A primer set and analysis method for constructing high-throughput interaction protein analysis

Abstract

The present invention discloses a primer set and analysis method for constructing high-throughput interaction protein analysis, creating plate barcode primers and block barcode primers, designing 12 plate front-end primers and 10 back-end primers, and 20 block front-end and back-end primers. The primer set is then used to construct a high-throughput interaction protein analysis method, and the interaction combination is randomly screened by "library to library" mating. The bait and target gene fragments in the protein interaction positive clones are subjected to two rounds of PCR amplification, so that each PCR amplification product is accompanied by a unique combination of four barcodes; finally, the Pacbio high read length and high-throughput sequencing method are used to mix and sequence the PCR products of all positive clones; carrying the same four barcode combinations. The present invention has the advantages of high throughput, high efficiency, low cost and wide application of species.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set for constructing high-throughput sequencing of interacting proteins and a method for constructing a high-throughput analysis of interacting proteins. Background Art

[0002] According to the central dogma, proteins are the ultimate executors of life functions. The vast majority of proteins perform their functions by interacting with each other to form protein complexes, or by regulating each other's functional activities through interactions, thereby precisely regulating various biological processes. Therefore, the identification and analysis of interacting proteins has become an important technical means for studying the functions of target proteins in life sciences. With the rapid development of biotechnology, especially the continuous innovation of DNA sequencing technology, high-throughput identification of protein interaction combinations and drawing of interaction maps can provide an important reference for the analysis of protein-level regulatory mechanisms for the growth and development of living organisms.

[0003] Traditional yeast two-hybrid technology is usually used for single "bait vs prey" screening of interacting proteins, with low identification throughput, low efficiency, and high cost. The yeast two-hybrid library screening technology that was subsequently developed can use a bait protein to screen the entire cDNA library, and can screen all proteins in the cDNA library that interact with the bait protein. The target protein screened by this technology still needs to be Sanger sequenced, which is labor-intensive, costly, and inefficient. Subsequently, yeast two-hybrid array (Y2H array) screening was developed to improve throughput and specificity by constructing a large number of bait vectors and target vectors.

[0004] At present, a strategy combining the principles of next-generation sequencing (NGS) and stitch PCR is used to screen interacting proteins with high throughput through yeast two-hybrid arrays. However, stitch PCR requires three PCR amplifications to connect the bait and target genes using a 40bp fragment. In this process, there are many difficulties, such as the read length limitation of NGS sequencing, the difficulty of PCR amplification, the low detection efficiency, the large workload, and the low accuracy of interaction information.

[0005] Therefore, in order to create a large-scale protein interaction network map and significantly improve scientific research efficiency, it is urgent to find a primer set and analysis method for constructing high-throughput identification of protein interactions. Summary of the invention

[0006] In view of this, the present invention provides a primer set and analysis method for constructing high-throughput interacting proteins, which has high throughput, high efficiency and low cost, and can be widely used in high-throughput identification of protein interactions and drawing of protein interaction maps in species such as humans, animals, plants, and microorganisms, providing important clues and information for the study of protein functions.

[0007] In order to achieve the above object, the present invention provides a primer set for constructing high-throughput interaction protein analysis, including plate barcode primers and blockbarcode primers;

[0008] The plate barcode primer consists of three parts: a vector backbone sequence, an 8 bp barcode sequence, and an 18 bp bridge sequence;

[0009] The vector backbone sequence includes: backbone-T7-F: as shown in SEQ ID NO.1; backbone-AD-R, as shown in SEQ ID NO.2; and backbone-BD-R, as shown in SEQ ID NO.3;

[0010] The 8 bp barcode sequence includes: barcode-F1: CGCTGCTC; barcode-F2: CTACTCGA; barcode-F3: TGTGTGCC; barcode-F4: AAGTTATC; barcode-F5: CAATGA TG; barcode-F6: GTCGCTGT; barcode-F7: GATGCCGG; barcode-F8: AGCAGAGA; barcode-F9: GGTGTCCG; barcode-F10: CCGTGCCA; barcode-F11: TTGTAGAT; barcode-F12: TAATAGGA; barcode-R1: GATCTGCC; barcode-R2: GTTCATCT; barcode-R3: TGACCGAG; barcode-R4: GATTCACA; barco de-R5: CGTCCGTG; barcode-R6: CTCTAGTG; barcode-R7: CCATCTGC; barcode-R8: ACCTGATG; barcode-R9: TGGTCTCC; barcode-R10: GCTGATCG;

[0011] The 18 bp bridge sequence includes: Bridge-F, as shown in SEQ ID NO.4; and Bridge-R, as shown in SEQ ID NO.5;

[0012] The blockbarcode primer consists of two parts: an 18 bp bridge sequence and a 16 bp barcode sequence; the 18 bp bridge sequence is shown in SEQ ID NO.4 and SEQ ID NO.5;

[0013] The 16bp barcode sequence includes:

[0014] Barcode-F1 is shown in SEQ ID NO.6; Barcode-F2 is shown in SEQ ID NO.7; Barcode-F3 is shown in SEQ ID NO.8; Barcode-F4 is shown in SEQ ID NO.9; Barcode-F5 is shown in SEQ ID NO.10; Barcode-F6 is shown in SEQ ID NO.11; Barcode-F7 is shown in SEQ ID NO.12; Barcode-F8 is shown in SEQ ID NO.13; Barcode-F9 is shown in SEQ ID NO.14; Barcode-F10 is shown in SEQ ID NO.15; Barcode-F11 is shown in SEQ ID NO.16; Barcode-F12 is shown in SEQ ID NO.17; Barcode-F13 is shown in SEQ ID NO.18; Barcode-F14 is shown in SEQ ID NO.19; Barcode-F15 is shown in SEQ ID NO.20; Barcode-F16 is shown in SEQ ID NO. NO.21; Barcode-F17, as shown in SEQ ID NO.22; Barcode-F18, as shown in SEQ ID NO.23; Barcode-F19, as shown in SEQ ID NO.24; Barcode-F20, as shown in SEQ ID NO.25; Barcode-R1, as shown in SEQ ID NO.26; Barcode-R2, as shown in SEQ ID NO.27; Barcode-R3, as shown in SEQ ID NO.28; Barcode-R4, as shown in SEQ ID NO.29; Barcode-R5, as shown in SEQ ID NO.30; Barcode-R6, as shown in SEQ ID NO.31; Barcode-R7, as shown in SEQ ID NO.32; Barcode-R8, as shown in SEQ ID NO.33; Barcode-R9, as shown in SEQ ID NO.34; Barcode-R10, as shown in SEQ ID NO.35; Barcode-R11, as shown in SEQ ID NO.36; Barcode-R12, as shown in SEQ ID NO. ID NO.37; Barcode-R13, as shown in SEQ ID NO.38; Barcode-R14, as shown in SEQ ID NO.39; Barcode-R15, as shown in SEQ ID NO.40; Barcode-R16, as shown in SEQ ID NO.41; Barcode-R17, as shown in SEQ ID NO.42; Barcode-R18, as shown in SEQ ID NO.43; Barcode-R19, as shown in SEQ ID NO.44; Barcode-R20, as shown in SEQ ID NO.45. .

