Three-plasmid yeast two-hybrid strain as well as construction method and application thereof

Abstract

The invention relates to the field of yeast double hybrid in molecular biology, in particular to a three-plasmid yeast double hybrid strain as well as a construction method and application thereof. According to the invention, an AH109 strain is taken as a chassis strain, an endogenous URA3 gene is knocked out, a URA3 screening site is released, and a NatMX gene resistance marker is introduced, so that a three-plasmid yeast two-hybrid strain is obtained. The strain can be compatible with three independent plasmids TRP1, LEU2 and URA3 at the same time, the limitation that only binary interaction can be researched in traditional yeast two-hybridization is broken through on the basis that the normal screening function is not affected, the cost and difficulty of binary interaction research are close to those of binary interaction research, more complex ternary interaction research is achieved, and the application prospect is broad. The method greatly widens the research field, improves the research efficiency and success rate, saves the time cost for researching the three-factor interaction regulation, is stable in plasmid expression level and good in experiment repeatability, and provides a powerful, efficient and low-cost new technical platform for researching the complex life process.

Description

Technical Field

[0001] This invention relates to the field of yeast two-hybrids in molecular biology, specifically to a three-plasmid yeast two-hybrid strain, its construction method, and its application. Background Technology

[0002] Protein-protein interactions are a crucial foundation for understanding life processes. Therefore, optimizing methods for investigating protein interactions and elucidating the mechanisms of interaction between multiple proteins are essential for understanding complex regulatory processes. Yeast two-hybrid assays are a traditional technique for studying protein-protein interactions, with wide applicability, low technical barriers, and ease of use. The AH109 background strain is a commonly used yeast two-hybrid strain, with genotypes including MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, MEL1GAL2UAS-GAL2TATA-ADE2, and URA3::MEL1UAS-MEL1TATA-lacZ. The auxotrophic selection markers for this strain are trp1 and leu2, and the reporter genes are lacZ, HIS3, ADE2, and MEL1. However, due to limitations in the selection markers, this strain cannot support three-plasmid co-transformation experiments and can only explore the interaction between two proteins, but cannot elucidate the regulatory mechanisms of multiple factors (such as competition / co-operation, complex formation).

[0003] Although yeast three-hybrid systems have been developed based on yeast two-hybrid systems to study the effect of a third component on the interaction between two proteins (promoting, inhibiting, or having no effect), the commonly used yeast three-hybrid systems currently on the market involve fusing the third component with one of the proteins and expressing it on the same plasmid to construct a dual-expression plasmid. Specifically, the N-terminus of the protein inserted at the MCSI site is still fused with the BD-binding domain amino acid polypeptide fragment in the Gal4 system, while the protein inserted at the MCSII site does not fuse with any tag, but requires a Pmet25 promoter upstream of it. This promoter is characterized by its ability to function only when methionine (Met) is lacking, enabling the expression of downstream protein genes. Therefore, experiments must be conducted in methionine-deficient media. Consequently, yeast strains need to be streaked at SD / -Met medium more than three times to ensure that Y2HGold or AH109 can stably synthesize methionine in order to obtain yeast three-hybrid strains that can grow in SD / -Met medium. Therefore, yeast three-hybrid experiments have several drawbacks, including long experimental time; complex and inefficient construction of expression vectors containing two factors; and unstable expression levels of the third factor, which are induced by methionine, leading to poor reproducibility of experimental results.

[0004] Based on this, the present invention aims to construct a yeast two-hybrid strain that is time-efficient, simple to process, highly efficient, and has a stable expression level, capable of simultaneously accommodating three different screening marker plasmids, without affecting the normal screening function, so as to explore the competitive or synergistic interaction of the three factors (protein, RNA or small molecules). Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a three-plasmid yeast two-hybrid strain to address the shortcomings of the prior art.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the three-plasmid yeast two-hybrid strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the aforementioned three-plasmid yeast two-hybrid strain.

[0008] The final technical problem to be solved by this invention is to provide a three-plasmid yeast two-hybrid system.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a three-plasmid yeast two-hybrid strain obtained by knocking out the endogenous URA3 gene, releasing the URA3 selection site, and introducing the NatMX gene resistance marker.

[0011] in, Figure 1 A schematic diagram showing the knockout of the URA3 gene and the insertion of the NatMX resistance marker by homologous recombination in the yeast genome is presented.

