Method for constructing interaction plasmid library, method for identifying interactions using cdna library, and use

Abstract

The present application relates to the technical field of yeast two-hybridization, and in particular, to a method for constructing an interaction plasmid library, a method for identifying interactions using a cDNA library, and use. A yeast double expression vector is used to construct two cDNA libraries in the same vector and express the two cDNA libraries separately. Moreover, the expression of the two cDNA libraries does not interfere with each other, and interaction identification can be accurately performed in the same yeast strain. This reduces the workload associated with constructing and extracting the interaction plasmid library as well as constructing and culturing strains, thereby improving the identification efficiency.

Description

Methods for constructing interaction plasmid libraries, methods for identifying interactions in cDNA libraries, and their applications. Technical Field This application relates to the field of yeast two-hybrid technology, specifically to methods for constructing interaction plasmid libraries, methods for identifying interactions in cDNA libraries, and their applications. Background Technology A cDNA plasmid library is a collection of clones formed by reverse transcription of all mRNA transcribed from an organism at a specific developmental stage, resulting in cDNA fragments ligated to a vector. A cDNA plasmid library is created by using specific tissue or cell mRNA as a template, reverse transcribing complementary DNA (cDNA) to a suitable vector (commonly bacteriophages or plasmid vectors), and then transforming the recipient bacteria to form a recombinant DNA clone group. This collection of cDNA clones containing all the mRNA information of the cell is called the interaction plasmid library of that tissue or cell. Interaction plasmid libraries specifically reflect the coding genes of proteins expressed at a specific developmental stage in a particular tissue or cell; therefore, interaction plasmid libraries are tissue- or cell-specific. The yeast two-hybrid system is an important method widely used in proteomics research. Its principle is that when a target protein and a bait protein specifically bind, the bait protein binds to the promoter of a reporter gene, initiating the expression of the reporter gene in yeast cells. If the expression product of the reporter gene is detected, it indicates an interaction between the two proteins; otherwise, there is no interaction. This technique can be miniaturized and arrayed for large-scale studies of interactions between proteins, such as cDNA files. In practice, one-hybrid, three-hybrid, and reverse hybridization systems have been developed to meet specific needs. Currently used yeast two-hybrid vectors are mainly based on enzyme digestion-ligation and Gateway cloning techniques, which suffer from problems such as cumbersome operation or expensive reagents. However, existing yeast two-hybrid techniques for identifying protein interactions require the exogenous gene under study to be constructed onto at least two vectors, such as a prey vector and a bait vector. These two vectors are then transformed into two separate yeast strains for expression and hybridization culture to determine whether hybridization has occurred. When multiple protein pairs need to be verified, multiple vectors need to be constructed accordingly. Furthermore, modern research systems are more complex, often requiring discussions and studies of multiple proteins. This increases the workload of traditional techniques, making them unsuitable for identifying multiple protein pairs. Summary of the Invention The inventors of this application have creatively constructed two cDNA libraries from yeast two-hybrid systems into a single vector to obtain an interaction plasmid library. This interaction plasmid library can express two cDNA libraries from different or the same source separately in yeast, and their expression does not interfere with each other. Furthermore, it allows for accurate interaction identification within the same yeast strain, significantly reducing the workload of interaction plasmid library construction and extraction, strain construction and culture, and substantially improving identification efficiency. The technical solution provided in this application assists in improving the efficiency of point-to-point, point-to-library, and library-to-library protein interaction identification. Therefore, this application discloses at least the following technical solutions: One approach provides a method for constructing an interacting plasmid library. This method includes: obtaining a yeast two-hybrid vector having a first multiple cloning site and a second multiple cloning site; obtaining a first cDNA library and a second cDNA library; obtaining a linearized yeast two-hybrid vector; and inserting the first cDNA library and the second cDNA library into the first multiple cloning site and the second multiple cloning site of the linearized yeast two-hybrid vector, respectively. The yeast two-hybrid vector further comprises a first sequence and a second sequence, which are linked to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to that of the second sequence. The first sequence includes, in sequence, the first multiple cloning site, a coding region of the GAL4 activation domain, a first promoter, a first operon, and a base vector initiation region. 