[0015] Preferably, the plate barcode primer specifically includes the following sequence information: plate-T7F1, as shown in SEQ ID NO.46; plate-T7F2, as shown in SEQ ID NO.47; plate-T7F3, as shown in SEQ ID NO.48; plate-T7F4, as shown in SEQ ID NO.49; plate-T7F5, as shown in SEQ ID NO.50; plate-T7F6, as shown in SEQ ID NO.51; plate-T7F7, as shown in SEQ ID NO.52; plate-T7F8, as shown in SEQ ID NO.53; plate-T7F9, as shown in SEQ ID NO.54; plate-T7F10, as shown in SEQ ID NO.55; plate-T7F11, as shown in SEQ ID NO.56; plate-T7F12, as shown in SEQ ID NO.57; plate-ADR1, as shown in SEQ ID NO.58; plate-ADR2, as shown in SEQ ID NO. NO.59; plate-ADR3, as shown in SEQ ID NO.60; plate-ADR4, as shown in SEQ ID NO.61; plate-ADR5, as shown in SEQ ID NO.62; plate-ADR6, as shown in SEQ ID NO.63; plate-ADR7, as shown in SEQ ID NO.64; plate-ADR8, as shown in SEQ ID NO.65; plate-ADR9, as shown in SEQ ID NO.66; plate-ADR10, as shown in SEQ ID NO.67; plate-BDR1, as shown in SEQ ID NO.68; plate-BDR2, as shown in SEQ ID NO.69; plate-BDR3, as shown in SEQ ID NO.70; plate-BDR4, as shown in SEQ ID NO.71; plate-BDR5, as shown in SEQ ID NO.72; plate-BDR6, as shown in SEQ ID NO.73; plate-BDR7, as shown in SEQ ID NO.74; plate-BDR8, as shown in SEQ ID NO.75; plate-BDR9, as shown in SEQ ID NO.76; plate-BDR10, as shown in SEQ ID NO.77.

[0016] Preferably, the blockbarcode primer specifically includes the following sequence information: Block-F1, as shown in SEQ ID NO.78; Block-F2, as shown in SEQ ID NO.79; Block-F3, as shown in SEQ ID NO.80; Block-F4, as shown in SEQ ID NO.81; Block-F5, as shown in SEQ ID NO.82; Block-F6, as shown in SEQ ID NO.83; Block-F7, as shown in SEQ ID NO.84; Block-F8, as shown in SEQ ID NO.85; Block-F9, as shown in SEQ ID NO.86; Block-F10, as shown in SEQ ID NO.87; Block-F11, as shown in SEQ ID NO.88; Block-F12, as shown in SEQ ID NO.89; Block-F13, as shown in SEQ ID NO.90; Block-F14, as shown in SEQ ID NO.91; Block-F15, as shown in SEQ ID NO. NO.92; Block-F16, as shown in SEQ ID NO.93; Block-F17, as shown in SEQ ID NO.94; Block-F18, as shown in SEQ ID NO.95; Block-F19, as shown in SEQ ID NO.96; Block-F20, as shown in SEQ ID NO.97; Block-R1, as shown in SEQ ID NO.98; Block-R2, as shown in SEQ ID NO.99; Block-R3, as shown in SEQ ID NO.100; Block-R4, as shown in SEQ ID NO.101; Block-R5, as shown in SEQ ID NO.102; Block-R6, as shown in SEQ ID NO.103; Block-R7, as shown in SEQ ID NO.104; Block-R8, as shown in SEQ ID NO.105; Block-R9, as shown in SEQ ID NO.106; Block-R10, as shown in SEQ ID NO. NO.107; Block-R11, as shown in SEQ ID NO.108; Block-R12, as shown in SEQ ID NO.109; Block-R13, as shown in SEQ ID NO.110; Block-R14, as shown in SEQ ID NO.111; Block-R15, as shown in SEQ ID NO.112; Block-R16, as shown in SEQ ID NO.113; Block-R17, as shown in SEQ ID NO.Block-R18 is shown in SEQ ID NO.115; Block-R19 is shown in SEQ ID NO.116; Block-R20 is shown in SEQ ID NO.117.

[0017] Preferably, the plate barcode primer and the blockbarcode primer are connected together via a bridging sequence;

[0018] Wherein, a bridging sequence 5'-GAGTTGGATGCTGGATGG-3' is added to the 5'-end of the reverse primer sequence of the plate barcode primer, and a bridging sequence 5'-GGAGTGAGTACGGTGTGC-3' is added to the 5'-end of the forward primer;

[0019] A bridging sequence 5'-GAGTTGGATGCTGGATGG-5' is added to the 3'-end of the reverse primer sequence of the block barcode primer, and a bridging sequence 5'-GGAGTGAGTACGGTGTGC-5' is added to the 3'-end of the forward primer.

[0020] Both the plate barcode and the blockbarcode contain a bridge sequence. The plate barcode and the blockbarcode are connected together through the bridge sequence in the middle, which is equivalent to an overlapping PCR.