[0012] The three-plasmid yeast two-hybrid strain can simultaneously transfect three independent plasmids carrying selection markers TRP1, LEU2, and URA3, respectively.

[0013] Although the three-plasmid yeast two-hybrid strain has been modified, it does not affect the original function of the AH109 strain, that is, it retains the AH109 strain reporter gene system (HIS3 / ADE2 / lacZ) and does not require reconstruction of the screening system.

[0014] The URA3 gene has the nucleotide sequence shown in SEQ ID NO.18; the NatMX gene has the nucleotide sequence shown in SEQ ID NO.19.

[0015] Secondly, the present invention provides a method for constructing the aforementioned three-plasmid yeast two-hybrid strain. AH109 strain cells are resuspended in a transformation medium containing the NatMX gene fragment, vortexed to mix, placed in ice water and then heat-shocked, centrifuged and the transformation medium discarded. The cells are then revived in YPD liquid medium and plated on YPD plates containing 100 μg / mL of Norilskine resistance. Positive clone verification yields the aforementioned three-plasmid yeast two-hybrid strain.

[0016] The AH109 strain cells were prepared by inoculating the AH109 strain seed culture into YPD liquid medium and culturing it until OD500. 600 =1.0~1.5, then centrifuged, washed with sterile water, centrifuged again, washed with lithium acetate buffer solution to a final concentration of 100 mmol / L, and centrifuged again.

[0017] Specifically, the seed culture of the AH109 strain needs to be cultured overnight.

[0018] Specifically, the inoculation is performed at a dosage of 5-15% v / v. In some embodiments of the present invention, the inoculation dosage is 10% v / v.

[0019] The transformation solution containing the NatMX gene fragment has a total volume of 360 μL and its formula is as follows: 240 μL 50% PEG3350, 50 μL 2 mg / mL ssDNA, 36 μL 1 mol / L LiAC, 10 μL NatMX gene fragment, and the remainder is sterile water.

[0020] The NatMX gene fragment was obtained by PCR amplification using plasmid pFA6a-NatMX as a template and specific primers.

[0021] Specifically, the 5' end of the specific primer contains sequences homologous to the upstream and downstream of the URA3 gene.

[0022] In some embodiments of the present invention, the specific primers are URA3 dele F1 and URA3 dele R1, the 5' end of which contains a 39bp sequence homologous to the upstream and downstream of the URA3 gene.

[0023] The heat shock conditions are: 42℃ for 45 minutes; the centrifugation conditions are: 3000g for 30 seconds.

[0024] The YPD plate is a YPD plate containing 100 μg / mL of Norscin resistance.

[0025] The cultivation conditions are as follows: 30℃ for 3 to 5 days.

[0026] The positive clone verification includes PCR verification and phenotypic verification.

[0027] Thirdly, the application of the aforementioned three-plasmid yeast two-hybrid strain in multifactor interaction research is also within the scope of protection of this invention.

[0028] The interaction is either a competitive interaction or a complex interaction; the multifactor is a protein, RNA, and / or a small molecule.

[0029] In some embodiments of the present invention, since SLD3 interacts with both MCM2 and CDC45, MCM2 and CDC45 should compete to bind to SLD3. Using the triplasmid yeast strain constructed in this invention, it was successfully verified that the interaction between MCM2 and SLD3 was weakened to some extent after being transformed into CDC45, proving that there is a competitive interaction among the three.

[0030] Fourthly, the present invention provides a three-plasmid yeast two-hybrid system.

[0031] The three-plasmid yeast two-hybrid system uses the three-plasmid yeast two-hybrid strain described in this invention as the chassis strain.

[0032] Beneficial effects:

[0033] (1) This invention successfully released the URA3 selection site for screening a third plasmid by knocking out the URA3 gene in strain AH109 with NatMX through homologous recombination and introducing the NatMX resistance marker. Without affecting the original function of strain AH109, ​​the modified strain can be transfected with independent plasmids with three selection markers, namely TRP1, LEU2 and URA3, respectively. This makes it possible to study the interaction of three independent factors in the same yeast cell simultaneously, providing a research tool for analyzing complex protein interaction networks.

