGATCCcccgggGGTTGGTCTTGGAGTTGCCC-3′.
[0108] The bold sequence is the homologous sequence of the pNH605-ADH1pr-NYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0109] 1-3. Construction of yeast expression vector for AtRPL11 (B protein) and NYFP protein fusion
[0110] After removing the stop codon, the AtRPL11 gene (SEQ ID NO: 6) was cloned into the pNH605-ADH1pr-NYFP vector between the XhoI and XmaI restriction sites by homologous recombination to obtain the yeast expression vector pNH605-ADH1pr-AtRPL11-NYFP fused with AtRPL11 and NYFP proteins (vector map as shown in Fig.11 The primer sequences used to amplify the CDS of the AtRPL11 gene are as follows:
[0111] Upstream primer pNH605-ADH1pr-AtRPL11-NYFP-F: 5′-AGCATACAATCAACTctcgagATGGCGTCTTCTTCTCTATC-3′;
[0112] Downstream primer pNH605-ADH1pr-AtRPL11-NYFP-R: 5′-GCTCACCATGGATCCcccgggCAATAAAACTGCTTTCTTTT-3′.
[0113] The bold sequence is the homologous sequence of the pNH605-ADH1pr-NYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0114] 1-4. Construction of yeast expression vector of TaHSFB1 (A protein) and CYFP protein fusion
[0115] RNA was extracted from wheat variety Chinese Spring and reverse transcribed into cDNA. CDS sequences of TaHSFB1, TaWRKY70 and TaMYB4 were amplified using wheat cDNA as template.
[0116] The TaHSFB1 gene (SEQ ID NO: 7) was cloned into the pNH603-ADH1pr-CYFP vector between the XhoI and XmaI restriction sites by homologous recombination after the termination codon was removed to obtain the yeast expression vector pNH603-ADH1pr-TaHSFB1-CYFP (vector map as shown in Fig.12 The primer sequences used to amplify the CDS of the TaHSFB1 gene are as follows:
[0117] Upstream primer pNH603-ADH1pr-TaHSFB1-CYFP-F: 5′-AGCATACAATCAACTctcgagATGGCCGGGGCGGCGGCGCA-3′;
[0118] Downstream primer pNH603-ADH1pr-TaHSFB1-CYFP-R: 5′-CTGCTTGTCGGATCCcccgggGTTCCCGCCGCCGCAGCGCG-3′.
[0119] The bold sequence is the homologous sequence of the pNH603-ADH1pr-CYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0120] 1-5. Construction of yeast expression vector of TaWRKY70 (B protein) and NYFP protein fusion
[0121] The TaWRKY70 gene (SEQ ID NO: 8) was cloned into the pNH605-ADH1pr-NYFP vector between the XhoI and XmaI restriction sites by homologous recombination after the stop codon was removed to obtain the yeast expression vector pNH605-ADH1pr-TaWRKY70-NYFP containing the fusion of TaWRKY70 and NYFP proteins (see vector map). Fig.13 The primer sequences used to amplify the CDS of TaWRKY70 gene are as follows:
[0122] Upstream primer pNH605-ADH1pr-TaWRKY70-NYFP-F: 5′-AGCATACAATCAACTctcgagATGATAGTATATAGCACGAA-3′;
[0123] Downstream primer pNH605-ADH1pr-TaWRKY70-NYFP-R: 5′-GCTCACCATGGATCCcccgggATGATCGAGGACGTAGGACA-3′.
[0124] The bold sequence is the homologous sequence of the pNH605-ADH1pr-NYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0125] 1-6. Construction of yeast expression vector of TaMYB4 (B protein) and NYFP protein fusion
[0126] The TaMYB4 gene (SEQ ID NO: 9) was cloned into the pNH605-ADH1pr-NYFP vector between the XhoI and XmaI restriction sites by homologous recombination after the termination codon was removed to obtain the yeast expression vector pNH605-ADH1pr-TaMYB4-NYFP fused with TaMYB4 and NYFP proteins (vector map as shown in Fig.14 The primer sequences used to amplify the CDS of TaMYB4 gene are as follows:
[0127] Upstream primer pNH605-ADH1pr-TaMYB4-NYFP-F: 5′-AGCATACAATCAACTctcgagATGGGGAGGTCCCCGTGCTG-3′;
[0128] Downstream primer pNH605-ADH1pr-TaMYB4-NYFP-R: 5′-GCTCACCATGGATCCcccgggTTGGCGTCCGCCCTCTAGCG-3′.
[0129] The bold sequence is the homologous sequence of the pNH605-ADH1pr-NYFP vector after digestion with restriction endonucleases; the lowercase sequence is the restriction enzyme cutting site sequence.
[0130] 2. Yeast transformation
[0131] The vector plasmid constructed in step 1 above was linearized with PmeI endonuclease. The enzyme digestion system is shown in Table 1.
[0132] Table 1. Linearization enzyme digestion system
[0133]
[0134] 10 μL of the digested products were co-transfected into the W303a yeast strain according to the following combinations.
[0135] Combination 1: pNH603-ADH1pr-AtHSFB1-CYFP / pNH605-ADH1pr-AteIF3G1-NYFP;
[0136] Combination 2: pNH603-ADH1pr-AtHSFB1-CYFP / pNH605-ADH1pr-AtRPL11-NYFP;
[0137] Combination 3: pNH603-ADH1pr-TaHSFB1-CYFP / pNH605-ADH1pr-TaWRKY70-NYFP;
[0138] Combination 4: pNH603-ADH1pr-TaHSFB1-CYFP / pNH605-ADH1pr-TaMYB4-NYFP;
[0139] Negative control: pNH603-ADH1pr-CYFP / pNH605-ADH1pr-NYFP.