[0021] This is equivalent to using the PCR product containing the plate barcode as a template and performing an overlapping PCR through the bridge sequence to connect the plate barcode and the blockbarcode together when amplifying with the blockbarcode primer. The final PCR product contains 4 barcodes.

[0022] (block-barcode-F1-F10)x(block-barcode-R1-R10)x(plate-barcode-F1-F10)x(plate-barcode-R1-R10)=10000

[0023] Preferably, the ratio of the plate barcode primer concentration to the blockbarcode primer concentration is 1:25.

[0024] The beneficial effects of the above technical solution include at least: the above method realizes high throughput and low cost, and only requires 10 pairs of plate and 10 pairs of block primers to identify 10x10x10x10=1000 clones, while the traditional method requires 1000 pairs of primers.

[0025] The present invention also provides a method for constructing a high-throughput analysis of interacting proteins, which specifically comprises:

[0026] 1) Screening yeast interacting proteins by constructing a DNA library;

[0027] 2) Using the yeast interactive cloning protein cell lysis product as a template, the first round of PCR amplification is performed to obtain the PCR amplification product, i.e., a mixture of target gene and bait gene fragments;

[0028] 3) Using the product of the first round of PCR amplification as a template, add plate barcode primers and blockbarcode primers at the same time to perform a second round of Barcode-PCR amplification to obtain the tag sequence; adding two primers at the same time can increase the amplification amount and reduce the use of primers. As long as the amplification can be achieved, theoretically only the target fragment containing four tags can be obtained after adding two primers;

[0029] 4) The PCR products with barcodes in the tag sequences are mixed, purified, sequenced, and analyzed to obtain the genetic information of the interacting proteins.

[0030] Preferably, the specific operations of step 1) are:

[0031] 11) Using the yeast two-hybrid library-to-library strategy, 0.8 ml of the AD library and BD library were placed in 2xYPDA medium containing kanamycin for incubation;

[0032] 12) After the incubation is completed, observe the yeast strain under a microscope to see if the conjugation is successful. If the conjugation is successful, proceed to step 13). If the conjugation is not successful, continue incubating for 4 hours and then observe. Mating is the conjugation of the AD library MATα strain and the BD library MATa strain. The interaction is that the two genes are close in space, which activates the expression of the reporter gene. At the same time, generally observe the yeast strain under a microscope after incubating for 20 hours to see if the conjugation is successful.

[0033] 13) Centrifuge at 1000 g for 10 min, then suspend the cells in 50 ml of 0.5xYPDA medium containing kanamycin; centrifuge at 1000 g for 10 min again, and finally suspend the culture in 5 ml of 0.5xYPDA medium containing kanamycin to obtain a bacterial solution;

[0034] 14) Dilute the bacterial solution 50 times, take 200 μL of the bacterial solution and spread it on SD / -Leu-Trp-His-Ade / +5mM 3-AT medium, and culture it in a 30°C incubator for 5-7 days;

[0035] 15) After the culture is completed, screening is performed to obtain interacting proteins.

[0036] Preferably, the concentration of kanamycin in step 11) is 50ug / mL;

[0037] The volume of the 2xYPDA medium is 50 ml;

[0038] The incubation conditions are 30°C, 30-50 rpm / min for 20 hours;

[0039] Step 13) The concentration of kanamycin is 50ug / mL;

[0040] Step 15) The diameter of the interacting protein is 0.2-1 mm.

[0041] Preferably, in step 2), yeast interaction cloned protein bacterial lysis products are specifically prepared as follows: 17 interaction proteins with a diameter of 0.2-1 mm are placed in a 200 μL PCR tube, 10 μL of 1x yeast lysis buffer is added, the mixture is mixed, and then treated with a PCR instrument at 100° C. for 5 min.

[0042] Preferably, the first round of PCR amplification in step 2) is specifically as follows: using the yeast interactive cloning protein bacterial lysis product as a template, adding the front-end and back-end common primers of the AD and BD vectors, and amplifying the bait and target gene fragments at the same time; wherein the common primers are: AD-F, as shown in SEQ ID NO.118; AD-R, as shown in SEQ ID NO.119; BD-F, as shown in SEQ ID NO.120; BD-R, as shown in SEQ ID NO.121.

[0043] Preferably, step 3) specifically includes: using the first round PCR product as a template, performing PCR amplification of four barcode sequences; that is, adding plate barcode primers and blockbarcode primer sequences to the bait and target genes at the same time.

[0044] The beneficial effects of the above technical solution include at least: high throughput and low cost. Only 10 pairs of plate primers and 10 pairs of block primers are needed to identify 10x10x10x10=1000 clones. The traditional method requires 1000 pairs of primers. Plate primers and block primers are reused. As long as one of the four types has a different barcode, it can be considered as a different interaction combination.

[0045] Preferably, step 4) specifically includes sequencing the purified PCR product using a PacBio PE150 sequencing platform, with a sample sequencing depth of 5G to obtain a high-throughput sequencing result.

[0046] Preferably, the gene information of the interacting proteins in step 4) is as shown in Table 5.

[0047] In summary, the technical effects achieved by the present invention at least include:

[0048] (1) The present invention designs plate barcode primers and block barcode primers containing different barcode sequences. Since the barcode sequences at the F end and the R end of the plate primer and the block primer are different and reused, if there are 10 pairs of plate primers and 10 pairs of block primers, there are 10x10x10x10=10,000 primer combinations that can be used.