[0034] (2) Using the modified strain of this invention, the competitive interaction among CDC45, MCM2, and SLD3 was successfully verified. The three-plasmid yeast two-hybrid system constructed in this invention is not limited to competitive studies, but can also be widely used for: synergistic interactions, verifying whether the third factor enhances the binding of two proteins; complex formation, studying whether the three proteins form a ternary complex; and non-protein factor regulation, studying the interaction between RNA-protein or small molecule-protein-protein by expressing RNA or small molecules.

[0035] (3) This invention employs a homologous recombination strategy, performing precise URA3 site replacement only on the AH109 genome, completely preserving all the original reporter gene systems (HIS3, ADE2, lacZ, MEL1) of the strain. It allows for seamless upgrading to a three-plasmid yeast two-hybrid research system without altering existing experimental procedures, vector systems, or detection methods, resulting in low technology migration costs. The newly added NatMX resistance screening and auxotrophic selection markers (TRP1, LEU2, URA3) do not interfere with each other, forming a multi-layered, orthogonal screening guarantee, ensuring the high purity of the co-transfected strain and the reliability of experimental results.

[0036] (4) Compared with existing yeast three-hybrid systems, this invention does not require the construction of complex fusion vectors. It only requires cloning the third factor into the URA3 screening vector and utilizing yeast two-hybrid technology to conduct more complex ternary interaction studies at a cost and difficulty approaching that of binary interaction studies. This greatly improves research efficiency and success rate, while saving time costs in exploring the regulation of three-factor interactions. The plasmid expression level is stable, and the experiment has good reproducibility. Furthermore, the strength of the promoter can be changed to adjust the expression level of the inserted factor in the URA3 screening plasmid, thus addressing level-mediated interaction differences. Attached Figure Description

[0037] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0038] Figure 1 A schematic diagram of knocking out the URA3 gene and inserting the NatMX resistance marker through homologous recombination of the yeast genome.

[0039] Figure 2 To verify the URA3 knockout strain by PCR.

[0040] Figure 3 The growth phenotype of URA knockout was confirmed by streaking. The left image shows the growth phenotype on complete medium, and the right image shows the growth phenotype on URA-deficient medium.

[0041] Figure 4 To investigate the effect of URA3 knockout on the interaction ability using known interacting proteins. From top to bottom, the proteins are AD-ECO1 / BD-MMS22, AD-CdC45 / BD-SLD3, and AD-MCM2 / BD-SLD3.

[0042] Figure 5 To detect the competitive relationship of three factors using two pairs of known interacting proteins. The left panel shows the growth phenotype on the -Leu-Trp-Ura synthetic medium, and the right panel shows the growth phenotype on the -Leu-Trp-Ura-His selection medium.

[0043] Figure 6 This is a diagram showing the experimental types that can be studied using this system. Among them, (1) represents competitive interactions between protein factors, (2) represents cooperative interactions between protein factors, and (3) represents competitive or cooperative interactions between nucleic acid types. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0046] In the following examples, the plasmid pFA6a-NatMX of the Norsmin resistance gene NatMX was donated by Professor Lou Huiqiang of China Agricultural University.

[0047] The primer information used in the following embodiments is shown in Table 1 below.

[0048] Table 1 Primer information for this invention

[0049]

[0050]

[0051] In the following examples, the culture medium used for yeast culture is as follows:

[0052] (1) YPD liquid culture medium, the formula is: 10g yeast extract, 20g peptone, 20g glucose, make up to 1L, sterilize at 115℃ for 30min.

[0053] (2) SC medium is prepared as follows: 1.7g yeast nitrogen base (excluding amino acids and ammonium sulfate), 20g glucose (or galactose or raffinose), 5g ammonium sulfate, 2g amino acid deficiency mixture, and 20g agar are dissolved in 800mL distilled water. The pH is adjusted to 6.0–6.5 with 1mol / L NaOH, and then the volume is brought to 1L. The mixture is then sterilized at 115℃ for 30min. The amino acid deficiency mixture can be prepared without one or more amino acids as needed, to create the corresponding SC nutrient deficiency medium, which is stored at room temperature. The specific composition of the amino acid deficiency mixture is shown in Table 2.

[0054] Table 2. Selective Composition of Amino Acid Defect Mixture

[0055]

[0056]

[0057] In the following examples, the transformation solution has a total volume of 360 μL and consists of 240 μL of 50% PEG3350, 50 μL of 2 mg / mL ssDNA, 36 μL of 1 mol / L LiAC, X / 10 μL of plasmid / PCR product, and the remainder is sterile water.

