Yeast double expression vector, construction method thereof, protein interaction identification method and application

Abstract

The invention relates to the technical field of protein interaction in yeast, in particular to a yeast double-expression vector, a construction method thereof, a protein interaction identification method and application. According to the vector, two exogenous genes can be cloned in the same vector, and the two exogenous genes can be expressed in yeast at the same time, so that interaction identification can be carried out. The work of plasmid construction and extraction, library preparation, yeast hybridization, screening culture and the like of an original yeast two-hybrid system is greatly reduced, and the identification efficiency of protein interaction is remarkably improved.

Description

Technical Field

[0001] The present application relates to the technical field of protein interaction in yeast, and specifically to a yeast dual expression vector and a construction method thereof, a protein interaction identification method and application thereof. Background Art

[0002] The interaction between proteins constitutes a major component of the cellular biochemical reaction network. The protein-protein interaction network and transcriptional regulatory network are of great significance in regulating cells and their signals.

[0003] The yeast two-hybrid system is an important method currently widely used in protein interactome research. The principle is that when the target protein and the bait protein specifically bind, the bait protein binds to the promoter of the reporter gene and starts the expression of the reporter gene in the yeast cell. If the expression product of the reporter gene is detected, it means that there is an interaction between the two, otherwise there is no interaction between the two. After this technology is miniaturized and arrayed, it can be used for large-scale protein interaction research. The yeast two-expression vectors currently used are mainly based on two cloning technologies, enzyme digestion-ligation and Gateway, which have problems such as cumbersome operation or expensive reagents.

[0004] However, the yeast two-hybrid system provided by the prior art needs to construct the exogenous gene to be studied on at least two vectors, such as a capture vector and a bait vector, when identifying protein interactions. These two vectors are transferred into two yeast strains for expression and hybridization culture to determine whether the two are hybridized. When it is necessary to identify the interaction of multiple pairs of proteins, it is necessary to construct multiple vectors accordingly. However, the traditional yeast two-hybrid system will dramatically increase the workload of identifying the interaction of multiple proteins or protein libraries, and the work efficiency is severely limited. Summary of the invention

[0005] The inventors of this application creatively cloned two exogenous genes into the same vector and simultaneously expressed the two exogenous genes in a yeast strain, so that interaction identification can be performed. This greatly reduces the work of plasmid construction and extraction, library preparation, yeast hybridization, and screening and cultivation of the original yeast two-hybrid system, and significantly improves the efficiency of protein interaction identification. The technical solution provided by this application provides assistance and improves efficiency for point-to-point, point-to-library, and library-to-library protein interaction identification.

[0006] To this end, this application discloses at least the following technical solutions:

[0007] On the one hand, a yeast dual expression vector is provided. The yeast dual expression vector has a first sequence and a second sequence. The first sequence includes a first multiple cloning site, a coding region of a GAL4 activation domain, a first promoter, a first gene operon, and a basic vector initiation region connected in sequence, the first multiple cloning site is used to insert at least one first target gene, the first promoter, the coding region of the GAL4 activation domain and the first multiple cloning site have the same transcription direction, and the first promoter is used to start the expression of the coding region of the GAL4 activation domain and / or the expression of the first target gene inserted in the first multiple cloning site. The second sequence includes a second multiple cloning site, a coding region of a GAL4 binding domain, a second promoter, a second gene operon, and a yeast 2U replication initiation region connected in sequence, the second multiple cloning site is used to insert at least one second target gene, the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second gene operon and the yeast 2U replication initiation region have the same transcription direction, and the second promoter is used to start the expression of the coding region of the GAL4 binding domain and / or the expression of the second target gene inserted in the second cloning site. The first sequence and the second sequence are connected to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to the transcription direction of the second sequence.

[0008] On the one hand, a method for constructing the yeast dual expression vector is provided. The method comprises: obtaining a basic vector composed of the first sequence and the second sequence by seamless cloning; synthesizing the terminator; and inserting the terminator between the transcription end point of the first multiple cloning site and the transcription end point of the second multiple cloning site.

[0009] In the present application, the term "yeast dual expression vector" refers to a vector that can simultaneously express two genes in yeast cells, and then utilize the GAL4 system in yeast to identify the interaction between the two expression products. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A schematic diagram of the structure of a yeast dual expression vector provided in one embodiment.