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 cDNA library inserted into 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 operon, and a yeast 2U replication initiation region, which are sequentially linked. The transcription directions of the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second 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 cDNA library inserted at the second cloning site. One aspect provides a method for identifying interactions between cDNA libraries. This method includes providing an interaction plasmid library constructed as described above, transforming the interaction plasmid library into yeast to obtain recombinant yeast, and determining whether the first cDNA library and the second cDNA library interact based on the growth status of the recombinant yeast on a selective culture medium. On the one hand, it provides the application of the interaction plasmid library obtained by the construction method described above in protein-interaction of the interaction plasmid library. Attached Figure Description Figure 1 is a schematic diagram of the structure of a yeast two-hybrid vector provided in one implementation scheme. Figure 2 is a schematic diagram of the interaction identification method for cDNA libraries provided in one implementation scheme. Figure 3 shows electrophoresis images of the AD and BD libraries provided in one implementation scheme. Figure 4 is an electrophoresis diagram of the linearized pGAB-Dual Drive-IF-1 plasmid fragment provided in one implementation scheme. Figure 5 shows a PCR electrophoresis diagram of transformant colonies of a transformation interaction plasmid library provided in one implementation scheme. Figure 6 is an electrophoresis diagram of the linearized pGAB-Dual Drive-IF-2 plasmid fragment provided in one implementation scheme. Figure 7 is a transformation plate diagram of a transformation self-activating plasmid library provided by one implementation scheme. Figure 8 shows the growth status of 40 quadruple-deficient plates of the recombinant transformants of the interaction plasmid library provided in one implementation scheme after 3 days. Figure 9 shows the growth status of eight quadruple-deficient plates of the recombinant transformants of the self-activated plasmid library provided by one implementation scheme after 3 days. Figure 10 is an electrophoresis diagram of PCR amplification products from an extracted interaction plasmid library or self-activating plasmid library provided by one implementation scheme. Detailed Implementation To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art. Methods for constructing interaction plasmid libraries Some implementations provide a method for constructing an interacting plasmid library. This method includes: obtaining a yeast two-hybrid vector having a first multiple cloning site and a second multiple cloning site; obtaining a first cDNA library and a second cDNA library; obtaining a linearized yeast two-hybrid vector; and inserting the first cDNA library and the second cDNA library into the first multiple cloning site and the second multiple cloning site of the linearized yeast two-hybrid vector, respectively. The yeast two-hybrid vector further comprises a first sequence and a second sequence, which are linked to form a circular DNA molecule, and the transcription direction of the first sequence is opposite to that of the second sequence. The first sequence includes, in sequence, the first multiple cloning site, a coding region of the GAL4 activation domain, a first promoter, a first operon, and a base vector initiation region. 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 cDNA library inserted into 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 operon, and a yeast 2U replication initiation region, which are sequentially linked. The transcription directions of the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second 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 cDNA library inserted at the second cloning site. In some embodiments, the yeast dual-expression vector further includes a termination region. The termination region is located between the transcriptional terminal of the first sequence and the transcriptional terminal of the second sequence. In some embodiments, the termination region, as in SEQ ID NO:11, comprises two synthetic termination sequences. These 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 