[0049] (2) The present invention also designs a method for constructing a high-throughput analysis of interacting proteins, and a high-throughput interaction combination identification method based on the "library to library" yeast two-hybrid strategy. First, a yeast two-hybrid system was constructed, and the interaction combination was randomly screened by the "library to library" mating method. Then, the primer set was used to perform two rounds of PCR amplification on the bait and target gene fragments in the protein interaction positive clones, so that each PCR amplification product was accompanied by a unique combination of four barcodes; finally, the Pacbio high-read length and high-throughput sequencing method was used to mix and sequence the PCR products of all positive clones; proteins carrying the same four barcode combinations (amplified from the same positive yeast clone) and derived from the bait and prey vectors were identified as interacting protein combinations; the four barcodes are the QR codes of the Bait gene and the prey gene. The QR codes of the bait and prey contained in an interacting colony are the same. The reason for selecting 4 is that the barcode sequences at the F end and R end of the plate primer and the block primer are different and reused. If they have 10 pairs of plate primers and 10 pairs of block primers, then there are 10x10x10x10=10,000 primer combinations available. The technical solution designed by the present invention has the advantages of high throughput, high efficiency, low cost and wide application of species. It can not only be applied to high-throughput identification of protein interactions and protein interaction mapping in species such as humans, animals, plants, and microorganisms, but also provide important clues and information for protein function research;

[0050] (3) The present invention ensures full coverage of cDNA length of 1000bp-30kb, can obtain complete protein interaction information, and ensures the identification of accurate interaction information of high similarity sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0052] Picture 1 is a schematic diagram of the Barcode PCR method of Example 1 of the present invention;

[0053] Picture 2 The yeast mating successful strain of Example 1 of the present invention contains a characteristic image of a clover leaf and a Mickey Mouse head;

[0054] Picture 3 This is a picture of positive colonies screened in SD / -Leu-Trp-His-Ade / +5mM 3-AT medium in Example 1 of the present invention;

[0055] Picture 4 This is the first round PCR detection diagram of 17 protein interaction combinations in Example 1 of the present invention;

[0056] Picture 5 This is a second round PCR detection diagram of 17 protein interaction combinations in Example 1 of the present invention;

[0057] Picture 6 It is the sequence structure of the interacting Bait and Prey genes with four barcodes in Example 1 of the present invention. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] A primer set for constructing high-throughput interaction protein analysis, comprising a platebarcode primer and a blockbarcode primer;

[0061] The plate barcode primer is composed of three parts: a vector backbone sequence, an 8 bp barcode sequence, and an 18 bp bridge sequence; the vector backbone sequence includes: backbone-T7-F: as shown in SEQ ID NO.1; backbone-AD-R, as shown in SEQ ID NO.2; and backbone-BD-R, as shown in SEQ ID NO.3;

[0062] The 8bp barcode sequence includes: barcode-F1: CGCTGCTC; barcode-F2: CTACTCGA; barcode-F3: TGTGTGCC; barcode-F4: AAGTTATC; barcode-F5: CAATGATG ; barcode-F6: GTCGCTGT; barcode-F7: GATGCCGG; barcode-F8: AGCAGAGA; barcode-F9: GGTGTCCG; barcode-F10: CCGTGCCA; barcode-F11: T TGTAGAT; barcode-F12: TAATAGGA; barcode-R1: GATCTGCC; barcode-R2: GTTCATCT; barcode-R3: TGACCGAG; barcode-R4: GATTCACA; barcod e-R5: CGTCCGTG; barcode-R6: CTCTAGTG; barcode-R7: CCATCTGC; barcode-R8: ACCTGATG; barcode-R9: TGGTCTCC; barcode-R10: GCTGATCG;

[0063] The 18 bp bridge sequence includes: Bridge-F, as shown in SEQ ID NO.4; and Bridge-R, as shown in SEQ ID NO.5;

[0064] The block barcode primer consists of two parts: an 18 bp bridge sequence and a 16 bp barcode sequence; the 18 bp bridge sequence is shown in SEQ ID NO.4 and SEQ ID NO.5;

[0065] The 16bp barcode sequence includes: Barcode-F1, as shown in SEQ ID NO.6; Barcode-F2, as shown in SEQ ID NO.7; Barcode-F3, as shown in SEQ ID NO.8; Barcode-F4, as shown in SEQ ID NO.9; Barcode-F5, as shown in SEQ ID NO.10; Barcode-F6, as shown in SEQ ID NO.11; Barcode-F7, as shown in SEQ ID NO.12; Barcode-F8, as shown in SEQ ID NO.13; Barcode-F9, as shown in SEQ ID NO.14; Barcode-F10, as shown in SEQ ID NO.15; Barcode-F11, as shown in SEQ ID NO.16; Barcode-F12, as shown in SEQ ID NO.17; Barcode-F13, as shown in SEQ ID NO.18; Barcode-F14, as shown in SEQ ID NO.19; Barcode-F15, as shown in SEQ ID NO.20; Barcode-F16, as shown in SEQ ID NO.21; Barcode-F17, as shown in SEQ ID NO.22; Barcode-F18, as shown in SEQ ID NO.23; Barcode-F19, as shown in SEQ ID NO.24; Barcode-F20, as shown in SEQ ID NO.25; Barcode-R1, as shown in SEQ ID NO.26; Barcode-R2, as shown in SEQ ID NO.27; Barcode-R3, as shown in SEQ ID NO.28; Barcode-R4, as shown in SEQ ID NO.29; Barcode-R5, as shown in SEQ ID NO.30; Barcode-R6, as shown in SEQ ID NO.31; Barcode-R7, as shown in SEQ ID NO.32; Barcode-R8, as shown in SEQ ID NO.33; Barcode-R9, as shown in SEQ ID NO.34; Barcode-R10, as shown in SEQ ID NO.35; Barcode-R11, as shown in SEQ ID NO.36; Barcode-R12, as shown in SEQ ID NO. ID NO.37; Barcode-R13, as shown in SEQ ID NO.38; Barcode-R14, as shown in SEQ ID NO.39; Barcode-R15, as shown in SEQ ID NO.40; Barcode-R16, as shown in SEQ ID NO.41; Barcode-R17, as shown in SEQ ID NO.42; Barcode-R18, as shown in SEQ ID NO.43; Barcode-R19, as shown in SEQ ID NO.44; Barcode-R20, as shown in SEQ ID NO.45. .