[0011] Figure 2 This is a schematic diagram of the structure of a yeast dual expression vector provided by an embodiment. In the figure, "1" is the first segment, "2" is the second segment, "3" is the third segment, and "4" is the fourth segment.

[0012] Figure 3 The electrophoresis result diagram of the yeast dual expression vector provided by one embodiment. Lane 1 is Figure 1 The electrophoresis bands of the provided vector, lane 2 is HindIII digestion Figure 1The electrophoresis bands of the provided vector are 1.1Kbp, 1.5Kbp and 8.2Kbp, respectively, and lane 3 is the marker.

[0013] Figure 4 The plate image of yeast transformants carrying yeast dual expression vectors containing P53 and / or T genes provided for a test example was coated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade after being diluted 10, 100, and 1000 times. In the positive control group, yeast transformants (BD-P53AD-T) containing both P53 and T genes showed an interaction state. Yeast transformants containing only P53 gene dual expression vectors (BD-P53AD) or only T gene (AD-T) failed to show an interaction state. As a negative control, the empty dual expression vector also did not show an interaction state.

[0014] Figure 5 A test example provides a plate image of yeast transformants carrying a yeast dual expression vector containing both a first gene and a second target gene, which are coated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade at 10, 100, and 1000 times dilutions, respectively. The interaction between the first target gene BIF1 and the second target gene BIF4, the interaction between the first target gene KRN2 and the second target gene DUF1644, the interaction between the first target gene GIF1 and the second target gene GRF1, the interaction between the first target gene MSCA1 and the second target gene FEA4, and the interaction between the first target gene KNR6 and the second target gene AGAP.

[0015] Figure 6 A plate image of a yeast transformant carrying a yeast dual expression vector containing both the BD library and the AD library provided in a test example, which was coated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade at 10, 100, and 1000 times dilutions, respectively. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0017] Yeast dual expression vector

[0018] Some embodiments provide a yeast dual expression vector. The yeast dual expression vector has a first sequence and a second sequence. The first sequence includes a first multiple cloning site, a coding region of a GAL4 activation domain, a first promoter, a first gene operon, and a basic vector initiation region connected in sequence, the first multiple cloning site is used to insert at least one first target gene, the first promoter, the coding region of the GAL4 activation domain and the first multiple cloning site have the same transcription direction, and the first promoter is used to start the expression of the coding region of the GAL4 activation domain and / or the expression of the first target gene inserted in the first multiple cloning site. The second sequence includes a second multiple cloning site, a coding region of a GAL4 binding domain, a second promoter, a second gene operon, and a yeast 2U replication initiation region connected in sequence, the second multiple cloning site is used to insert at least one second target gene, the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second gene operon and the yeast 2U replication initiation region have the same transcription direction, and the second promoter is used to start the expression of the coding region of the GAL4 binding domain and / or the expression of the second target gene inserted in the second cloning site. The first sequence and the second sequence are connected to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to the transcription direction of the second sequence.

[0019] In some embodiments, the yeast dual expression vector also has a termination region. The termination region is located between the transcription terminal of the first sequence and the transcription terminal of the second sequence. In some embodiments, the termination region is such as SEQ ID NO: 11, and the termination region includes two synthetic termination sequences. The two termination sequences are used to terminate the expression of the coding region of the GAL4 activation domain in the first sequence and / or the expression of the first target gene inserted in the first multiple cloning site, and to terminate the expression of the coding region of the GAL4 binding domain in the second sequence and / or the expression of the second target gene inserted in the second cloning site. The underlined sequence is a sequence connecting two terminators and has a higher specificity.

[0020] In some embodiments, the first cloning site and the second cloning site each have at least two restriction endonuclease sites.

[0021] In some embodiments, the yeast dual expression vector further has two T7 promoters inserted between the first multiple cloning site and the coding region of the GAL4 activation domain and between the second multiple cloning site and the coding region of the GAL4 binding domain, respectively.

[0022] In some embodiments, the first promoter and the second promoter are both promoters suitable for yeast.