at the first multiple cloning site, respectively, 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 at the second cloning site. In some implementations, both the first cloning site and the second cloning site have at least two restriction endonuclease sites. In some embodiments, the yeast dual expression vector further comprises two T7 promoters, one inserted between the first multiple cloning site and the coding region of the GAL4 activation domain, and the other inserted between the second multiple cloning site and the coding region of the GAL4 binding domain. In some implementations, both the first promoter and the second promoter are promoters suitable for yeast. In some embodiments, both the first promoter and the second promoter are selected from: GK (glycerol phosphate kinase) promoters, GAP (glyceraldehyde-3-phosphate dehydrogenase) promoters, ADH (alcohol dehydrogenase) promoters, G3P (glyceraldehyde-3-phosphate dehydrogenase) promoters, ICL1 (isocitrate lyase) promoters, AOX1 (alcohol oxidase 1) promoters, TEF (transcription elongation factor EF-1a) promoters, GAL1 (galactokinase) promoters, GAL1 (galactokinase gene) promoters, Trp1 (tryptophan operon) promoters, or promoters derived therefrom. In some implementations, the first promoter is the ADH1 promoter. In some implementations, the second promoter is a truncated ADH1 promoter. In some implementations, the first gene operon includes a first operating gene and a promoter of the first operating gene, and the second gene operon includes the first operating gene and a promoter of the second operating gene. In some embodiments, both the first and second operator genes are selected from LEU2, TRP1, or HIS. The first operator gene is the LEU gene, and its promoter is selected from the LEU2 promoter. The second operator gene is selected from the TRP gene, and its promoter is selected from the TRP1 promoter. 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 that of the second multiple cloning site; an f1 initiation region connected between the transcriptional endpoint of the resistance gene cassette and the second promoter; and a resistance gene connected to the transcriptional initiation terminal of the yeast 2U replication initiation region. In some embodiments, the resistance gene is selected from the coding genes of ampicillin, tetracycline, chloramphenicol, streptomycin, hygromycin, spectinomycin, kanamycin, blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin, or neomycin. In some implementations, as shown in Figure 1, 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 the GAL4 activation domain (GAL4 activation domain, SEQ ID NO:8), an ADH1 promoter (ADH1 Promoter, SEQ ID NO:7), a LEU2 operon (including the LEU2 promoter and the LEU2 gene, SEQ ID NO:6), and a pUC origin (SEQ ID NO:5). The second sequence of the yeast dual expression vector includes a second multiple cloning site (MCS2, SEQ ID NO:12), a T7 promoter (T7 RNA Polymerase Promoter, SEQ ID NO:9), a coding region of the GAL4 binding domain (GAL4 DNA binding domain, SEQ ID NO:13), a Truncated ADH1 Promoter (SEQ ID NO:14), an f1 origin (SEQ ID NO:1), a TRP1 operon (including the TRP1 gene and TRP1 promoter, SEQ ID NO:2), a yeast 2U replication origin (SEQ ID NO:3), and a kanamycin resistance gene (SEQ ID NO:4). As shown in Figure 1, the nucleotide sequence of the yeast dual expression vector starts with the f1 origin shown in SEQ ID NO:1, and proceeds sequentially through 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, 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 ADH1 Promoter shown in SEQ ID NO:14. All the nucleotide sequences shown in the sequence listing are forward strand sequences in this order. As shown in Figure 1, the direction of transcription of each region sequence on the yeast dual expression vector is indicated by its arrows. As shown in Figure 1, the yeast dual expression vector also 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 operon and the ADH1 promoter. Construction of yeast dual expression vector Some implementations provide a method for constructing a yeast dual-expression vector. This method includes: obtaining a base vector consisting of a first sequence and a second sequence via seamless cloning; synthesizing the termination region; and inserting the termination region between the transcriptional endpoint at the first multiple cloning site and the transcriptional endpoint at the second multiple cloning site. In some implementations, the construction of the base vector includes: synthesizing a first fragment, the first fragment including a first multiple cloning site, a coding region of the GAL4 activation domain, and a first promoter; synthesizing a second sequence, the second sequence including a first gene operon; synthesizing a third fragment, the third fragment including a base vector initiation region and a first yeast 2U replication initiation region; synthesizing a fourth fragment, the fourth fragment including