Yeast dual-expression vector and construction method therefor, protein-protein interaction identification method and use

Abstract

A yeast dual-expression vector and a construction method therefor, a protein-protein interaction identification method and the use. The yeast dual-expression vector allows two exogenous genes to be cloned into the same vector and expressed simultaneously in yeast, thereby enabling interaction identification. The present invention greatly reduces the procedures in original yeast two-hybrid systems, such as plasmid construction and extraction, library preparation, yeast mating and screening culture, and significantly improves the identification efficiency of protein-protein interaction.

Description

Yeast dual expression vector, construction method thereof, protein interaction identification method and application TECHNICAL FIELD

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

[0002] Protein-protein interaction constitutes a major component of the biochemical reaction network of cells, and the protein-protein interaction network and the transcriptional regulatory network are of great significance to the regulation of cells and signals.

[0003] The yeast two-hybrid system is an important method widely used in the current protein interaction omics 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 the expression of the reporter gene in the yeast cell is started. If the expression product of the reporter gene is detected, it indicates that there is interaction between the two, otherwise there is no interaction between the two. After the technology is miniaturized and arrayed, it can be used for large-scale research on the interaction between proteins. The yeast dual expression vectors currently used are mainly based on enzyme digestion-ligation and Gateway cloning technology, which have problems such as complicated operation or expensive reagents.

[0004] However, the yeast two-hybrid provided by the prior art needs to construct at least two vectors for the studied exogenous genes when identifying protein interaction, for example, a capture vector and a bait vector. These two vectors are respectively transformed into two yeast strains for expression and hybridization culture, so as to determine whether the two are hybridized. When multiple pairs of proteins need to be identified for interaction, multiple vectors need to be constructed accordingly. However, the traditional yeast two-hybrid system dramatically increases the workload of identifying multiple proteins or protein libraries for interaction, and severely limits the work efficiency. SUMMARY

[0005] The present application creatively clones two exogenous genes in the same vector and simultaneously expresses the two exogenous genes in a yeast strain, so as to identify the interaction. This greatly reduces the plasmid construction and extraction, library preparation, yeast hybridization and screening culture of the original yeast two-hybrid system, and significantly improves the identification efficiency of protein interaction. The technical solutions provided by the present application help and improve the efficiency of point-to-point, point-to-library and library-to-library protein interaction identification.

[0006] Therefore, the present application discloses at least the following technical solutions:

[0007] In one aspect, a yeast two-hybrid vector is provided. The yeast two-hybrid vector has a first sequence and a second sequence. The first sequence comprises, in order, a first multiple cloning site for inserting at least one first gene of interest, a coding region of a GAL4 activation domain, a first promoter, a first gene operon, and a basic vector initiation region, wherein the first promoter, the coding region of the GAL4 activation domain, and the first multiple cloning site have the same transcriptional orientation, and the first promoter is operable to initiate expression of the coding region of the GAL4 activation domain and / or the first gene of interest inserted in the first multiple cloning site. The second sequence comprises, in order, a second multiple cloning site for inserting at least one second gene of interest, a coding region of a GAL4 binding domain, a second promoter, a second gene operon, and a yeast 2U replication initiation region, wherein 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 transcriptional orientation, and the second promoter is operable to initiate expression of the coding region of the GAL4 binding domain and / or the second gene of interest inserted in the second cloning site. The first sequence and the second sequence are linked to form a circular DNA molecule, and the transcriptional orientation of the first sequence is opposite to that of the second sequence.

[0008] In one aspect, a method for constructing the yeast two-hybrid vector is provided. The method comprises: obtaining a basic vector comprising the first sequence and the second sequence by seamless cloning; synthesizing the termination region; and inserting the termination region between the transcription termination point of the first multiple cloning site and the transcription termination point of the second multiple cloning site.

[0009] In this application, the term "yeast two-hybrid vector" refers to a vector that is capable of expressing two genes simultaneously in a yeast cell, and then using the GAL4 system in the yeast to identify the interaction between the two expression products. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a schematic diagram of the structure of a yeast two-hybrid vector according to one embodiment.