[0023] In some embodiments, the first promoter and the second promoter are both selected from; GK (phosphoglycerol kinase) type promoter, GAP (3-phosphoglyceraldehyde dehydrogenase) type promoter, ADH (alcohol dehydrogenase) type promoter, G3P (glyceraldehyde 3-phosphate dehydrogenase) type promoter, ICL1 (isocitrate lyase) type promoter, AOX1 (alcohol oxidase 1) type promoter, TEF (transcription elongation factor EF-1a) type promoter, GAL1 (galactokinase) type promoter, GAL1 (galactokinase gene) type promoter, Trp1 (tryptophan operon) promoter or its derivative promoters.

[0024] In some embodiments, the first promoter is ADH1 promoter (ADH1Promoter).

[0025] In some embodiments, the second promoter is a truncated ADH1 promoter.

[0026] In some embodiments, the first gene operon includes a first operator and a promoter of the first operator, and the second gene operon includes the first operator and a promoter of the second operator.

[0027] In some embodiments, the first operator gene and the second operator gene are both selected from LEU2, TRP1 or HIS. The first operator gene is a LEU gene, and its promoter is selected from a LEU2 promoter (LEU2 promoter). The second operator gene is selected from a TRP gene, and its promoter is selected from a TRP1 promoter (TRP1 promoter).

[0028] In some embodiments, the second sequence further includes: a resistance gene expression cassette located between the second promoter and the yeast 2U replication initiation region, the transcription direction of the resistance gene expression cassette being the same as the second multiple cloning site; an f1 initiation region connected between the transcription end point of the resistance gene cassette and the second promoter; and a resistance gene connected to the transcription start end of the yeast 2U replication initiation region.

[0029] In some embodiments, the resistance gene is selected from one of the genes encoding ampicillin, tetracycline, chloramphenicol, streptomycin, hygromycin, spectinomycin, kanamycin, blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin or neomycin.

[0030] In some embodiments, Figure 1 As shown, the first sequence of the yeast dual expression vector includes a first multiple cloning site (MCS1, SEQ ID NO: 10), a T7 promoter (T7 RNA Polymerase Promoter, SEQ ID NO: 9), a coding region of a GAL4 activation domain (GAL4 activation domain, SEQ ID NO: 8), an ADH1 Promoter (SEQ ID NO: 7), a LEU2 operator (including LEU2 promoter and LEU2 gene, SEQ ID NO: 6) and a pUC origin (SEQ ID NO: 5) connected sequentially. The second sequence of the yeast dual expression vector includes a sequentially connected second multiple cloning site (MCS2, SEQ ID NO: 12), a T7 promoter (T7 RNA Polymerase Promoter, SEQ ID NO: 9), a coding region of a GAL4 binding domain (GAL4 DNA binding domain, SEQ ID NO: 13), a Truncated ADH1 Promoter (SEQ ID NO: 14), a f1 origin (SEQ ID NO: 1), a TRP1 operon (including a TRP1 gene and a TRP1 promoter, SEQ ID NO: 2), a yeast 2U replication origin (Yeast 2U replication origin, SEQ ID NO: 3) and a kanamycin resistance gene (Kanamycin resistance gene, SEQ ID NO: 4).

[0031] like Figure 1The nucleotide sequence of the yeast dual expression vector is arranged in sequence with the f1origin shown in SEQ ID NO.1 as the starting nucleotide sequence, through the TRP1 operator shown in SEQ ID NO:2, the Yeast2U replication origin shown in SEQ ID NO:3, the Kanamycin resistance gene shown in SEQ ID NO:4, the pUC origin shown in SEQ ID NO:5, the LEU2 operator shown in SEQ ID NO:6, the ADH1Promoter shown in SEQ ID NO:7, the GAL4 activation domain shown in SEQ ID NO:8, the T7 promoter shown in SEQ ID NO:9, the MCS1 shown in SEQ ID NO:10, the termination region shown in SEQ ID NO:11, the MCS2 shown in SEQ ID NO:12, the GAL4 DNA binding domain shown in SEQ ID NO:13, and the Truncated ADH1Promoter shown in SEQ ID NO:14, and the nucleotide sequences shown in the sequence table are all forward chain sequences in this order.

[0032] like Figure 1 As shown in , the directions in which the sequences in each region of the yeast dual expression vector are transcribed are shown by the arrows.