[0066] Further, the plate barcode primer specifically includes the following sequence information: plate-T7F1, as shown in SEQ ID NO.46; plate-T7F2, as shown in SEQ ID NO.47; plate-T7F3, as shown in SEQ ID NO.48; plate-T7F4, as shown in SEQ ID NO.49; plate-T7F5, as shown in SEQ ID NO.50; plate-T7F6, as shown in SEQ ID NO.51; plate-T7F7, as shown in SEQ ID NO.52; plate-T7F8, as shown in SEQ ID NO.53; plate-T7F9, as shown in SEQ ID NO.54; plate-T7F10, as shown in SEQ ID NO.55; plate-T7F11, as shown in SEQ ID NO.56; plate-T7F12, as shown in SEQ ID NO.57; plate-ADR1, as shown in SEQ ID NO.58; plate-ADR2, as shown in SEQ ID NO.59; plate-ADR3, as shown in SEQ ID NO.60; plate-ADR4, as shown in SEQ ID NO.61; plate-ADR5, as shown in SEQ ID NO.62; plate-ADR6, as shown in SEQ ID NO.63; plate-ADR7, as shown in SEQ ID NO.64; plate-ADR8, as shown in SEQ ID NO.65; plate-ADR9, as shown in SEQ ID NO.66; plate-ADR10, as shown in SEQ ID NO.67; plate-BDR1, as shown in SEQ ID NO.68; plate-BDR2, as shown in SEQ ID NO.69; plate-BDR3, as shown in SEQ ID NO.70; plate-BDR4, as shown in SEQ ID NO.71; plate-BDR5, as shown in SEQ ID NO.72; plate-BDR6, as shown in SEQ ID NO.73; plate-BDR7, as shown in SEQ ID NO.74; plate-BDR8, as shown in SEQ ID NO.75; plate-BDR9, as shown in SEQ ID NO.76; plate-BDR10, as shown in SEQ ID NO.77.

[0067] Further, the blockbarcode primer specifically includes the following sequence information: Block-F1, as shown in SEQ ID NO.78; Block-F2, as shown in SEQ ID NO.79; Block-F3, as shown in SEQ ID NO.80; Block-F4, as shown in SEQ ID NO.81; Block-F5, as shown in SEQ ID NO.82; Block-F6, as shown in SEQ ID NO.83; Block-F7, as shown in SEQ ID NO.84; Block-F8, as shown in SEQ ID NO.85; Block-F9, as shown in SEQ ID NO.86; Block-F10, as shown in SEQ ID NO.87; Block-F11, as shown in SEQ ID NO.88; Block-F12, as shown in SEQ ID NO.89; Block-F13, as shown in SEQ ID NO.90; Block-F14, as shown in SEQ ID NO.91; Block-F15, as shown in SEQ ID NO. NO.92; Block-F16, as shown in SEQ ID NO.93; Block-F17, as shown in SEQ ID NO.94; Block-F18, as shown in SEQ ID NO.95; Block-F19, as shown in SEQ ID NO.96; Block-F20, as shown in SEQ ID NO.97; Block-R1, as shown in SEQ ID NO.98; Block-R2, as shown in SEQ ID NO.99; Block-R3, as shown in SEQ ID NO.100; Block-R4, as shown in SEQ ID NO.101; Block-R5, as shown in SEQ ID NO.102; Block-R6, as shown in SEQ ID NO.103; Block-R7, as shown in SEQ ID NO.104; Block-R8, as shown in SEQ ID NO.105; Block-R9, as shown in SEQ ID NO.106; Block-R10, as shown in SEQ ID NO. NO.107; Block-R11, as shown in SEQ ID NO.108; Block-R12, as shown in SEQ ID NO.109; Block-R13, as shown in SEQ ID NO.110; Block-R14, as shown in SEQ ID NO.111; Block-R15, as shown in SEQ ID NO.112; Block-R16, as shown in SEQ ID NO.113; Block-R17, as shown in SEQ ID NO.Block-R18 is shown in SEQ ID NO.115; Block-R19 is shown in SEQ ID NO.116; Block-R20 is shown in SEQ ID NO.117.

[0068] Further, the plate barcode primer and the blockbarcode primer are connected together via a bridging sequence;

[0069] Wherein, a bridging sequence 5'-GAGTTGGATGCTGGATGG-3' is added to the 5'-end of the reverse primer sequence of the plate barcode primer, and a bridging sequence 5'-GGAGTGAGTACGGTGTGC-3' is added to the 5'-end of the forward primer;

[0070] A bridging sequence 5'-GAGTTGGATGCTGGATGG-5' was added to the 3'-end of the reverse primer sequence of the block barcode primer, and a bridging sequence 5'-GGAGTGAGTACGGTGTGC-5' was added to the 3'-end of the forward primer.

[0071] In order to further optimize the above technical solution, the ratio of plate barcode primer concentration to blockbarcode primer concentration was 1:25.

[0072] 12 Plate front-end primers, 10 Plate back-end primers, 20 Block front-end primers, and 20 Block back-end primers were designed, which can form 48,000 combinations.

[0073] A method for constructing a high-throughput analysis of interacting proteins is disclosed using the above primer set, specifically comprising:

[0074] 1) Screening yeast interacting proteins by constructing a DNA library;

[0075] 2) Using the yeast interactive cloning protein cell lysis product as a template, the first round of PCR amplification is performed to obtain the PCR amplification product, i.e., a mixture of target gene and bait gene fragments;

[0076] 3) Using the first round of PCR amplification product as a template, add plate barcode primers and blockbarcode primers to perform a second round of Barcode-PCR amplification to obtain the tag sequence, such as Picture 1 As shown;

[0077] 4) The PCR products with barcodes in the tag sequences are mixed, purified, sequenced, and analyzed to obtain the genetic information of the interacting proteins.