[0011] Figure 2 is a schematic diagram of the structure of a yeast two-hybrid vector according to one embodiment. In the figure, "1" is a first fragment, "2" is a second fragment, "3" is a third fragment, and "4" is a fourth fragment.

[0012] Figure 3 is an electrophoresis result of a yeast two-hybrid vector according to one embodiment. Lane 1 is an electrophoresis band of the vector provided in Figure 1, lane 2 is an electrophoresis band of the vector provided in Figure 1 digested by HindIII, which has bands of 1.1 Kbp, 1.5 Kbp, and 8.2 Kbp, respectively, and lane 3 is a Marker.

[0013] Figure 4 is a plate map of dilution of 10, 100, 1000 times of yeast transformants carrying yeast dual expression vectors containing P53 and (or) T genes in a test example, respectively plated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade.

[0014] The plate map of plating on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade, respectively. The yeast transformants carrying yeast dual expression vectors containing both P53 and T genes (BD-P53 AD-T) in the positive control group can exhibit the interaction state. The yeast transformants carrying yeast dual expression vectors containing only P53 gene (BD-P53 AD) or only T gene (AD-T) fail to exhibit the interaction state, and the empty dual expression vector also fails to exhibit the interaction state as a negative control.

[0015] Figure 5 is a plate map of dilution of 10, 100, 1000 times of yeast transformants carrying yeast dual expression vectors containing both the first and second target genes in a test example, respectively plated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade. The interaction of the first target gene BIF1 and the second target gene BIF4, the interaction of the first target gene KRN2 and the second target gene DUF1644, the interaction of the first target gene GIF1 and the second target gene GRF1, the interaction of the first target gene MSCA1 and the second target gene FEA4, and the interaction of the first target gene KNR6 and the second target gene AGAP.

[0016] Figure 6 is a plate map of dilution of 10, 100, 1000 times of yeast transformants carrying yeast dual expression vectors containing both the BD library and the AD library in a test example, respectively plated on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade. DETAILED DESCRIPTION

[0017] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from the prior art.

[0018] Yeast dual expression vector

[0019] Some embodiments provide a yeast dual expression vector. The yeast dual expression vector has a first sequence and a second sequence. The first sequence comprises, in order, a first multiple cloning site for inserting at least one first gene of interest, a coding region of a GAL4 activation domain, a first promoter, a first gene operon, and a basic vector origin of replication, wherein the first promoter, the coding region of the GAL4 activation domain, and the first multiple cloning site have the same direction of transcription, and the first promoter is used to initiate expression of the coding region of the GAL4 activation domain and / or the first gene of interest inserted in the first multiple cloning site. The second sequence comprises, in order, a second multiple cloning site for inserting at least one second gene of interest, a coding region of a GAL4 binding domain, a second promoter, a second gene operon, and a yeast 2U replication origin, wherein 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 have the same direction of transcription, and the second promoter is used to initiate expression of the coding region of the GAL4 binding domain and / or the second gene of interest inserted in the second cloning site. The first sequence and the second sequence are linked to form a circular DNA molecule, and the direction of transcription of the first sequence is opposite to the direction of transcription of the second sequence.

[0020] In some embodiments, the yeast dual expression vector further has a termination region. The termination region is located between the transcriptional termination of the first sequence and the transcriptional termination of the second sequence. In some embodiments, the termination region is as set forth in SEQ ID NO: 11, which comprises two synthetic termination sequences. The two termination sequences are used to terminate expression of the coding region of the GAL4 activation domain in the first sequence and / or the first gene of interest inserted in the first multiple cloning site, and to terminate expression of the coding region of the GAL4 binding domain in the second sequence and / or the second gene of interest inserted in the second cloning site, respectively. The underlined sequence is the sequence connecting the two terminators, which has a higher specificity.

[0021] In some embodiments, the first cloning site and the second cloning site each has at least two restriction enzyme sites.

[0022] 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.

[0023] In some embodiments, the first promoter and the second promoter are promoters suitable for use in yeast.