[0033] like Figure 1 As shown in , the yeast dual expression vector further has a sequence such as SEQ ID NO: 15 between the Kanamycin resistance gene and the pUC origin, and a sequence such as SEQ ID NO: 16 between the LEU2 operator and the ADH1 promoter.

[0034] Construction of yeast dual expression vector

[0035] Some embodiments provide a method for constructing a yeast dual expression vector, which comprises: obtaining a basic vector consisting of the first sequence and the second sequence by seamless cloning; synthesizing the termination region; and inserting the termination region between the transcription end point of the first multiple cloning site and the transcription end point of the second multiple cloning site.

[0036] In some embodiments, the construction of the basic vector includes: synthesizing a first fragment, the first fragment includes the first multiple cloning site, the coding region of the GAL4 activation domain and the first promoter; synthesizing a second sequence, the second sequence includes a first gene operon; synthesizing a third fragment, the third fragment includes a basic vector initiation region and a first yeast 2U replication initiation region; synthesizing a fourth fragment, the fourth fragment includes a second yeast 2U replication initiation region, the second gene operon, the second promoter, and a coding region of the GAL4 binding domain; homologously recombine the first fragment, the second fragment, the third fragment and the fourth fragment; transforming the homologous recombination product into Escherichia coli, screening positive colonies, and extracting and recovering the basic vector from the culture of the positive colonies.

[0037] In some embodiments, the step of synthesizing the first fragment includes: using pGADT7 (Coollebo Company) as a template, F1 (SEQ ID NO: 17) and R1 (SEQ ID NO: 18) as a primer pair, performing high-fidelity enzyme PCR amplification, performing gel electrophoresis and recovering the amplified product to obtain the first fragment (SEQ ID NO: 19).

[0038] In some embodiments, the step of synthesizing the second fragment includes: using pGADT7 as a template and F2 (SEQ ID NO: 20) and R2 (SEQ ID NO: 21) as a primer pair, performing high-fidelity enzyme PCR amplification, performing gel electrophoresis and recovering the amplified product to obtain the second fragment (SEQ ID NO: 22).

[0039] In some embodiments, the step of synthesizing the third fragment includes: using pGBKT7 (Coollebo Company) as a template, F3 (SEQ ID NO: 23) and R3 (SEQ ID NO: 24) as a primer pair, performing high-fidelity enzyme PCR amplification, performing gel electrophoresis and recovering the amplified product to obtain the third fragment (SEQ ID NO: 25).

[0040] In some embodiments, the step of synthesizing the fourth fragment includes: using pGBKT7 as a template, F4 (SEQ ID NO: 26) and R4 (SEQ ID NO: 27) as a primer pair, performing high-fidelity enzyme PCR amplification, performing gel electrophoresis and recovering the amplified product to obtain the fourth fragment (SEQ ID NO: 28).

[0041] Among them, PCR amplification used Novazonics' 2×Phanta Max Master Mix high-fidelity enzyme and kit instructions. Please refer to the official website manual https: / / www.vazyme.com / product / 120.html to recover and purify the PCR product. This experiment used Novazonics' FastPure Gel DNA Extraction Mini Kit purification kit. Please refer to the official website manual https: / / www.vazyme.com / companyfile / 2149.html for instructions.

[0042] In some embodiments, the step of homologously recombinating the first fragment, the second fragment, the third fragment, and the fourth fragment comprises: taking 40 ng of the first fragment, 36 ng of the second fragment, 70 ng of the third fragment, and 66 ng of the fourth fragment, 10 μL of 2×CE Mix (from ClonExpress μLtra One Step Cloning Kit V2 of Norvegicus), and controlling the final reaction system to a total volume of 20 μL; performing the recombination reaction at 50°C for 30 min; cooling to 4°C or immediately placing on ice; then performing a traditional E. coli transformation experiment to obtain a positive clone; performing bacterial propagation of the positive clone and plasmid extraction; and testing the successfully constructed basic vector. The results are as follows Figure 2 shown.

[0043] In some embodiments, the termination region is synthesized using known methods.

[0044] In some embodiments, the termination region is cloned into the yeast dual expression vector using two restriction endonuclease sites in both the first cloning site and the second cloning site for enzyme ligation.