a second yeast 2U replication initiation region, a second gene operon, a second promoter, and a coding region of the GAL4 binding domain; performing homologous recombination on the first fragment, the second fragment, the third fragment, and the fourth fragment; transforming the homologous recombination product into *E. coli*, screening for positive colonies, and extracting and recovering the base vector from the culture of the positive colonies. In some implementations, the steps for synthesizing the first fragment include: using pGADT7 (Coollab) as a template, using F1 (SEQ ID NO:17) and R1 (SEQ ID NO:18) as primer pairs, performing high-fidelity enzyme PCR amplification, and performing gel electrophoresis on the amplification product and recovering it to obtain the first fragment (SEQ ID NO:19). In some implementations, the steps for synthesizing the second fragment include: using pGADT7 as a template and F2 (SEQ ID NO:20) and R2 (SEQ ID NO:21) as primer pairs, performing high-fidelity enzyme PCR amplification, and performing gel electrophoresis on the amplification product and recovering it to obtain the second fragment (SEQ ID NO:22). In some implementations, the steps for synthesizing the third fragment include: using pGBKT7 (Coollab) as a template, and using F3 (SEQ ID NO:23) and R3 (SEQ ID NO:24) as primer pairs, performing high-fidelity enzyme PCR amplification, and performing gel electrophoresis on the amplification product and recovering it to obtain the third fragment (SEQ ID NO:25). In some implementations, the steps for synthesizing the fourth fragment include: using pGBKT7 as a template, using F4 (SEQ ID NO:26) and R4 (SEQ ID NO:27) as primer pairs, performing high-fidelity enzyme PCR amplification, and performing gel electrophoresis on the amplification product and recovering it to obtain the fourth fragment (SEQ ID NO:28). The PCR amplification used Novizan's 2 × Phanta Max Master Mix high-fidelity enzyme and kit instructions (see official website: https: / / www.vazyme.com / product / 120.htmL). The PCR products were then purified using Novizan's FastPure Gel DNA Extraction Mini Kit (see official website: https: / / www.vazyme.com / companyfile / 2149.htmL). In some implementations, the steps for homologous recombination of the first fragment, the second fragment, the third fragment, and the fourth fragment include: 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, and 10 μL of 2×CE Mix (from Novizan ClonExpress μLtra One Step Cloning Kit V2), with the final reaction system controlled at a total volume of 20 μL; carrying out the recombination reaction at 50°C for 30 min; cooling to 4°C or immediately placing on ice for cooling; then performing a conventional E. coli transformation experiment to obtain a positive clone; performing positive clone bacterial culture propagation and plasmid extraction; and detecting the successfully constructed basic vector. In some implementations, the termination region is synthesized using known methods. In some implementations, the termination region is cloned into the yeast dual expression vector by using two restriction endonuclease sites, both at the first and second cloning sites, for enzyme digestion and ligation. Construction and interaction of interaction plasmid libraries In some implementations, a first cDNA library (used as an AD library) and a second cDNA library (used as a BD library) are simultaneously inserted into the yeast dual-expression vector provided in the examples to construct a plasmid library carrying both the first and second cDNAs. These steps specifically include: 1. Extract total RNA Total RNA was extracted from maize SAM tissue using the Trizol method. Reverse recording to obtain AD library and BD library This step involves reverse transcription of the extracted total RNA to obtain first-strand cDNA, followed by PCR amplification of the first-strand cDNA to obtain a cDNA library. In some embodiments, purified total RNA (0.2-4.0 μg), 1.0 μL CDS primers, 1.0 μL dNTPs, and the remainder deionized water were mixed to prepare a 7.5 μL solution. This solution was incubated at 65°C for 5 min, followed by an ice bath for 2-3 min to obtain a reverse transcription sample solution. Then, 7.88 μL of the sample solution, 1.0 μL SuperScript IV reverse transcriptase (2000 U / μL), 1.0 μL RNase inhibitor (40 U / μL), 4.0 μL SuperScript IV first-strand buffer (5×), 1 μL DTT (100 mM), 4 μL betaine (5M), and 0.12 μL MgCl₂ were added. 