[0140] The conversion steps are as follows:
[0141] 1) Streak the W303a strain on YPDA solid medium and culture it upside down in a constant temperature incubator at 30°C for about 3 days;
[0142] 2) Pick a single colony and place it in 3 mL YPDA liquid medium, culture overnight at 30°C and 220 rpm;
[0143] 3) Centrifuge at 700 × g for 5 min, discard the supernatant, resuspend the cells in 100 mL YPDA liquid medium, and culture at 30°C, 220 rpm until OD 600 is 0.5;
[0144] 4) Centrifuge at 700 × g for 5 min, discard the supernatant and resuspend in 60 mL of sterile deionized water;
[0145] 5) Centrifuge at 700×g for 5 min, discard the supernatant, resuspend the cells in 3 mL 1.1×TE / LiAc, and pre-denature the salmon sperm DNA twice;
[0146] 6) Centrifuge at 700 × g for 5 min, discard the supernatant, and resuspend the cells in 600 μL 1.1 × TE / LiAc;
[0147] 7) Take a 1.5 mL sterile centrifuge tube, add 600 μL 1.1×TE / LiAc / PEG4000 solution, 100 μL yeast competent cell, 10 μL pre-denatured Carrier DNA, and linearized product in sequence, mix by gentle pipetting, and heat shock in a 30°C water bath for 45 min (shake every 15 min);
[0148] 8) Add 20 μL DMSO, mix gently, and heat shock in a 42°C water bath for 20 min;
[0149] 9) Centrifuge at 700 × g for 5 min, resuspend the precipitate in 0.9% saline, apply to solid defective medium SD / -His-Leu lacking histidine and leucine, and culture at 30°C for about 3 days. Observe the growth of colonies and identify whether the recombinant vector has been successfully transferred into the yeast strain by bacterial liquid PCR.
[0150] 3. Microscopic observation
[0151] Pick the positive monoclonal clone into 300μL PBS buffer, centrifuge at 700×g for 30s, discard the supernatant and wash once with 300μL PBS. Finally, resuspend the bacteria in 100μL 1×PBS buffer. Apply 10μL of concanavalin A (ConA) evenly on the slide and cover slip, respectively. The working concentration of concanavalin A is 2.5mg / μL (dissolved in 1×PBS). Wash the slide and cover slip coated with concanavalin with double distilled water once and leave to dry at room temperature (about 5min). Pipette 10μL of bacterial solution, apply it to the slide, and observe it under a laser confocal microscope. The excitation wavelength is 488nm and the emission wavelength is 507nm. When fluorescence appears, it means that there is an interaction between the genes A and B to be tested. The results are as follows Fig.15As shown, luminescent cells can be observed in yeast transformed with the vector plasmids shown in combination 1, combination 2, combination 3 and combination 4, which indicates that the yeast expression vector system (pNH603-ADH1pr-CYFP vector and pNH605-ADH1pr-NYFP vector) designed and constructed by the present invention is well suited for protein interaction research in bimolecular fluorescence complementation experiment (BiFC).
[0152] In summary, the present invention provides a vector tool for realizing efficient detection of interacting gene pairs. After adopting the technical solution provided by the present invention, the interaction verification of the proteins to be tested in vivo can be efficiently completed.
[0153] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. A yeast expression vector system, characterized in that: The yeast expression vector system comprises expression vector 1 and expression vector 2, wherein expression vector 1 contains a gene encoding a fluorescent protein C-terminal residue and a promoter operably connected thereto, and expression vector 2 contains a gene encoding a fluorescent protein N-terminal residue and a promoter operably connected thereto, wherein the promoter is a yeast ADH1 promoter.
2. The yeast expression vector system according to claim 1, characterized in that The nucleotide sequence of the promoter is shown in SEQ ID NO:
1.
3. The yeast expression vector system according to claim 1 or 2, characterized in that: The expression vector 1 is a recombinant expression vector obtained by cloning the promoter described in claim 1 or 2 and the gene encoding the C-terminal residue of the fluorescent protein into the pNH603 vector; the expression vector 2 is a recombinant expression vector obtained by cloning the promoter described in claim 1 or 2 and the gene encoding the N-terminal residue of the fluorescent protein into the pNH605 vector.
4. The yeast expression vector system according to any one of claims 1 to 3, characterized in that: The fluorescent protein is yellow fluorescent protein.
5. The yeast expression vector system according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the gene encoding the C-terminal residue of the fluorescent protein is shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the N-terminal residue of the fluorescent protein is shown in SEQ ID NO:
3.
6. The yeast expression vector system according to any one of claims 1 to 5, characterized in that: The nucleotide sequence of the expression vector 1 is shown in SEQ ID NO:10, and the nucleotide sequence of the expression vector 2 is shown in SEQ ID NO:
11.
7. Use of the yeast expression vector system according to any one of claims 1 to 6, wherein the use is to detect protein interactions in a bimolecular fluorescence complementation experiment.
8. A method for preparing a yeast expression vector system, characterized in that: The method comprises the following steps: A1) cloning the gene encoding the C-terminal residue of yellow fluorescent protein and the promoter driving its expression into the pNH603 vector to obtain expression vector 1; A2) cloning the gene encoding the N-terminal residue of yellow fluorescent protein and the promoter driving its expression into the pNH605 vector to obtain expression vector 2; The promoter is the promoter described in claim 1 or 2.
9. The method according to claim 8, characterized in that The nucleotide sequence of the gene encoding the C-terminal residue of the yellow fluorescent protein is shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the N-terminal residue of the yellow fluorescent protein is shown in SEQ ID NO:
3.
10. A method for detecting protein interaction based on bimolecular fluorescence complementation experiment, characterized in that: The method comprises detecting a pair of proteins to be detected using the yeast expression vector system described in any one of claims 1 to 6.