[0045] Identification of protein interactions

[0046] Some embodiments provide a method for identifying the interaction between two different proteins. The identification method comprises: respectively amplifying the target genes of the two different proteins; respectively inserting the target genes of the two different proteins into the first multiple cloning site and the second multiple cloning site to obtain a recombinant vector carrying the target genes of the two different proteins; transferring the recombinant vector into yeast to obtain recombinant yeast; and judging whether the two proteins interact based on the growth state of the recombinant yeast on the selection medium.

[0047] In some embodiments, the yeast is a Y2H type yeast.

[0048] In some embodiments, the selection medium is selected from SD / -Leu / -Trp (Product No.: PM2220, Coolbo), SD / -His / -Leu / -Trp (Product No.: PM2112, Coolbo), SD / -Leu / -Trp / -His / -Ade (Product No.: PM2112, Coolbo), SD / -Leu / -Trp / -His / -Ade+3-AT. Aureobasidin A (3-AT) Product No.: CA2332G. SD / -Leu / -Trp / -His / -Ade+3-AT is SD / -Leu / -Trp / -His / -Ade medium supplemented with 10-20 mM 3A-T. In some embodiments, SD / -Leu / -Trp / -His / -Ade+3-AT is SD / -Leu / -Trp / -His / -Ade medium supplemented with 20 mM 3A-T.

[0049] In some embodiments, the construction process of the recombinant vector includes:

[0050] 1. Linearized yeast dual expression vector

[0051] In some steps, the yeast dual expression vector is double-digested with BspDI and NdeⅠ to obtain a linearized yeast dual expression vector, and the double-digestion reaction system contains 5μg yeast dual expression vector, 5μLBspDI (NEB), 5μLNdeⅠ (NEB), 10μL 10×cutsmartbuffer and 85μL deionized water in 100μL. The mixture of the double-digestion reaction system is incubated at 30°C for 4h, and the digestion product is detected to be consistent with the band size, and the digestion product is purified using the Cycle PureKit purification kit produced by Omega Bio-tek to obtain a linearized yeast dual expression vector.

[0052] 2. The first target gene sequence and the second target gene sequence

[0053] PCR amplification was performed using the primers for amplifying the first target gene and the second target gene shown in Table 1, and the PCR amplification products were subjected to gel electrophoresis, and the sequences of the first target gene and the second target gene were recovered using the FastPure Gel DNA Extraction Mini Kit of Novazon. Among them, the PCR reaction system contained 1.0 μL template, 22 μL deionized water, 1.0 μL upstream primer, 1.0 μL downstream primer, 25 μL and 2×Phanta Max Master Mix in 50 μL. The PCR reaction amplification steps included: 1 cycle (95°C, 3 min), 34 cycles (98°C, 20 s; 56°C, 2 min; 72°C, 3 min), 1 cycle (72°C, 5 min) and 1 cycle (4°C, 1 min).

[0054] Table 1 The first target gene, the second target gene and primers

[0055]

[0056] 3. Recombinant vector carrying both the first target gene sequence and the second target gene sequence

[0057] This experiment used the ClonExpress μLtra One Step Cloning Kit V2 from Novozymes, and prepared the multi-fragment recombination reaction system as shown in Table 2. The first target gene sequence (Bait) and the second target gene sequence (Pery) were inserted into the first multiple cloning site and the second cloning site, respectively, to connect with the basic vector to obtain a recombinant vector. The multi-fragment recombination reaction system was pipetted and mixed at 50°C for 30 minutes; then cooled to 4°C or immediately placed on ice for cooling.