2 A reverse transcription mixture consisting of 1 M TSO primer (10 μM) and 1 μL TSO primer (10 μM) was prepared and incubated at 55℃ for 10 min and 80℃ for 10 min to obtain first-strand cDNA. In some embodiments, a PCR reaction system consisting of 1.0–5.0 μL of first-strand cDNA, 1.0 μL of AD-F, 1.0 μL of AD-R, 25 μL of KAPA hot-start HiFi high-fidelity enzyme premix (Roche, KK2602), and the remainder deionized water, totaling 50 μL, was used for PCR. The PCR products were detected by electrophoresis. The optimal number of cycles was selected based on the brightness and size distribution of the bands, and PCR amplification was performed to obtain a 200 μL cDNA library product. The PCR reaction program included: one cycle at 95°C for 3 min; 11, 13, 15, 17, or 19 cycles at 98°C for 20 s, 62–67°C for 15 s, and 72°C for 3 min, with annealing stability determined based on the primers; one cycle at 72°C for 5 min; and one cycle at 4°C for 1 min. In some embodiments, a PCR reaction system consisting of 1.0–5.0 μL of first-strand cDNA, 1.0 μL of BD-F, 1.0 μL of BD-R, 25 μL of KAPA hot-start HiFi high-fidelity enzyme premix (Roche, KK2602), and the remainder deionized water, totaling 50 μL, was used for PCR reaction. The PCR products were detected by electrophoresis, and the optimal number of cycles was selected based on the brightness and size distribution of the bands. PCR amplification was then performed to obtain a 200 μL cDNA library product. The PCR reaction program included: one cycle at 95°C for 3 min; 11, 13, 15, 17, or 19 cycles at 98°C for 20 s, 62–67°C for 15 s, and 72°C for 3 min, with annealing stability determined based on the primers; one cycle at 72°C for 5 min; and one cycle at 4°C for 1 min. Table 1 lists the primers involved, where r represents ribonucleotide, N is A, C, G, or T, and V is A, G, or C. "AD" indicates primers used for synthesizing AD libraries, and "BD" indicates primers used for synthesizing BD libraries. Table 1 Primers In some embodiments, the obtained cDNA library products were purified using KAPA purification magnetic beads (Roche, KK8000), and sorted in three proportions: 0.4 (>2000bp), 0.4-0.5 (2000bp~1000bp), and 0.5-0.6 times the product volume, to obtain AD and BD libraries, respectively. Electrophoresis of the obtained AD and BD libraries, as shown in Figure 2, revealed good diffuse bands, indicating a high degree of homogenization of the fragments in both libraries. Constructing an interaction plasmid library This embodiment provides steps for constructing an interaction plasmid library, including obtaining a linearized yeast dual-expression vector fragment, performing a multi-fragment recombination reaction with the sequence shown in SEQ ID NO:11, the AD library, and the BD library, transforming the resulting recombinant fragment into *E. coli*, screening for positive clones, and extracting the interaction plasmid library from the culture of the positive clones. The embodiment also specifies the primers used for colony PCR detection. This embodiment provides a plasmid (named pGAB-Dual Drive-IF-1) lacking the termination region shown in SEQ ID NO:11 compared to the vector shown in Figure 1, and the vector shown in Figure 1 (named pGAB-Dual Drive-IF-2) as the base plasmids for preparing the interaction plasmid library. The steps for obtaining linearized yeast dual expression vector fragments include: transforming pGAB-Dual Drive-IF-1 or pGAB-Dual Drive-IF-2 into E. coli, incubating upside down in a 37 ℃ incubator for one day, then inoculating single clones into LB medium containing kanamycin and culturing for 12-16 h, and extracting the yeast dual expression vector (concentration > 100 ng / μL) using the Plasmid DNA Mini Kit 1 kit manufactured by Omega Bio-tek. A reaction system was prepared by mixing 5 μg of the yeast dual expression vector (pGAB-Dual Drive-IF-1), 5 μL of BamH Ⅰ-HF, 5 μL of NdeⅠ, 10 μL of 10× Cutsmart buffer, and 85 μL of deionized water. The mixture was incubated at 30°C for 4 h for double enzyme digestion. The digestion products were detected by electrophoresis and purified using the Cycle Pure Kit (Omega Bio-tek) to obtain the linearized yeast dual expression vector (pGAB-Dual Drive-IF-1). As shown in Figures 4 and 6, the linearized products of pGAB-Dual Drive-IF-1 and pGAB-Dual Drive-IF-2 both showed a single band upon electrophoresis, indicating that linearized fragments of pGAB-Dual Drive-IF-1 and pGAB-Dual Drive-IF-2 were obtained. The multi-fragment recombination reaction system, in 10 μL, contains 30–60 μg (1 μL) of AD library, 30–60 μg (1 μL) of BD library product, 10 μg (1 μL) of the sequence shown in SEQ ID NO:11, 200 μg (1–2 μL) of linearized yeast dual expression vector (pGAB-Dual Drive-IF-1), 5 μL of 2× CE Mix, and the remainder of deionized water. The multi-fragment recombination reaction conditions include reacting at 50 °C for 30 min and then cooling to 4 °C. Each plasmid in the interaction plasmid library obtained in the examples carries two cDNA fragments, one for the AD acting element and the other for the BD acting element, to facilitate interaction detection in yeast. As shown in Figure 5, the obtained transformants were transformed into 30 plates, and 96 single clones were randomly selected. 72 single clones were able to amplify bands of varying sizes, preliminarily indicating that each plasmid in the interaction plasmid library carries both AD and BD sequences. In some comparative examples, the yeast dual expression vector (pGAB-Dual Drive-IF-2) was double-digested with BspdⅠ and SacⅠ in the same manner to obtain linearized pGAB-Dual Drive-IF-2. 