[0024] In some embodiments, the first promoter and the second promoter are selected from a GK (glycerol kinase) type promoter, a GAP (glyceraldehyde 3-phosphate dehydrogenase) type promoter, an ADH (alcohol dehydrogenase) type promoter, a G3P (glyceraldehyde 3-phosphate dehydrogenase) type promoter, an ICL1 (isocitrate lyase) type promoter, an AOX1 (alcohol oxidase 1) type promoter, a TEF (transcription elongation factor EF-1a) type promoter, a GAL1 (galactokinase) type promoter, a GAL1 (galactokinase gene) type promoter, a Trp1 (tryptophan operon) promoter, or a derivative promoter thereof.

[0025] In some embodiments, the first promoter is an ADH1 promoter.

[0026] In some embodiments, the second promoter is a truncated ADH1 promoter.

[0027] In some embodiments, the first gene operon comprises a first operon gene and a promoter of the first operon gene, and the second gene operon comprises a first operon gene and a promoter of the second operon gene.

[0028] In some embodiments, the first operon gene and the second operon gene are selected from LEU2, TRP1, or HIS. The first operon gene is a LEU gene, and the promoter thereof is selected from a LEU2 promoter. The second operon gene is selected from a TRP gene, and the promoter thereof is selected from a TRP1 promoter.

[0029] In some embodiments, the second sequence further comprises: a resistance gene expression cassette between the second promoter and the yeast 2U replication initiation region, the transcription direction of the resistance gene expression cassette being the same as that of the second multiple cloning site; an f1 initiation region connected between the transcription end of the resistance gene cassette and the second promoter; and a resistance gene connected to the transcription initiation end of the yeast 2U replication initiation region.

[0030] In some embodiments, the resistance gene is selected from one of the genes encoding ampicillin (Ampicillin), tetracycline (Tetracycline), chloramphenicol (Chloramphenicol), streptomycin (Streptomycin), hygromycin (Hygromycin), spectinomycin (Spectinomycin), kanamycin (Kanamycin), blasticidin (Blasticidin), geneticin (Geneticin), hygromycin B (Hygromycin B), mycophenolic acid (Mycophenolic Acid), puromycin (Puromycin), zeocin (Zeocin), or neomycin (Neomycin).

[0031] In some embodiments, as shown in FIG. 1, the first sequence of the yeast dual expression vector includes, in order, a first multiple cloning site (MCS1, SEQ ID NO: 10), a T7 promoter (T7 RNA Polymerase Promoter, SEQ ID NO: 9), a coding region for a GAL4 activation domain (GAL4 activation domain, SEQ ID NO: 8), an ADH1 Promoter (SEQ ID NO: 7), a LEU2 operon (including LEU2 promoter and LEU2 gene, SEQ ID NO: 6), and a pUC origin (SEQ ID NO: 5). The second sequence of the yeast dual expression vector includes, in order, a second multiple cloning site (MCS2, SEQ ID NO: 12), a T7 promoter (T7 RNA Polymerase Promoter, SEQ ID NO: 9), a coding region for a GAL4 DNA binding domain (GAL4 DNA binding domain, SEQ ID NO: 13), a Truncated ADH1 Promoter (SEQ ID NO: 14), a fl origin (SEQ ID NO: 1), a TRP1 operon (including TRP1 gene and 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).

[0032] The nucleotide sequence of the yeast dual expression vector in FIG. 1 is sequentially arranged with the f1 origin shown in SEQ ID NO. 1 as the starting nucleotide sequence, the TRP1 operon shown in SEQ ID NO: 2, the Yeast 2U 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 operon shown in SEQ ID NO: 6, the ADH1 Promoter 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, MCS1 shown in SEQ ID NO: 10, the termination region shown in SEQ ID NO: 11, MCS2 shown in SEQ ID NO: 12, the GAL4 DNA binding domain shown in SEQ ID NO: 13, and the Truncated ADH1 Promoter shown in SEQ ID NO: 14, and the nucleotide sequences shown in the sequence listing are all forward strand sequences in this order.

[0033] As shown in FIG. 1, the direction of transcription of each region sequence on the yeast dual expression vector is shown by the arrow thereof.

[0034] As shown in FIG. 1, the yeast dual expression vector also has the sequence shown in SEQ ID NO: 15 between the Kanamycin resistance gene and the pUC origin, and the sequence shown in SEQ ID NO: 16 between the LEU2 operon and the ADH1 Promoter.