[0058] For 50% PEG3350: Take 50g of PEG3350 powder, dissolve it in deionized water, bring the volume to 100mL, autoclave, and store at 4℃. Note: 3350 refers to the average molecular weight. Heating can be used to aid dissolution during the process.

[0059] 2 mg / mL ssDNA: Dissolve ssDNA (Sigma, D1626) in 10 mmol / L Tris-HCl (pH 8.0) and 1.0 mmol / L EDTA (pH 8.0) to a final concentration of 2 mg / mL. After thorough stirring on a magnetic stirrer, repeatedly pipette the solution about 10 times using a syringe with a needle to help break the ssDNA into different lengths. Before use, boil the ssDNA in water for 5–10 minutes, then immediately place it in ice water to prevent annealing.

[0060] 1 mol / L LiAC: Weigh 6.599 g of LiAC, dissolve it in deionized water, bring the volume to 100 mL, filter to remove bacteria, and store at 4 °C.

[0061] In the following examples, the Norlesmycin (antibiotic Nat) was purchased from Taobao and prepared as follows: a certain mass of Norlesmycin powder was weighed and dissolved in a certain volume of deionized water to make the stock solution concentration 100 mg / mL. After filtration and sterilization, it was dispensed into EP tubes and stored at -20°C.

[0062] Example 1: Construction of the AH109 modified strain, namely AH109 ura3Δ::NatMX strain

[0063] I. PCR amplification of the NatMX gene fragment

[0064] Using plasmid pFA6a-NatMX, containing the nourescin resistance gene NatMX, as a template, specific amplification primers URA3 dele F1 (SEQ ID NO.1) and URA3 dele R1 (SEQ ID NO.2) were designed at both ends of the NatMX gene. The 5' ends of these primers contain 39 bp sequences homologous to the upstream and downstream ends of URA3. Under the action of high-fidelity DNA polymerase, PCR amplification yielded the NatMX gene fragment with homologous arms of URA3 at both ends. The sequence of the URA3 gene is shown in SEQ ID NO.18; the sequence of the NatMX gene is shown in SEQ ID NO.19.

[0065] The PCR amplification reaction system consisted of 50 μL: 25 μL 2× buffer, 1 μL dNTPs, 1 μL primer F, 1 μL primer R, 1 μL template, 1 μL enzyme, and 20 μL ddH2O; the reaction conditions were: 95℃ for 5 min, (95℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min and 30 s) × 29 cycles, and 72℃ for 5 min.

[0066] II. Transformation and Screening of Lithium Acetate (LiAC) Yeast

[0067] 1. Yeast Conversion

[0068] The overnight cultured AH109 strain seed culture was inoculated into YPD liquid medium at an inoculation rate of 10% v / v and cultured at 30°C with shaking for 6–8 hours. At this time, the cells are in the logarithmic growth phase, and the OD... 600 =1.0~1.5. Centrifuge at 3000g for 30s to collect cells, repeat 1~2 times, and completely aspirate any remaining culture medium with a pipette tip during the last centrifugation. Next, wash the cells with 1mL of sterile water, centrifuge at 3000g for 30s to collect cells, aspirate any remaining liquid with a pipette tip, and then wash the cells once more with 1mL of lithium acetate buffer with a final concentration of 100mmol / L, centrifuge at 3000g for 30s to collect cells, and aspirate any remaining liquid with a pipette tip.

[0069] Resuspend the cells in the transformation medium, shake well to mix, incubate in ice water for 10 min, heat shock at 42℃ for 45 min, centrifuge at 3000g for 30 s and discard the transformation medium, add 1 mL of YPD liquid medium to recover for 3 h, then spread on YPD plates containing 100 μg / mL of Norilsk resistant cells and incubate at 30℃ for 3–5 days.

[0070] 2. Validation of positive clones

[0071] (1) Yeast genome extraction: After transformants grow on YPD plates (selection plates) containing norethin resistance, pick single-clone transformants and streak them onto new selection plates, then incubate them in an incubator at the corresponding temperature for 10 h. Pick single colonies the size of rice grains and resuspend them thoroughly in 100 μL of genome extraction buffer (final concentration 200 mmol / L LiAC, 1 wt% SDS), and incubate at 75 °C for 10 min. Add 300 μL of anhydrous ethanol, shake thoroughly, centrifuge at 14000 g for 5 min, and discard the supernatant. Add 1 mL of 75% ethanol, shake thoroughly, centrifuge at 14000 g for 5 min, discard the supernatant, and after a quick high-speed shake, aspirate as much residual liquid as possible with a pipette tip. Place the open end in a 65 °C oven to dry any residual ethanol. Dissolve the DNA in 100 μL of deionized water, centrifuge at 14000 g for 5 min, and take 2 μL as PCR template.