200 μg (1~2 μL) of linearized pGAB-Dual Drive-IF-2, 60 μg (1 μL) of BD library product, 5 μL of 2× CE Mix, and the remainder deionized water, for a total of 10 μL, were transformed into *E. coli* using the same method as in the previous examples. Positive clones were screened, and self-activating plasmid libraries were extracted from the cultures of the positive clones. Each plasmid in the obtained self-activating plasmid library carried only one cDNA fragment, which was a BD acting element. Compared with the previous examples, genes capable of self-activation in this cDNA library were identified. Figure 7 shows the growth status of the self-activating transformants 16 hours after transformation on 6 plates. 4. cDNA library interaction test The method for cDNA library interaction testing includes: preparing competent yeast Y2HGold cells; transforming the interaction plasmid library and the self-activation plasmid library obtained in the above examples into the competent Y2HGold cells; plating the transformants onto a four-deficient selection medium SD / -Leu / -Trp / -Ade / -His, and detecting positive colonies by colony PCR; extracting cDNA plasmids and self-activation plasmids from the positive colonies; performing PCR amplification on the interaction plasmid library and the self-activation plasmid library; and constructing and sequencing the PCR amplification products. In some embodiments, the steps for preparing competent yeast Y2HGol include: 1) Yeast Y2HGold strain (Weidi Bio: CAT#: YC1002) was streaked on YPDA solid plates (Coolaber, PM2011-500g + agar, CA1331-500g) and fresh single colonies were picked and transferred to 5mL YPDA liquid medium (using a 50mL centrifuge tube). The culture was then incubated at 30℃ and 250rpm for 12h. 50μL of the bacterial culture was then inoculated into 50mL YPDA liquid medium (using a 250mL Erlenmeyer flask) and incubated at 250rpm until the OD600 reached approximately 0.15-0.3 (16-20h). 2) Collect the bacterial culture, centrifuge at 700g for 5min to collect the bacterial cells, discard the supernatant, resuspend in 100mL YPDA liquid culture medium, and shake at 30℃ and 250rpm until OD600=0.4-0.5. 3) Collect the bacterial culture. Centrifuge at 700g for 5 minutes to collect the bacterial cells. Discard the supernatant and resuspend in 50mL of sterile ddH2O. 4) Collect the bacterial culture, centrifuge at 700g for 5min to collect the bacterial cells, discard the supernatant, and resuspend in 15mL of 1.1XTE / liAC (Weidi Bio: YC4210L) pre-cooled at 4℃. 5) Collect the bacterial culture, centrifuge at 1500g for 30s, discard the supernatant, and resuspend in 0.6mL of 1.1×TE / liAC to obtain the solution of competent yeast Y2HGol. In some embodiments, the step of transferring the interaction plasmid library and the self-activation plasmid library obtained in the above embodiments into the competent Y2HGold includes: 1) Place a phosphate buffer solution containing 10 mg / mL salmon sperm DNA (Klamar) at pH 7.0 in a 95°C water bath for 3 min, then in an ice bath for 3 min; 2) Take 5-15 μg of the interaction plasmid library or self-activation plasmid library obtained above, mix it with 40 μL salmon sperm DNA solution, 600 μL yeast competent cells and 2.5 mL PEG / LiAc (Weidi Bio: YC5001M), incubate at 30℃ for 45 min, and invert 6-8 times every 15 min to mix. 3) Add 160 μL of sterile DMSO (Weidi Bio: YC6020S); 4) Incubate in a 42℃ water bath for 20 minutes, turning the container 6-8 times every 10 minutes to mix thoroughly; 5) Centrifuge at 4000 rpm for 40s, discard the supernatant, and resuspend in 3mL of 2×YPDA or YPD Plus (Weidi Bio: YC6006S); 6) After shaking the bacteria at 30℃ and 200rpm for 1.5h, centrifuge for 30s and discard the supernatant; 7) The collected bacterial cells were resuspended in 18 mL of 0.9% NaCl, and 300 μL of the resuspended bacterial solution was plated and incubated at 30℃ for 48~72 h. 8) Take 300 μL of bacterial culture, 300 μL of a 10-fold dilution (double-distilled water), and 300 μL of a 100-fold dilution (double-distilled water) of bacterial culture, and plate them onto SD / -Leu-Trp plates with two-deficient cells to test transformation efficiency. Additionally, take 300 μL of transformants from the interaction plasmid library and 300 μL of transformants from the self-activating plasmid library, and plate them onto SD / -Leu / -Trp / -Ade / -His plates with four-deficient cells to screen for interacting genes and self-activating genes in the cDNA library. Figure 8 shows the growth status of 40 recombinant transformants from the interaction plasmid library on four-deficient plates after 3 days. Figure 9 shows the growth status of 8 recombinant transformants from the self-activating plasmid library on four-deficient plates after 3 days. In some embodiments, the step of detecting positive colonies by colony PCR includes: TaKaRa