[0035] Construction of the yeast dual expression vector

[0036] Some embodiments provide a method for constructing a yeast dual expression vector. The method for constructing the yeast dual expression vector comprises: obtaining a base 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 of the first multiple cloning site and the transcription end of the second multiple cloning site.

[0037] In some embodiments, the constructing of the base vector comprises: synthesizing a first fragment comprising the first multiple cloning site, the coding region of the GAL4 activation domain, and the first promoter; synthesizing a second sequence comprising a first gene operon; synthesizing a third fragment comprising a base vector start region and a first yeast 2U replication origin; synthesizing a fourth fragment comprising a second yeast 2U replication origin, the second gene operon, the second promoter, the coding region of the GAL4 binding domain; homologously recombining the first fragment, the second fragment, the third fragment, and the fourth fragment; transforming the homologously recombined product into E. coli, screening positive colonies, extracting and recovering the base vector from a culture of the positive colonies.

[0038] In some embodiments, the step of synthesizing the first fragment comprises: performing high-fidelity enzyme PCR amplification using pGADT7 (Clontech) as a template and F1 (SEQ ID NO: 17) and R1 (SEQ ID NO: 18) as a primer pair, subjecting the amplification product to gel electrophoresis and recovering, to obtain the first fragment (SEQ ID NO: 19).

[0039] In some embodiments, the step of synthesizing the second fragment comprises: performing high-fidelity enzyme PCR amplification using pGADT7 as a template and F2 (SEQ ID NO: 20) and R2 (SEQ ID NO: 21) as a primer pair, subjecting the amplification product to gel electrophoresis and recovering, to obtain the second fragment (SEQ ID NO: 22).

[0040] In some embodiments, the step of synthesizing the third fragment comprises: performing high-fidelity enzyme PCR amplification using pGBKT7 (Clontech) as a template and F3 (SEQ ID NO: 23) and R3 (SEQ ID NO: 24) as a primer pair, subjecting the amplification product to gel electrophoresis and recovering, to obtain the third fragment (SEQ ID NO: 25).

[0041] In some embodiments, the step of synthesizing the fourth fragment comprises: performing high-fidelity enzyme PCR amplification using pGBKT7 as a template and F4 (SEQ ID NO: 26) and R4 (SEQ ID NO: 27) as a primer pair, subjecting the amplification product to gel electrophoresis and recovering, to obtain the fourth fragment (SEQ ID NO: 28).

[0042] PCR amplification uses Novozyme 2 × Phanta Max Master Mix high-fidelity enzyme and reagent kit. Refer to the official website instructions https: / / www.vazyme.com / product / 120.html. The PCR product is recovered and purified. The experiment uses Novozyme FastPure Gel DNA Extraction Mini Kit purification kit. Refer to the official website instructions https: / / www.vazyme.com / companyfile / 2149.html.

[0043] In some embodiments, the step of homologous recombination of 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, 66 ng of the fourth fragment, 10 μL of 2 × CE Mix (from Novozyme ClonExpress μLtra One Step Cloning Kit V2), and controlling the final reaction system at 20 μL total volume; performing recombination reaction at 50°C for 30 min; reducing to 4°C or immediately cooling on ice; then performing traditional E. coli transformation experiment to obtain positive clones; performing positive clone liquid amplification and plasmid extraction; and performing detection of the successfully constructed base vector. The results are shown in FIG. 2.

[0044] In some embodiments, the termination region is synthesized using known methods.

[0045] In some embodiments, the termination region is cloned into the yeast dual expression vector by enzyme digestion and ligation using two restriction enzyme sites of the first and second cloning sites.

[0046] Identification of protein interaction

[0047] Some embodiments provide a method for identifying two different protein interactions. The identification method comprises: respectively amplifying the target genes of the two different proteins; inserting the target genes of the two different proteins into the first and second multiple cloning sites, respectively, to obtain a recombinant vector carrying the target genes of the two different proteins; transforming the recombinant into yeast to obtain a recombinant yeast; and determining whether the two proteins interact according to the growth state of the recombinant yeast on a selective medium.

[0048] In some embodiments, the yeast is Y2H type yeast.