[0078] In order to further optimize the above technical solution, the specific operation of step 1) is as follows:

[0079] 11) Using the yeast two-hybrid library-to-library strategy, 0.8 ml of the AD library and BD library were placed in 2xYPDA medium containing kanamycin for incubation;

[0080] 12) After the incubation is complete, observe the yeast strain under a normal optical microscope to see if it has successfully reproduced, such as a cloverleaf or Mickey Mouse head shape (such as Picture 2 If two yeast strains have completed conjugation reproduction, proceed to step 13). If reproduction is unsuccessful, continue incubation for 4 hours and observe under an optical microscope;

[0081] 13) Centrifuge at 1000 g for 10 min, then suspend the cells in 50 ml of 0.5xYPDA medium containing kanamycin; centrifuge at 1000 g for 10 min again, and finally suspend the culture in 5 ml of 0.5xYPDA medium containing kanamycin to obtain a bacterial solution;

[0082] 14) Dilute the bacterial solution 50 times, take 200 μL of the bacterial solution and spread it on SD / -Leu-Trp-His-Ade / +5mM 3-AT medium, and culture it in a 30°C incubator for 5-7 days;

[0083] 15) After the culture, screening was performed to obtain interacting proteins (such as Picture 3 shown).

[0084] In order to further optimize the above technical solution, the concentration of kanamycin in step 11) is 50ug / mL;

[0085] The volume of 2xYPDA medium is 50 ml;

[0086] The incubation conditions are 30°C, 30-50 rpm / min for 20 hours;

[0087] Step 13) The concentration of kanamycin is 50ug / mL;

[0088] Step 15) The diameter of the interacting protein is 0.2-1 mm.

[0089] In order to further optimize the above technical solution, step 2) yeast interaction cloning protein bacterial lysis products are specifically prepared as follows: 17 interaction proteins with a diameter of 0.2-1 mm are dropped into a 200 μL PCR tube, 10 μL 1x yeast lysis buffer is added, the mixture is mixed and then treated with a PCR instrument at 100°C for 5 minutes.

[0090] In order to further optimize the above technical solution, the first round of PCR amplification in step 2) is specifically as follows: using the yeast interactive cloning protein bacterial lysis product as a template, adding the front-end and rear-end common primers of the AD and BD vectors, and amplifying the bait and target gene fragments at the same time; wherein the common primers are: AD-F, as shown in SEQ ID NO.118; AD-R, as shown in SEQ ID NO.119; BD-F, as shown in SEQ ID NO.120; BD-R, as shown in SEQ ID NO.121. The first round PCR reaction system of step 2) is shown in Table 1, and the first round PCR reaction procedure is shown in Table 2:

[0091] Table 1

[0092]

[0093] Table 2

[0094]

[0095] In order to further optimize the above technical solution, the first round PCR product of step 2) was detected by 1% agarose gel. Picture 4 shown.

[0096] In order to further optimize the above technical solution, step 3) is specifically as follows: using the first round PCR product as a template, PCR amplification of four barcode sequences is performed; that is, plate barcode primers and blockbarcode primer sequences are added to the bait and target genes at the same time.

[0097] In order to further optimize the above technical solution, in step 3), the second round of Barcode-PCR amplification adds the same Barcode sequence to the interacting target gene and bait gene, and the second round PCR reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.

[0098] Table 3

[0099]

[0100]

[0101] Table 4

[0102]

[0103] In order to further optimize the above technical solution, the second round of PCR products in step 3) were detected by 1% agarose gel. Picture 5 shown.

[0104] In order to further optimize the above technical solution, the sequences of the bait gene (bait) and the target gene (prey) are allowed to have a 2 bp mismatch in the aligned sequences.

[0105] (-) indicates that the sequence is 3'-5'

[0106] (+) indicates that the sequence is 5'-3'

[0107] The sequence information of AD-LOC_Os01g43520-1434bp(-) is as follows:

[0108]

[0109] The sequence information of BD-LOC_Os06g01450-695bp(+) is as follows:

[0110] ACACTGACGTCGCGACGGAGTGAGTACGGTGTGCCGCTGCTCTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGGGCCATTACGGCCCCAGATTACGCTCATATGACAAGTTTGTACAAAAAAGTTGGGAGGTGGAAGAGCCAAATCATGGACCATGAGTCCGGTCCATTCAACACTGACAAGATCCTAGTGGCTGTCAAGACTTACTGGACCGGGAAGCTCGTGCAAAAACAATAATCGTCGCCTTGTTACTGTTTTTCGTCTGCGAAAATGTAGGCATGACTTGCCTGTCAGTTCTGTCTTCATTGCCATTTTTTTATCCTAACTTCAGGATATCTGTATATAGGAGGCATGTGAACCATGTAGCTGGAATTGGTATCAAACATCATTAGCCACGTAGCGTATGTGACACATTGCAATGGTGTATAAGATGTTCACTCATATATTTGGTGTGATATTGCAATCTAATAACATGATGCTTGGTGTGAAAAAAAAAAAAAAAAAAAAACCCAACTTTCTTGTACAAAGTGGGGCCGCCTCGGCCGGATCCGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGCATCAGGTCCATCCAGCATCCAACTCATATCAGTCATGCATA, as shown in SEQ ID NO.123;

[0111] The specific information of the AD-LOC_Os01g39000-1109bp(-) sequence is:

[0112]

[0113] The sequence information of BD-LOC_Os02g43330-706bp(-) is specifically as follows:

[0114] CGAGACTGTCGATCTCGAGTTGGATGCTGGATGGGATCTGCCCTCAAGACCCAGTTTAGAGGCACTTGCGGGTTTTTCAGTATCTACGATCAGCTGCAGCTCGAGGCTCGGCCGAGGCGGCCTTTTGTTTTTTCTTTTTTTTTTTCACGAACATGAATAATTAATGTAGCATCCTAGCATAACAAGCAGCAAGACTCGGCGATCGACCGGCCGGAGGATTTCTATCTGTGCAACGCAATGCATGTGTGGATTTGCGTTGTTGCATGGTTTGCCGGCCGGCCGGTTGCATCCTCCATCTCTAATCTGCTCTCCCGTCTAGCTTACGAGCGCCGCGGCTCTAGTCGATCTCTCCGGACCAACTGACTGCACAAGCCAGCAGCAGCAACAGCAGCAAAATATCTATATAGACCCAATACAACACGTACTCATACACATATATATTTATATACTGTATGCTACGCTGATTGATTAGCATGATCATGCTACTGCGTTCCATTCCACCATTGGCCATATCTCCCACAGATCCGGCATGCCGCAGAACGGATCGGGCAGCTGGCCGCCGCCCAAGTAGTCTACCCGGCCGTAATGGCCCATATGCAGGTCCTCCTCTGAGATCAGCTTCTGCTCCTCCATGATGGCGGCTCGCCCTATAGTGAGTCGTATTATCGAGTAGGCACACCGTACTCACTCCGTCGCGACGTCAGTGT, as shown in SEQ ID NO.125;