[0072] (2) PCR verification: Since the selection markers used for gene knockout or tagging contain universal upstream or downstream primers, only paired downstream or upstream primers need to be designed for the target gene. If the knockout is correct, PCR using URA3 internal primers will not yield the theoretically sized fragment, but using NatMX internal primers will amplify the fragment to the specific size.

[0073] Specifically, using SEQ ID NO.5 (URA3 ORF Chk F) and SEQ ID NO.4 (URA3 ter Chk R) as primers (named primer 1), and SEQ ID NO.3 (NatMX Chk F) and SEQ ID NO.4 (URA3 ter Chk R) as primers (named primer 2), PCR was used to verify whether the endogenous URA3 gene was successfully knocked out. The results are as follows: Figure 2 As shown. From Figure 2 As can be seen, primer 1 can only identify PCR products using the wild-type AH109 genome as a template, while the URA3 knockout strain does not amplify any PCR products, indicating that the URA3 knockout was successful. To further prove this, we used primer 2 for further PCR, and we can see that only PCR products using the URA3 knockout strain genome as a template can be amplified, indicating that URA3 was successfully knocked out by NatMX.

[0074] (3) Phenotypic verification (screening marker line identification)

[0075] To further confirm the acquisition of the strain, the strain before modification (AH109 strain) and the strain after modification (AH109URA3dele strain) were streaked on YPD plates (control plates) and -Ura screening plates (to verify the auxotrophic phenotype) respectively, and then observed after incubation at 30°C for 48 hours.

[0076] The results are as follows Figure 3 As shown, because URA3 is present in strain AH109, ​​strain AH109 can grow on a medium lacking Ura, while strain AH109ura3Δ::NatMX cannot grow, indicating that the genetic phenotype of the modified strain AH109ura3Δ::NatMX is not problematic.

[0077] Example 2: Interaction study of modified strain AH109 ura3Δ::NatMX

[0078] To demonstrate the usability of the modified strain AH109 ura3Δ::NatMX, it was first necessary to confirm that it would not affect the original interaction screening function. Therefore, based on literature reports, this embodiment selected three known interacting protein pairs: AD-ECO1 / BD-MMS22, AD-CdC45 / BD-SLD3, and AD-MCM2 / BD-SLD3. Yeast transformation was used to transform the above plasmids and their corresponding control plasmids into AH109 and AH109 ura3Δ::NatMX strains. After transformants grew, interaction phenotypes were detected using serial dilution phenotypic analysis.

[0079] The process of identifying interaction phenotypes through gradient dilution phenotyping is as follows:

[0080] (1) Pick yeast colonies and put them into 3-5 mL of culture medium and culture overnight with shaking.

[0081] (2) Dilute the yeast culture to OD level. 600 =0.2. According to the calculation formula X×Y×Vx=1mL×0.2 (where X represents the OD value measured by the spectrophotometer, Y represents the dilution factor, and Vx represents the required volume of bacterial solution), the volume of yeast solution used is obtained, and the remainder is made up to 1mL with sterile water.

[0082] (3) Take 200 μL of diluted yeast culture and place it in the first row of a 96-well plate. Then, take 200 μL of sterile water in the next four rows and perform serial dilutions of 5 times.

[0083] (4) Using a pipette, take 7 μL of serially diluted bacterial solution and spot it onto the corresponding plate from low concentration to high concentration.

[0084] (5) After the plate is dried, invert it into an incubator at the corresponding temperature.

[0085] (6) Incubate for 72 hours, take a picture and save the results.

[0086] The specific process is as follows:

[0087] 1. Interacting proteins AD-ECO1 / BD-MMS22

[0088] Using the AH109 genome as a template, the MMS22 gene fragment was amplified by PCR using primers BD-MMS22F (SEQ ID NO.10) and BD-MMS22R (SEQ ID NO.11). The nucleotide sequence of the MMS22 gene is shown in SEQ ID NO.20. The PCR product and pGBKT7 vector were digested with BamH1 and Sal1 enzymes, ligated overnight at 16°C using T4 ligase, and transformed into *E. coli*. Sequencing confirmed the presence of the pGBKT7-BD-MMS22 plasmid.