[0049] In some embodiments, the selection medium is selected from the group consisting of SD / -Leu / -Trp (Product No. PM2220, Clontech), SD / -His / -Leu / -Trp (Product No. PM2112, Clontech), SD / -Leu / -Trp / -His / -Ade (Product No. PM2112, Clontech), and SD / -Leu / -Trp / -His / -Ade+3-AT.

[0050] Aurantiamycin A (3-AT) Product No. CA2332G. SD / -Leu / -Trp / -His / -Ade+3-AT is SD / -Leu / -Trp / -His / -Ade medium with 10-20 mM 3A-T, and in some embodiments, SD / -Leu / -Trp / -His / -Ade+3-AT is SD / -Leu / -Trp / -His / -Ade medium with 20 mM 3A-T.

[0051] In some embodiments, the construction of the recombinant vector comprises:

[0052] 1. Linearization of the yeast dual expression vector

[0053] In some steps, the yeast dual expression vector is double-digested with BspDI and NdeI to obtain a linearized yeast dual expression vector. The double-digested reaction system, in a total volume of 100 μL, comprises 5 μg of the yeast dual expression vector, 5 μL of BspDI (NEB), 5 μL of NdeI (NEB), 10 μL of 10×cutsmart buffer, and 85 μL of deionized water. The mixture of the double-digested reaction system is incubated at 30°C for 4 h. The digested product is detected for the band size, and the digested product is purified using a Cycle Pure Kit purification kit produced by Omega Bio-tek to obtain the linearized yeast dual expression vector.

[0054] 2. First and second target gene sequences

[0055] PCR amplification was performed using the primers shown in Table 1 for amplifying the first and second target genes, and the PCR amplification products were subjected to gel electrophoresis, and the sequences of the first and second target genes were recovered using the Novagen FastPure Gel DNA Extraction Mini Kit. The PCR reaction system contained 1.0 μL of template, 22 μL of deionized water, 1.0 μL of upstream primer, 1.0 μL of downstream primer, 25 μL of 2 × Phanta Max Master Mix, and 50 μL in total.

[0056] Table 1 First target gene, second target gene, and primer

[0057]

[0058] 3. Recombinant vector carrying the first target gene sequence and the second target gene sequence

[0059] The ClonExpress μLtra One Step Cloning Kit V2 kit from Novagen was used in this experiment, and the multi-fragment recombination reaction system was prepared as shown in Table 2. The first target gene sequence (Bait) and the second target gene sequence (Pery) were inserted into the first and second cloning sites, respectively, to be connected with the basic vector to obtain the recombinant vector. The multi-fragment recombination reaction system was mixed by pipetting and homogenized at 50℃ for 30 min, and then reduced to 4℃ or immediately placed on ice for cooling.

[0060] Table 2 Multi-fragment recombination reaction system

[0061]

[0062] 4. Transformation of yeast

[0063] 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 (Y2H Gold Chemically Competent Cell product specification (Eubichina CAT#: YC1002)), gently shaking the tube wall to mix (do not shake to mix), and placing it on ice for 30 min. After 30 s of 42°C water bath heat shock, immediately place on ice for 2-3 min. Add 900 μL of SOC or LB liquid medium (without adding antibiotics), and shake the bacteria at 37°C for 1 h (at a speed of 200-250 rpm). The corresponding resistant LB solid medium plate is preheated in a 37°C incubator. Centrifuge at 5000 rpm for 5 min, and discard 900 μL of supernatant. Resuspend the bacterial cells with the remaining medium, and gently spread them on the plate containing the correct resistance with a sterile spreader, and incubate them in a 37°C incubator for 12-16 h. Pick single colonies on the culture plate for PCR detection, and expand the positive clone strain and extract the plasmid for subsequent yeast transformation and verification test.

[0064] 5. Point-to-point experiment of exogenous genes

[0065] Some embodiments test the interaction effect of known interacting genes P53 and SV40gp6-T by using the yeast dual expression vector provided in the embodiments of the present application (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 Y2H yeast cells into which the yeast dual expression vector is transformed are plated on selective deficiency medium SD / -Leu / -Trp and selective deficiency medium SD / -Leu / -Trp / -His / -Ade as a positive control. In addition, Y2H yeast cells transformed with a yeast dual expression vector that does not contain the first target gene and the second target gene are plated on selective deficiency medium SD / -Leu / -Trp and selective deficiency medium SD / -Leu / -Trp / -His / -Ade as a negative control.