[9164] The transformants obtained in the above examples were lysed using a rapid DNA preparation lysis buffer for bacteria, fungi, and other microorganisms to prepare a lysis buffer; The lysis buffer was denatured at 80°C for 15 min, centrifuged at low speed, and 1-5 μL of supernatant template was taken. PCR was performed using CX-Primer-F (SEQ ID NO:37) and CX-Primer-R (SEQ ID NO:38) as primer pairs. The PCR products were detected by electrophoresis on a 1% agarose gel, and positive colonies were identified based on the electrophoretic bands. In some embodiments, the steps of extracting cDNA plasmids and self-activating plasmids from positive colonies include: 1) Collect positive colonies on four-deficient interaction screening plates cultured for 2-4 days or self-activation plates cultured for 6-7 days. Centrifuge the obtained yeast culture at 12000 rpm at room temperature for 1 min, discard the supernatant, and the precipitate is the collected bacterial cells. 2) Resuspend the collected cells in the enzyme hydrolysate (classic yeast plasmid mini-preparation kit (containing lysin), PE054, Cooler Master; this kit includes package A and package B, package A is the enzyme hydrolysate, package B includes solution I, solution II, solution III, solution IV, elution buffer, matrix particle adsorption column) at 37℃, incubate at 37℃ on a shaker (200-250 rpm) for 1-2 h, centrifuge the resulting enzyme hydrolysate at 12000 rpm at room temperature for 1 min, discard the supernatant, and the resulting precipitate is the lysed cell. 3) Preheat solutions II and III in a 40°C water bath until the precipitate is completely dissolved, then let stand at room temperature until ready for use. 4) Add 180 μL of Solution I to the bacterial precipitate and mix by pipetting or vortexing. 5) Add 180 μL of solution II, invert 6-8 times to mix thoroughly, and let stand at room temperature for 5 min. 6) Add 440 μL of solution III and immediately invert to mix. 7) Centrifuge at 12,000 rpm at room temperature for 10 min. Carefully transfer the supernatant to the adsorption column, let stand at room temperature for 2 min, then centrifuge at 12,000 rpm at room temperature for 1 min. Discard the permeate. Note: White suspended matter may appear after centrifugation; simply avoid this area when using the supernatant. A small amount of suspended matter will not affect the extraction results. 8) Add 500 μL of washing buffer IV to the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the breakthrough liquid. 9) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm at room temperature for 2 min. 10) Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 30-50 μL of elution buffer (preheated to 65-70℃) to the center of the adsorption column, let stand at room temperature for 2 min, and centrifuge at 1,2000 rpm at room temperature for 1 min. 11) The breakthrough solution is the obtained interaction plasmid library or self-activating plasmid library. Perform PCR on it using detection steps such as colony PCR. Detect the amplification products by electrophoresis. If a single bright band without any drag is observed, it indicates that the extracted interaction plasmid library or self-activating plasmid library is intact and has not been degraded. In some embodiments, to improve the detection accuracy of plasmids in the extracted interacting plasmid libraries or activation plasmid libraries, the extracted plasmid-containing libraries are further subjected to PCR amplification, and the amplification products are sequenced for verification. Specifically, this includes: 1) Prepare the penetration buffer containing the interaction plasmid library or the self-activating plasmid library as shown in Table 2, and perform PCR amplification according to the procedure shown in Table 3. The cDNA library interaction test products and the self-activating test products in the cDNA library can be sorted by adding different combinations of barcodes. For example, different barcode combinations can be added during amplification, followed by sequencing (third-generation sequencing), and finally sorted and analyzed using the sequencing data. For example, the example provides 5 sets of primers containing barcodes to generate 25 combinations. AD+BD are used in combination; for example, AD-barcode1 + BD-barcode2 is used for amplifying interaction test products; AD-barcode1 + BD-barcode3 is used for amplifying self-activating products. As shown in Figure 10, the amplification product quality is good. AD-barcode1: GCGCTCTGTGTGCAGCcatacgacgtaccagattacg, SEQ ID NO:39 AD-barcode2: TCATGAGTCGACACTAcatacgacgtaccagattacg, SEQ ID NO:40 AD-barcode3: TATCTATCGTATACGCcatacgacgtaccagattacg, SEQ ID NO:41 AD-barcode4: ATCACACTGCATCTGAcatacgacgtaccagattacg, SEQ ID NO:42 AD-barcode5:ACGTACGCTCGTCATAcatacgacgtaccagattacg,SEQ ID NO:43 BD-barcode1: GCGCTCTGTGTGCAGCcatcatggaggagcagaagct, SEQ ID NO:44 BD-barcode2: TCATGAGTCGACACTAcatcatggaggagcagaagct, SEQ ID NO: 45 BD-barcode3: TATCTATCGTATACGCcatcatggaggagcagaagct, SEQ ID NO:46 BD-barcode4: ATCACACTGCATCTGAcatcatggaggagcagaagct, SEQ ID NO:47 BD-barcode5: ACGTACGCTCGTCATAcatcatggaggagcagaagct, SEQ ID NO:48 2) Take 5 μL of product from PCR amplification cycles 13, 15, and 17 and add it together with 0.1 μg of 5-kb DNA size marker into 1.5% agarose Golden View gel in 1X TAE buffer to determine the optimal number of cycles. The amplified product was purified using the KAPA Pure Beads kit. The purified 500bp product was then sequenced. Table 2 PCR reaction system Table 3 PCR reaction procedure The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for constructing an interaction plasmid library, comprising: A yeast two-hybrid vector with a first multiple cloning site and a second multiple cloning site was obtained; Obtain the first cDNA library and the second cDNA library; Obtain a linearized yeast two-hybrid vector; as well as The first cDNA library and the second cDNA library were respectively inserted into the first multiple cloning site and the second multiple cloning site of the linearized yeast two-hybrid vector; The yeast two-hybrid vector also has the following characteristics: A first sequence comprising a first multiple cloning site, a coding region of the GAL4 activation domain, a first promoter, a first operon, and a base vector initiation region, all sequentially linked. 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 cDNA library inserted into the first multiple cloning site. as well as The second sequence comprises a second multiple cloning site, a coding region of the GAL4 binding domain, a second promoter, a second operon, and a yeast 2U replication initiation region, which are sequentially linked. The transcription directions of the second multiple cloning site, the coding region of the GAL4 binding domain, the second promoter, the second 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 cDNA library 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. According to the construction method of claim 1, the yeast dual expression vector further has a termination region, which is located between the transcriptional terminal of the first sequence and the transcriptional terminal of the second sequence.