[0058] Table 2 Multi-fragment recombination reaction system

[0059] Element Dosage The first target gene sequence 40ng(1μL) The second target gene sequence 40ng(1μL) Double terminator fragment recovery product 10ng(1μL) Linearized multiple expression vector 200ng(1μL) Deionized water 85ng(1μL) 2×CE Mix 5μL Total 10μL

[0060] 4. Transformation of Yeast

[0061] In some steps, the recombinant vector obtained in the above steps is transformed into yeast. The transformation step includes: taking 5-10 μL of the recombinant product and adding it to 100 μL of competent Y2H yeast cells (Y2HGold Chemically CompetentCell product specifications (Weidi Bio CAT#: YC1002)), gently flicking the tube wall to mix (do not oscillate to mix), and standing on ice for 30 minutes. After heat shock in a 42°C water bath for 30 seconds, immediately place it on ice to cool for 2-3 minutes. Add 900 μL of SOC or LB liquid culture medium (without adding antibiotics), and shake the bacteria at 37°C for 1 hour (speed 200-250rpm). Preheat the LB solid culture medium plate with the corresponding resistance in a 37°C incubator. Centrifuge at 5000rpm for 5 minutes and discard 900 μL of supernatant. Resuspend the bacteria with the remaining culture medium, gently spread it on the plate containing the correct resistance with a sterile coating rod, and invert and culture it in a 37°C incubator for 12-16 hours. Single clones were picked from the culture plates for PCR testing, and the positive clones were expanded and plasmids were extracted for subsequent yeast transformation and verification tests.

[0062] 5. Point-to-point experiment of foreign genes

[0063] Some embodiments use the yeast dual expression vector provided in the embodiments of the present application to test the interaction effect of the known interacting gene P53 and SV40gp6-T (Li J, Li Y, Dang M, et al., 2022. Jasmonate-Responsive Transcription Factors NnWRKY70a and NnWRKY70b Positively Regulate Benzylisoquinoline Alkaloid Biosynthesis in Lotus (Nelumbo nucifera). Frontiers in Plant Science, 13: 862915.). For example, a yeast dual expression vector with P53 as the first target gene and SV40gp6-T as the second target gene is constructed, and the vector is transferred into Y2H yeast cells, and the transformants are coated on the selective defective medium SD / -Leu / -Trp and the selective defective medium SD / -Leu / -Trp / -His / -Ade as a positive control. In addition, a yeast dual expression vector without the first target gene and the second target gene was used to transform Y2H yeast cells, and the transformants were spread on the selective defective medium SD / -Leu / -Trp and the selective defective medium SD / -Leu / -Trp / -His / -Ade as negative controls.

[0064] The results are as follows Figure 4As shown, the transformants carrying the yeast dual expression vector provided in the above embodiment containing only the P53 sequence and the transformants carrying the yeast dual expression vector provided in the above embodiment containing only the SV40gp6-T sequence can grow on SD / -Leu / -Trp, but cannot grow on SD / -Leu / -Trp / -His / -Ade. The transformants carrying the yeast dual expression vector provided in the above embodiment containing both the P53 sequence and the T sequence can grow on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade. This shows that the yeast dual expression vector provided in the embodiment of the present application can achieve the simultaneous expression of two exogenous genes in the same yeast cell, which may produce an interaction. A new tool has been developed for the study of protein interactions, further improving the research efficiency.

[0065] 6. Other point-to-point experiments with exogenous genes

[0066] Using the yeast dual expression vector provided in the examples of the present application, some examples tested the interaction effect of the known interacting genes BIF1 and BIF4 (Galli M, Liu Q, Moss BL, et al. Auxin signaling modules regulate maize inflorescence architecture. Proc Natl Acad Sci USA. 2015; 112(43): 13372-13377. doi: 10.1073 / pnas.1516473112), the interaction effect of the known interacting genes KRN2 and DUF1644 (Chen W, Chen L, Zhang X, et al. Convergent selection of a WD40 protein that enhances grain yield in maize and rice.Science.2022;375(6587):eabg7985.doi:10.1126 / science.abg7985), the interaction effect of the known interacting genes GIF1 and GRF1 (Zhang D, Sun W, Singh R, et al. GRF-interacting factor1Regulates Shoot Architecture and Meristem Determinacy in Maize.Plant Cell.2018;30(2):360-374.doi:10.1105 / tpc.17.00791), the interaction effect of the known interacting genes MSCA1 and FEA4, and the interaction effect of the known interacting genes KNR6 and AGAP (Jia H, Li M, Li W, et al. A serine / threonine protein kinase encoding gene KERNEL NUMBER PER ROW6regulates maize grain yield.Nat Commun. 2020;11(1):988.doi:10.1038 / s41467-020-14746-7).