[0066] The results are shown in Figure 4. The transformants carrying the yeast two-expression vector provided in the above examples containing only P53 sequence and the transformants carrying the yeast two-expression vector provided in the above examples containing only SV40 gp6-T sequence can grow on SD / -Leu / -Trp, but cannot grow on SD / -Leu / -Trp / -His / -Ade. The transformants carrying the yeast two-expression vector provided in the above examples containing both P53 sequence and T sequence can grow on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade. Thus, it is shown that the yeast two-expression vector provided in the present application can realize the simultaneous expression of two exogenous genes in the same yeast cell, and further can produce interaction. A new tool is developed for protein interaction research, and the research efficiency is further improved.

[0067] 6. Other point-to-point experiments of exogenous genes

[0068] Using the yeast double expression vector provided in the embodiments of the present application, some embodiments test the interaction effect of 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 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 known interacting genes GIF1 and GRF1 (Zhang D, Sun W, Singh R, et al. GRF-interacting factor 1 Regulates Shoot Architecture and Meristem Determinacy in Maize. Plant Cell. 2018; 30(2): 360-374. doi: 10.1105 / tpc.17.00791), the interaction effect of known interacting genes MSCA1 and FEA4, and the interaction effect of known interacting genes KNR6 and AGAP (Jia H, Li M, Li W, et al. A serine / threonine protein kinase encoding gene KERNEL NUMBER PER ROW 6 regulates maize grain yield. Nat Commun. 2020; 11(1): 988. doi: 10.1038 / s41467-020-14746-7).

[0069] The embodiments respectively construct yeast dual expression vectors with the first target gene as BIF1 and the second target gene as BIF4, the first target gene as KRN2 and the second target gene as DUF1644, the first target gene as GIF1 and the second target gene as GRF1, the first target gene as MSCA1 and the second target gene as FEA4, and the first target gene as KNR6 and the second target gene as AGAP. The Y2H yeast cells transformed by the yeast dual expression vectors are plated on selective deficiency medium SD / -Leu / -Trp and selective deficiency medium SD / -Leu / -Trp / -His / -Ade.

[0070] As shown in FIG. 5, the transformants carrying the yeast dual expression vectors provided by the above embodiments containing the first target gene sequence and the second target gene sequence can grow on SD / -Leu / -Trp / -His / -Ade. Again, it is shown that the multi-expression vectors provided by the embodiments can successfully detect and verify the interaction of genes.

[0071] 7. Exogenous gene library vs. library experiment

[0072] Using the yeast dual expression vectors provided by the above embodiments carrying multiple first target gene sequences and multiple second target gene sequences, some embodiments also test the interaction effect of the target proteins expressed by the first target genes and the second target genes in yeast.

[0073] For example, the multiple first target genes are P53, BIF1, KRN2, GIF1, MSCA1 and KNR6, and the six gene sequences are mixed at equal proportions to form a BD library. The multiple second target genes are SV40gp6-T, BIF4, DUF1644, GRF1, FEA4 and AGAP, and the six gene sequences are mixed at equal proportions to form an AD library. In the same way as the above steps, the BD library and the AD library are cloned into the yeast dual expression vectors by the multi-fragment recombination reaction system shown in Table 2 to construct the recombination vectors carrying the BD library and the AD library. The recombination vectors are transformed into Y2H yeast cells, and the transformants are plated on selective deficiency medium SD / -Leu / -Trp and selective deficiency medium SD / -Leu / -Trp / -His / -Ade to test the interaction between the multiple target proteins expressed by the BD library and the multiple target proteins expressed by the AD library.

[0074] The results are shown in Figure 6. The growth of the transformants containing the recombinant vector on SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade indicates that the BD library and the AD library produce interactions. Thus, the multi-expression vector provided by the embodiments of the present application can not only be applied to the interaction of two target genes, but also can realize the interaction test between libraries composed of multiple genes, thereby facilitating the screening and verification of large-scale library and library protein interaction experiments.