3. The construction method according to claim 2, wherein the termination region is as shown in SEQ ID NO:

11.

4. The construction method according to claim 1, wherein both the first cloning site and the second cloning site have at least two restriction endonuclease sites.

5. The construction method 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 construction method according to claim 1, wherein the coding region of the GAL4 activation structure domain is shown in SEQ ID NO:8, and the coding region of the GAL4 binding structure domain is shown in SEQ ID NO:

13.

7. The construction method 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 construction method according to claim 1, wherein the first operon includes a first operon gene and a promoter of the first operon gene, and the second operon includes a first operon gene and a promoter of the second operon gene; Optionally, both the first manipulator and the second manipulator are selected from LEU2, TRP1, or HIS.

9. The construction method according to claim 1, wherein the basic vector initiation region is as shown in SEQ ID NO:5, and the yeast 2U replication initiation region is as shown in SEQ ID NO:

3.

10. The construction method 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 operon and the second promoter, as shown in SEQ ID NO:

1.

11. The construction method according to claim 10, wherein the resistance gene is selected from one of the resistance-encoding genes for ampicillin, tetracycline, chloramphenicol, streptomycin, hygromycin, spectinomycin, kanamycin, blastcin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin, or neomycin.

12. The construction method according to any one of claims 1 to 11, wherein the step of obtaining the linearized yeast two-hybrid vector includes digesting the yeast two-hybrid vector with BamH I-HF and Nde I.

13. The construction method according to any one of claims 1 to 11, wherein the step of inserting the first cDNA library and the second cDNA library into the first multiple cloning site and the second multiple cloning site of the linearized yeast two-hybrid vector, respectively, comprises: The first cDNA library, the second cDNA library, the sequence shown in SEQ ID NO:11, and the linearized yeast two-hybrid vector were subjected to multiplex PCR. The products of the multiplex PCR reaction were transformed into *E. coli*, and positive colonies were screened; and The interaction plasmid library was extracted from the positive colonies.

14. Methods for identifying interactions in cDNA libraries, including: The interaction plasmid library obtained by the construction method according to any one of claims 1 to 13; The interaction plasmid library was transferred into yeast to obtain recombinant yeast; The interaction between the first cDNA library and the second cDNA library 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 14, wherein the selective culture medium is selected from SD / -Leu / -Trp and / or SD / -Leu / -Trp / -His / -Ade.

17. The application of the interaction plasmid library obtained by any of the construction methods of claims 1 to 13 in protein-protein interactions of the interaction plasmid library.

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