[0089] Using the AH109 genome as a template, the ECO1 gene fragment was amplified by PCR using primers AD-ECO1 F (SEQ ID NO.12) and AD-ECO1 R (SEQ ID NO.13). The nucleotide sequence of the ECO1 gene is shown in SEQ ID NO.21. The PCR product and pGADT7 vector were digested with BamH1 and Xho1 enzymes, ligated overnight at 16°C using T4 ligase, and transformed into E. coli. The pGADT7-AD-ECO1 plasmid was extracted and verified by sequencing.

[0090] Following the LiAc yeast transformation method, the pGBKT7-BD-MMS22 and pGADT7-AD-ECO1 plasmids were co-transformed into the modified strain AH109 ura3Δ::NatMX, with the original strain AH109 serving as a control. The transformed strains were then plated onto SC-Trp / -Leu auxotrophic plates containing 100 μg / mL NatMX resistance and incubated at 30°C for 48 h.

[0091] Pick co-transformed colonies and inoculate them into 3–5 mL of SC-Trp / -Leu liquid medium, then incubate overnight with shaking. Adjust the yeast culture to OD200. 600 =0.2. Place 200 μL of yeast culture into the first row of a 96-well plate. For the subsequent four rows, use a multi-channel pipette to draw 200 μL of sterile water, performing a 5-fold serial dilution. Take 7 μL of each dilution gradient, from low to high concentration, and spot it onto -Leu-Trp and -Leu-Trp-His (AH109 background bacteria) or -Leu-Trp-Ura and -Leu-Trp-His-Ura (AH109 ura3Δ::NatMX background bacteria) plates. Incubate at 30°C for 72 h and observe the degree of interaction activation.

[0092] 2. Interacting proteins AD-CDC45 / BD-SLD3

[0093] Using the AH109 genome as a template, the SLD3 fragment was amplified by PCR using primers BD-SLD3 F (SEQ ID NO. 8) and BD-SLD3 R (SEQ ID NO. 9). The nucleotide sequence of the SLD3 gene is shown in SEQ ID NO. 23. The PCR product and pGBKT7 vector were digested with BamH1 and Not1, ligated overnight at 16°C using T4 ligase, and transformed into E. coli. Sequencing confirmed the extraction of the pGBKT7-BD-SLD3 plasmid.

[0094] Using the AH109 genome as a template, the PCR product CDC45 fragment was amplified by primers AD-CDC45 F (SEQ ID NO. 6) and AD-CDC45R (SEQ ID NO. 7). The nucleotide sequence of the CDC45 gene is shown in SEQ ID NO. 22. The PCR product and pGADT7 vector were digested with BamH1 and Xho1 enzymes, ligated overnight at 16°C using T4 ligase, and transformed into E. coli. The pGADT7-AD-CDC45 plasmid was extracted and verified by sequencing.

[0095] Following the LiAc yeast transformation method, pGBKT7-BD-SLD3 and pGADT7-AD-CDC45 plasmids were co-transformed into the modified strain AH109 ura3Δ::NatMX, with the original strain AH109 serving as a control. The transformed strains were then plated on SC-Trp / -Leu auxotrophic plates containing 100 μg / mL NatMX resistance and incubated at 30°C for 48 h.

[0096] Pick co-transformed colonies and inoculate them into 3–5 mL of SC-Trp / -Leu liquid medium, then incubate overnight with shaking. Adjust the yeast culture to OD200. 600 =0.2. Place 200 μL of yeast culture into the first row of a 96-well plate. For the subsequent four rows, use a multi-channel pipette to draw 200 μL of sterile water, performing a 5-fold serial dilution. Take 7 μL of each dilution gradient, from low to high concentration, and spot it onto -Leu-Trp and -Leu-Trp-His (AH109 background bacteria) or -Leu-Trp-Ura and -Leu-Trp-His-Ura (AH109 ura3Δ::NatMX background bacteria) plates. Incubate at 30°C for 72 h and observe the degree of interaction activation.