[0067] In the embodiment, yeast dual-epigene vectors with the first target gene being BIF1 and the second target gene being BIF4, yeast dual-epigene vectors with the first target gene being KRN2 and the second target gene being DUF1644, yeast dual-epigene vectors with the first target gene being GIF1 and the second target gene being GRF1, yeast dual-epigene vectors with the first target gene being MSCA1 and the second target gene being FEA4, and yeast dual-epigene vectors with the first target gene being KNR6 and the second target gene being AGAP were constructed. Y2H yeast cells transformed with these yeast dual expression vectors were coated with transformants on selective defective medium SD / -Leu / -Trp and selective defective medium SD / -Leu / -Trp / -His / -Ade.

[0068] The results are as follows Figure 5 As shown, the transformants carrying the yeast dual expression vector provided in the above embodiment containing both the first target gene sequence and the second target gene sequence can grow on SD / -Leu / -Trp / -His / -Ade. This once again shows that the multi-expression vector provided in the embodiment of the present application can successfully detect and verify the interaction of genes.

[0069] 7. Exogenous gene library comparison experiment

[0070] Using the yeast dual expression vector provided in the above embodiments carrying multiple first target gene sequences and multiple second target gene sequences, some embodiments also tested the interaction effect of the target proteins expressed by the first target genes and the second target genes respectively in yeast.

[0071] For example, multiple first target genes are P53, BIF1, KRN2, GIF1, MSCA1 and KNR6, and these six gene sequences are mixed in equal proportions to form a BD library. Multiple second target genes are SV40gp6-T, BIF4, DUF1644, GRF1, FEA4 and AGAP, and these six gene sequences are mixed in equal proportions to form an AD library. In the same manner as the above steps, the BD library and the AD library are cloned into a yeast dual expression vector through a multi-fragment recombination reaction system as shown in Table 2 to construct a recombinant vector carrying both the BD library and the AD library. The recombinant vector is transferred into Y2H yeast cells, and the transformants are coated on selective defective medium SD / -Leu / -Trp and selective defective medium SD / -Leu / -Trp / -His / -Ade to test the interaction between multiple target proteins expressed by the BD library and multiple target proteins expressed by the AD library.

[0072] The results are as follows Figure 6As shown, the growth of the transformants containing the recombinant vector on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade can indicate that the BD library and the AD library interact. This shows that the multi-expression vector provided in the embodiment of the present application can not only apply the interaction of two target genes but also realize the interaction test between libraries composed of multiple genes, which is a screening and verification work for protein interaction experiments between large-scale libraries.

[0073] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A yeast dual expression vector having: a first sequence, wherein the first sequence comprises a first multiple cloning site, a coding region of a GAL4 activation domain, a first promoter, a first gene operon and a basic vector initiation region connected in sequence, the first multiple cloning site is used to insert at least one first target gene, the first promoter, the coding region of the GAL4 activation domain and the first multiple cloning site have the same transcription direction, and the first promoter is used to promote the expression of the coding region of the GAL4 activation domain and / or the expression of the first target gene inserted in the first multiple cloning site; and a second sequence, wherein the second sequence comprises a second multiple cloning site, a coding region of a GAL4 binding domain, a second promoter, a second gene operon and a yeast 2U replication origin region connected in sequence, the second multiple cloning site is used to insert at least one second target gene, the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second gene operon and the yeast 2U replication origin region have the same transcription direction, and the second promoter is used to promote the expression of the coding region of the GAL4 binding domain and / or the expression of the second target gene inserted in the second cloning site; The first sequence and the second sequence are connected to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to the transcription direction of the second sequence. 2 . The yeast dual expression vector according to claim 1 , further comprising a termination region, wherein the termination region is located between the transcription terminus of the first sequence and the transcription terminus of the second sequence.

3. The yeast dual expression vector according to claim 2, wherein the termination region is shown as SEQ ID NO:

11. The yeast dual expression vector according to claim 1 , wherein the first cloning site and the second cloning site each have at least two restriction endonuclease sites.

5. The yeast dual expression vector according to claim 1, further comprising two T7 promoters inserted between the first multiple cloning site and the coding region of the GAL4 activation domain and between the second multiple cloning site and the coding region of the GAL4 binding domain, respectively.