[0115] The sequence information of AD-LOC_Os05g391101015bp(+) is specifically as follows:

[0116]

[0117] The specific sequence information of BD-LOC_Os04g53350-1398bp(+) is:

[0118]

[0119] The sequence information of AD-LOC-Os06g05100-1368bp(-) is as follows:

[0120]

[0121] The specific sequence information of BD-LOC_Os03g61620-1364bp(+) is:

[0122]

[0123] The sequence information of AD-LOC_Os01g60830-824bp(-) is specifically as follows:

[0124] AGATATGCGCGACACGGAGTTGGATGCTGGATGGCGTCCGTGATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATTCATCTGCAGCTCGAGCTCGGGCCGAGGCGGCCTTTTGTTTTTTCTTTTTTTTTTTTGTGGCAAAACTGAACACATAATATTGTTCAAATTCTTGGGGCTACAACACCACCTATGTATCTAAGCATGAGTAAAAATATGTCTTTGATTGTACAATTGAAGTTCATCTGACGAACAATTTGGCGAAGATGTTGGTCGGTTTTGGGTTCTCCAAGCATGCGACATGTGCATATCTTCCTAGCTTGTCGACCTGCCTGGGTTCCCCTCTAGTGTCCTTCTTGCAAACTTTGCAGGTCTTATCCACCCAACCTTCCTCTACGTATTCCCCGTCAACATCCACATATGACTTGAAGAAATCCTTGTAGGTGCCACCGATCTTCCTCCGAAGTGCTCCATACTGCTCCTTTGTCATCCTTCCTCCATCCTTTGTGGCCTCTCTGGCTTCATACAGAAGCTCCGCGTCCTTCTCCGACCGGACCTGCGAGAAGTCCAGCGCCTCGGTGATGCTCCCGAACAATGACTGCTTGCTGCCCTTCTCCTCATCTTCCTTTGCGTAGACCTTCACTGATCTGGTGGAGGCAGTGCTTGGCCTGAAGGAGGACGGCATGGTTTGGTGCCCGGCCGTAATGGCCACATATGAGCGTAATCTGGTACGTCGTATGGGTACTCCATGGCGGCGCTCGCCCTATAGTGAGTCGTATTAGGCACACAGCACACCGTACTCACTCCGTCGCGACGTCAGTGT, as shown in SEQ ID NO.130;

[0125] The sequence information of BD-LOC_Os04g51120-930bp(+) is specifically as follows:

[0126] ACACTGACGTCGCGACGGAGTGAGTACGGTGTGCTGTGTGCCTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGGGCCATTACGGCCGGGATCCAAAGGCTGATGCATCCGATTTTGGTGCCTTTGTATCGCACAGTGAGGAAGTAGCCAAGGATCCTTTTGATCTTTCTTTGAGTACCAGCTCTGGAAGGACAAACCAAGCACCTCTGGCGGCTCCCAAGTCAGATACCAAGAAAGAAAATTTTCAGGTCAAGTCTGGCATATGGGCTGACTCATTGAGCCGTGGACTGATTGATCTGAACATAACTGGACCAAAGAAGGTGAACTTAGCCGACGTTGGGATTGTCGGTGGCCTCGATGACGGGTCTGATGATAAAGCTCTGCCCTCATGGACCATGGGTGCAGGAGGTTCCGGCCTAGGAATGTCCGGAATTCCTTCATCTACACAAAGTGGTGGCATTGAGAGCTTAGCTAACTACAACAAGTACCAGTTTGGCTTTAAATGAGCTTTCATGCGAGGCTTTCTAGTATATGCCGGTTATTGTCGGAATGTCGGCTTGTGCCTTATTTATTTTTTTGTTGATTCTATTCTCCCCTCTTTTGATGCTTACATTCCTTTACAATCGCCTCCAAGGATCGTGATCTTGGTTCGTCTTGTCGCGCTTGTATTGTATTTATTGGATTTGCGGATGAGAGACTCCTACCTATATCACACCCTGATTCAATTTAACTGTACTGTTATTCCAAAAAAAAAAGAAAAAACAAAAGGCCGCCTCGGCCGGATCCGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGCACGGACGCCATCCAGCATCCAACTCCGTGTCGCGCATATCT, as shown in SEQ ID NO.131;

[0127] The sequence information of AD-LOC_Os08g04500-1249bp(-) is as follows:

[0128]

[0129] The sequence information of BD-LOC_Os01g63160-815bp(+) is specifically as follows:

[0130] TACTAGAGTAGCACTCGGAGTGAGTACGGTGTGCTGTGTGCCTAATACGACTCACTATAGGGCGAGCCGCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGGGCCATTACGGCCGGGGGGCATGGCAAATCGGGGATCAGAGTTGCAGAACTGAAGTTGTTGATCCACCTTATCTTGCCCGACCAGTTGCAAAGGTTGGTGCATTCAAACCATACAATGTAGGACCTGCTCAATCATCACAAAAGGAAATGCTGAGCTTTGCCACCAAGTTTGACTCAAACCTTCATCCTTTCAAGCCTGAAAATGGGGTCTGCAAGTTTGTTGATCCTACAAGTTTTGCTGCTGAGGTGCCAAATAAATGTGGGCATGGCTGCTGCAGTTCTCATGGGCAACTTCGTAAGAACTCATTGCTGGGTCCCGAGTTTAATGAGTTTGAAGATCATCCACCAATTTCAGATAGTAGTTTTGCTTCGTTGGTGTCAGAAATTAGCAGCATTGCCTGGATGAAAAGTGGTTTGCAAAGCAGTGACACCTTAGCGCGGTCTGTTCCACCAGCTTAACAGATTAGGATAATGTAAATTAGTGAGATGAAACTTTTGGAGTCAATTTTTTATATTAAGGTGTAATCAAGTCAAATCATGGTTATTGTTGTAAAAAAAAAAAAAAAGAAAAAACAAAAGGCCGCCTCGGCCGGATCCGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGCACTAGAGCCATCCAGCATCCAACTCCGTGTCGCGCATATCT, as shown in SEQ ID NO. 133;