[0097] 3. Interacting proteins AD-MCM2 / BD-SLD3

[0098] Using the AH109 genome as a template, the SLD3 fragment was amplified by PCR using primers BD-SLD3 F (SEQ ID NO.8) and BD-SLD3 R (SEQ ID NO.9). The PCR product and pGBKT7 vector were digested with BamH1 and Not1, ligated overnight at 16°C using T4 ligase, and transformed into *E. coli*. Sequencing confirmed the extraction of the pGBKT7-BD-SLD3 plasmid.

[0099] Using the AH109 genome as a template, the PCR product MCM2 fragment was amplified by PCR using primers AD-MCM2 F (SEQ ID NO.14) and AD-MCM2R (SEQ ID NO.15). The nucleotide sequence of the MCM2 gene is shown in SEQ ID NO.24. The PCR product and pGADT7 vector were digested with BamH1 and Xho1 enzymes, ligated overnight at 16°C using T4 ligase, and transformed into E. coli. The pGADT7-AD-MCM2 plasmid was extracted and verified by sequencing.

[0100] Following the LiAc yeast transformation method, pGBKT7-BD-SLD3 and pGADT7-AD-MCM2 plasmids were co-transformed into the modified strain AH109 ura3Δ::NatMX, with the original strain AH109 serving as a control. The transformed strains were then plated on SC-Trp / -Leu auxotrophic plates containing 100 μg / mL NatMX resistance and incubated at 30°C for 48 h.

[0101] Pick co-transformed colonies and inoculate them into 3–5 mL of SC-Trp / -Leu liquid medium, then incubate overnight with shaking. Adjust the yeast culture to OD200. 600 =0.2. Place 200 μL of yeast culture into the first row of a 96-well plate. For the subsequent four rows, use a multi-channel pipette to draw 200 μL of sterile water, performing a 5-fold serial dilution. Take 7 μL of each dilution gradient, from low to high concentration, and spot it onto -Leu-Trp and -Leu-Trp-His (AH109 background bacteria) or -Leu-Trp-Ura and -Leu-Trp-His-Ura (AH109 ura3Δ::NatMX background bacteria) plates. Incubate at 30°C for 72 h and observe the degree of interaction activation.

[0102] The results are as follows Figure 4 As shown, three pairs of protein interactions were identified in strain AH109, ​​which is consistent with previous reports. Simultaneously, equivalent interactions were also identified in the modified strain AH109 ura3Δ::NatMX constructed in this invention, indicating that the modification does not affect the original function.

[0103] Example 3: Study on the interaction of three plasmid systems in the modified strain AH109 ura3Δ::NatMX

[0104] To further demonstrate that the constructed strain can be used in a three-plasmid system, this invention constructs CDC45 into an expression vector containing the URA3 selection marker, based on the MCM2 / SLD3 interaction. Since SLD3 interacts with both MCM2 and CDC45, MCM2 and CDC45 should compete for binding to SLD3. To demonstrate this, this invention introduces an empty vector and overexpression of CDC45, respectively, based on the SLD3 / MCM2 interaction.

[0105] Specifically, using the AH109 genome as a template, the CDC45 fragment was amplified by PCR using primers AD-CDC45 F (SEQ ID NO. 6) and AD-CDC45R (SEQ ID NO. 7). The PCR product and the URA3-selective-labeled vector were digested with BamH1 and Xho1 enzymes, ligated overnight at 16°C using T4 ligase, and transformed into *E. coli*. Sequencing verification confirmed the presence of the pURA3-AD-CDC45 plasmid. An empty URA3-selective-labeled vector served as a control group.

[0106] Following the LiAc yeast transformation method, pGBKT7-BD-SLD3, pGADT7-AD-MCM2, and pURA3-AD-CDC45 plasmids were co-transformed into the modified strain AH109 ura3Δ::NatMX. The transformed strain was then plated on SC-Trp / -Leu / -Ura auxotrophic plates containing 100 μg / mL NatMX resistance and incubated at 30°C for 48 h.

[0107] Pick co-transformed colonies and inoculate them into 3–5 mL of SC-Trp / -Leu / -Ura liquid medium, and incubate overnight with shaking. Adjust the yeast culture to OD200. 600 =0.2. Place 200 μL of yeast culture into the first row of a 96-well plate. For the subsequent four rows, use a multi-channel pipette to draw 200 μL of sterile water, performing a 5-fold serial dilution. Take 7 μL of each dilution, from low to high concentration, and spot it onto -Leu-Trp-Ura and -Leu-Trp-His-Ura plates. Incubate at 30°C for 72 h and observe the degree of interaction activation.