6. The yeast dual expression vector according to claim 1, wherein the coding region of the GAL4 activation domain is shown as SEQ ID NO: 8, and the coding region of the GAL4 binding domain is shown as SEQ ID NO:

13.

7. The yeast dual expression vector according to claim 1, wherein the first promoter and the second promoter are promoters suitable for yeast; Optionally, the first promoter and the second promoter are both selected from: GK (phosphoglycerol kinase) type promoter, GAP (3-phosphoglyceraldehyde dehydrogenase) type promoter, ADH (alcohol dehydrogenase) type promoter, G3P (glyceraldehyde 3-phosphate dehydrogenase) type promoter, ICL1 (isocitrate lyase) type promoter, AOX1 (alcohol oxidase 1) type promoter, TEF (transcription elongation factor EF-1a) type promoter, GAL1 (galactokinase) type promoter, or GAL1 (galactokinase gene) type promoter, or TRP1 (tryptophan operon) promoter; Optionally, the first promoter is ADH1 promoter, and its nucleotide sequence is shown in SEQ ID NO:7; Optionally, the second promoter is a truncated ADH1 promoter, and its nucleotide sequence is shown in SEQ ID NO:

14.

8. The yeast dual expression vector according to claim 1, wherein the first gene operon comprises a first operator gene and a promoter of the first operator gene, and the second gene operon comprises the first operator gene and a promoter of the second operator gene; Optionally, the first operator gene and the second operator gene are both selected from LEU2, TRP1 or HIS.

9. The yeast dual expression vector according to claim 1, wherein the basic vector origin region is shown as SEQ ID NO:5, and the yeast 2U replication origin region is shown as SEQ ID NO:

3.

10. The yeast dual expression vector according to any one of claims 1 to 9, wherein the second sequence further comprises: a resistance gene located at the transcription start end of the second sequence; as well as The f1 initiation region is connected between the transcription end point of the second gene operon and the second promoter, and the f1 initiation region is shown in SEQ ID NO:

1.

11. The yeast dual expression vector according to claim 10, wherein the resistance gene is selected from one of the genes encoding ampicillin, tetracycline, chloramphenicol, streptomycin, hygromycin, spectinomycin, kanamycin, blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin or neomycin.

12. A method for constructing a yeast dual expression vector, comprising: Obtaining a basic vector consisting of the first sequence and the second sequence of any one of claims 1 to 11 through seamless cloning; synthesizing the termination region of any one of claims 1 to 11; and The termination region is inserted between the transcription end point of the first multiple cloning site and the transcription end point of the second multiple cloning site.

13. The construction method according to claim 12, wherein the construction of the basic vector comprises: Synthetically obtaining a first fragment, wherein the first fragment includes the first multiple cloning site, the coding region of the GAL4 activation domain and the first promoter; synthesizing a second sequence, wherein the second sequence includes the first gene operon; Synthetically obtaining a third fragment, wherein the third fragment includes a basic vector initiation region and a first yeast 2U replication initiation region; synthesizing a fourth fragment, wherein the fourth fragment includes the second yeast 2U replication origin, the second gene operon, the second promoter, and the coding region of the GAL4 binding domain; homologously recombine the first fragment, the second fragment, the third fragment and the fourth fragment; The homologous recombination product is transformed into Escherichia coli, positive colonies are screened, and the basic vector is extracted and recovered from the culture of the positive colonies.

14. Methods for identifying interactions between two different proteins, including: Amplifying the target genes of the two different proteins respectively; Inserting the target genes of the two different proteins into the first multiple cloning site and the second multiple cloning site of any one of claims 1 to 13, respectively, to obtain a recombinant vector carrying the target genes of the two different proteins; Transforming the recombinant yeast into yeast to obtain recombinant yeast; Whether the two proteins interact is determined based on the growth status of the recombinant yeast on the selection medium. The identification method according to claim 14 , wherein the yeast is a Y2H type yeast.

16. The identification method according to claim 15, wherein the selection medium is selected from SD / -Leu / -Trp, SD / -His / -Leu / -Trp, SD / -Leu / -Trp / -His / -Ade, and SD / -Leu / -Trp / -His / -Ade+3-AT.

17. Use of the yeast dual expression vector according to any one of claims 1 to 11 in protein interaction.

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