[0075] The above description is merely preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A yeast dual-expression vector, which has the following characteristics: A first sequence comprising a first multiple cloning site, a coding region of a GAL4 activation domain, a first promoter, a first gene operon, and a base vector initiation region, sequentially linked. The first multiple cloning site is used to insert at least one first target gene. The transcription directions of the first promoter, the coding region of the GAL4 activation domain, and the first multiple cloning site are the same. The first promoter is used to initiate the expression of the coding region of the GAL4 activation domain and / or the expression of the first target gene inserted at the first multiple cloning site. The second sequence comprises a second multiple cloning site, a coding region of the GAL4 binding domain, a second promoter, a second gene operon, and a yeast 2U replication initiation region, which are sequentially linked. The second multiple cloning site is used to insert at least one second target gene. The transcription directions of 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 are the same. The second promoter is used to initiate the expression of the coding region of the GAL4 binding domain and / or the expression of the second target gene inserted at the second cloning site. The first sequence and the second sequence are joined to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to that of the second sequence.

2. The yeast dual expression vector according to claim 1, wherein the yeast dual expression vector further comprises a termination region, the termination region being located between the transcriptional terminal of the first sequence and the transcriptional terminal of the second sequence.

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

11.

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

5. The yeast dual expression vector according to claim 1, wherein the yeast dual expression vector further comprises 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.

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

13.

7. The yeast dual expression vector according to claim 1, wherein both the first promoter and the second promoter are promoters suitable for yeast; Optionally, both the first promoter and the second promoter are selected from: GK (glycerol phosphate kinase) type promoter, GAP (glyceraldehyde-3-phosphate 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 the ADH1 promoter, the nucleotide sequence of which is shown in SEQ ID NO:7; Optionally, the second promoter is a truncated ADH1 promoter, the nucleotide sequence of which 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 a first operator gene and a promoter of the second operator gene; Optionally, both the first manipulator and the second manipulator are selected from LEU2, TRP1, or HIS.

9. The yeast dual expression vector according to claim 1, wherein the initiation region of the basic vector is shown in SEQ ID NO:5, and the yeast 2U replication initiation region is shown in 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: The resistance gene located at the transcription start terminus of the second sequence; as well as The f1 initiation region is connected between the transcriptional endpoint of the second gene operon and the second promoter, as shown in SEQ ID NO:

1.

11. The yeast dual-expression vector according to claim 10, wherein the resistance gene is selected from the encoding genes of 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: A base vector consisting of the first sequence and the second sequence of any one of claims 1 to 11 was obtained through seamless cloning; Synthesize the termination region as described in any one of claims 1 to 11; and The termination region is inserted between the transcriptional endpoint of the first multiple cloning site and the transcriptional endpoint of the second multiple cloning site.

13. The construction method according to claim 12, wherein the construction of the basic carrier includes: The first fragment is synthesized, and the first fragment includes the first multiple cloning site, the coding region of the GAL4 activation domain, and the first promoter; A second sequence was synthesized, the second sequence including the first gene operon; A third fragment was synthesized, which includes the base vector initiation region and the first yeast 2U replication initiation region; A fourth fragment was synthesized, which includes the second yeast 2U replication initiation region, the second gene operon, the second promoter, and the coding region of the GAL4 binding domain; Homologous recombination is performed on the first segment, the second segment, the third segment, and the fourth segment; The homologous recombination product was transferred into Escherichia coli, positive colonies were screened, and the basic vector was extracted and recovered from the culture of the positive colonies.

14. Methods for identifying interactions between two different proteins, including: The target genes of the two different proteins were amplified separately. The target genes of the two different proteins are respectively inserted into the first multiple cloning site and the second multiple cloning site of any one of claims 1 to 13 to obtain a recombinant vector carrying the target genes of the two different proteins. The recombinant was transferred into yeast to obtain recombinant yeast; Whether the two proteins interact is determined based on the growth status of the recombinant yeast on the selective culture medium.

15. 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 selective culture 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. The use of the yeast dual expression vector according to any one of claims 1 to 11 in protein interaction.

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