[0108] from Figure 5It can be seen that, compared with the control group containing the empty vector, the interaction between MCM2 and SLD3 was weakened to some extent after transformation into CDC45, proving that there is a competitive interaction among the three. These results demonstrate that this invention successfully constructed a plasmid that can fully support co-transformation and screening using a yeast two-hybrid system with three selection markers (TRP1, LEU2, and URA3), respectively. Compared with the yeast three-hybrid system, this invention saves time in exploring the regulation of three-factor (protein, RNA, or small molecule) interactions, and the plasmid expression levels are stable with good experimental reproducibility. Furthermore, the expression level of the inserted factor in the URA3 selection marker plasmid can be adjusted later by flexibly changing the promoter strength to address level-mediated interaction differences.

[0109] Based on this, the present invention can also verify the interaction relationship between complexes. For example, when X and Y interact, the interaction between Z and X is stronger, and Z and Y do not interact. Then, the three-plasmid yeast two-hybrid system of the present invention can be used to detect the competitive relationship between XYZ. Figure 6 (1) Furthermore, X and Y do not interact, but X and Y each interact with Z. Therefore, during XYZ co-rotation, the interaction between X and Y can be detected, proving that the XYZ complex is mediated by the Z subunit. Figure 6 (2) In addition, the three-plasmid yeast two-hybrid system of the present invention is not limited to protein, and can also be replaced with DNA or RNA sequences, the principle of which is as follows. Figure 6 As shown in Figure 3.

[0110] This invention provides a three-plasmid yeast two-hybrid strain, its construction method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A three-plasmid yeast two-hybrid strain, characterized in that, The strain AH109 was used as the chassis strain. The endogenous URA3 gene was knocked out to release the URA3 selection site, and the NatMX gene resistance marker was introduced. The three-plasmid yeast two-hybrid strain can simultaneously transfect three independent plasmids carrying selection markers TRP1, LEU2, and URA3, respectively.

2. The three-plasmid yeast two-hybrid strain according to claim 1, characterized in that, The nucleotide sequence of the URA3 gene is shown in SEQ ID NO.18; the nucleotide sequence of the NatMX gene is shown in SEQ ID NO.

19.

3. The method for constructing the three-plasmid yeast two-hybrid strain according to claim 1 or 2, characterized in that, The AH109 strain cells were resuspended in a transformation medium containing the NatMX gene fragment, vortexed to mix, incubated in ice water, then heat-shocked, centrifuged and the transformation medium was discarded. The cells were then revived in YPD liquid medium and plated on YPD plates containing 100 μg / mL of Norlesin resistance. Positive clone verification yielded the three-plasmid yeast two-hybrid strain.

4. The construction method according to claim 3, characterized in that, The AH109 strain cells were prepared by inoculating the AH109 strain seed culture into YPD liquid medium and culturing it until OD500. 600 =1.0~1.5, then centrifuged, washed with sterile water, centrifuged again, washed with lithium acetate buffer solution to a final concentration of 100 mmol / L, and centrifuged again.

5. The construction method according to claim 3, characterized in that, The transformation solution containing the NatMX gene fragment has a total volume of 360 μL and its formulation is as follows: 240 μL 50% PEG3350, 50 μL 2 mg / mL ssDNA, 36 μL 1 mol / L LiAC, 10 μL NatMX gene fragment, and the remainder is sterile water.

6. The construction method according to claim 3, characterized in that, The NatMX gene fragment was obtained by PCR amplification using a specific primer containing a sequence at the 5' end that is homologous to the upstream and downstream of the URA3 gene.

7. The construction method according to claim 3, characterized in that, The heat shock conditions are: 42℃ for 45 min; the centrifugation conditions are: 3000g for 30 s; the YPD plate is a YPD plate containing 100 μg / mL of Norscin resistance.

8. The application of the three-plasmid yeast two-hybrid strain according to claim 1 or 2 in multifactor interaction studies.

9. The application according to claim 8, characterized in that, The interaction is either a competitive interaction or a complex synergistic interaction; the multifactor is a protein, RNA, and / or a small molecule.

10. A three-plasmid yeast two-hybrid system, characterized in that, The three-plasmid yeast two-hybrid strain described in claim 1 or 2 is used as the chassis strain.

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