[0131] The sequence information of AD-LOC_Os03g11580-1122bp(-) is specifically as follows:

[0132]

[0133] The specific sequence information of BD-LOC_Os01g14950-1099bp(-) is:

[0134]

[0135] The sequence information of AD-LOC_Os10g10130-899bp(-) is as follows:

[0136] TGTGCGCGCGACGAGCGAGTTGGATGCTGGATGGGATTCACAATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATTCATCTGCAGCTCGAGCTCGGGCCGAGGCGGCCTTTTGTTTTTTCTTTTTTTTTTTTTTCAGAATTAGCTCCTTGTTGATGGCATATAGTATAATTTCTCATTTCAACATACATGACTGCAAGTACACAATACATATGCAAGTTAACTAAACACATCAGAAATAGGATAAACACATCTACCGTGGTAAGCTAGCAGATGACAAGAATTCTTGCTCCAAGCTATAGCATGATATTTGGAAGCGTTGAGATGCTGCATTAGCTCTATCAACATCCAAATTTGTTACTAATGGTTGTATCTCTGGGTCACTGGCTCCTTCAGTCAGAACAGTTTGAATCTCTTGGTCAATGGTTGGATCTACTTGAGATGAGTTCATCGTTTTTGTTCGCAGAAGCTGTAACGTCATCTCTACTTTCTTCATCGTGGGCCTTTCTTCACCTTTTAGCTTCAAGCACGTCTCAGCAAGGGATGCAACTTCTTTGATATCTTCCTCATTTGCTTCTTCGAGAACTTGAGCATCGACCATATCTGTAATTGGCCTGGTTTTAATCTCTGAAAGGAAGTAACTACACAAATTTTGTTTCATCCCAGAGTTTATTGTAAAAATGGGTTGCTTTCTCAGAAGAAGTTCTAGGAGTACCACACCAAAACTATAGACATCACTCTTCTCATTTAGCTGCCTAGTTCCCGGCCGTAATGGCCATCATATGAGCGTAATCTGGTACGTCGTATGGGTACTCCATGGCGGCGCTCGCCCTATAGTGAGTCGTATTAGAGCAGCGGCACACCGTACTCACTCCGTCGCGACGTCAGT, as shown in SEQ ID NO. 136;

[0137] The sequence information of BD-LOC_Os05g05950-761bp(-) is specifically as follows:

[0138] TGTGCGCGCGACGAGCGAGTTGGATGCTGGATGGGATTCACACTCAAGACCCGTTTAGAGGCCCCGGCCTTTTGTTTTTTCTTTTTTTTTTTTTGGTATATAGATCTAACAACATTATTCAGTTGAAGTTCCACATTAAATTTCACAACCCTCTTCAGCCAAAATTATGATAGGAACGATCTTCGGAGTACACTGGTAAAAGCAAAAACAGACAGAAAATTATGGTTCATATAAGATAGGAAAATTGCTAAGACGAAAGGAGGCGCAGCAGACATGAGTAAATTGTTCATCTTGATGCGAAGCTAATAGGCGAAGGAGGGTTCGCTGGGCCTGAAGCTCCTGTAAATGGAGGCTGCAACTGGGATAAGACCCAACAGCGCGATCTGGACCTGCTCCGAGGTGCTTGTCCTGACAGTGATCTGGCCACTTAGTTTGTTATTCAATCCAAGACGAACCACCATCTTCGAGCCCCTTCCAATCGAGAACTGGGACTGCAAATTCGCACCGAGAGCAAGGTCTCGGCGCCACTTCATCAAAGAAAGACCCAGGGTTGACAGGGACTGGCCAATCGGATAATCCCCGGCCGTAATGGCCCATATGCAGGTCCTCCTCTGAGATCAGCTTCTGCTCCTCCATGATGGCGGCTCGCCCTATAGTGAGTCGTATTAGAGCAGCGGCACACCGTACTCACTCCGTCGCGACGTCAGT, as shown in SEQ ID NO.137;

[0139] The sequence information of AD-LOC_Os02g04325-1246bp(+) is specifically:

[0140]

[0141] The specific sequence information of BD-LOC_Os01g58700-1082bp(-) is:

[0142]

[0143] The sequence information of AD-LOC_Os08g04840-1216bp(+) is as follows:

[0144]

[0145] The specific sequence information of BD-LOC_Os01g14950-1100bp(-) is:

[0146]

[0147] The sequence information of AD-LOC_Os10g02930-1113bp(-) is as follows:

[0148]

[0149] The specific sequence information of BD-LOC_Os03g22740-1490bp(-) is:

[0150]

[0151] The sequence information of AD-LOC_Os08g27010-1061bp(+) is as follows:

[0152]

[0153] The specific sequence information of BD-LOC_Os01g43630-977bp(-) is:

[0154] CACTATCTCTAGTCTCGAGTTGGATGCTGGATGGTGACCGAGCTCAAGACCCGTTTAGAGAATGCACAGTGTTGTGGTAAAGGCCAAGAAGGTCATCGAGACTGAAGTTTGATTGTGAGGAATATGTCCCACCAGTATTTACTAGTGAAGTTTCAGCTGGTGTTTGCGAGCTTATCAATGAAGGACTAATCAAATCGGAAGGAGCATTGTATGTAGAGGTGGAAGAACCATTGCTGGCTCTTGTACCATTGTCCTCTTTATCTGAAGTACTTAGCAAAAGATCATTATCATTTTCTTGATATCTTTGGGCAGAAATTACATTTTCTGGATCCTCAGTCAAGCCTGTAAGATCCTCTGAGTACTCAAATGGTCCGCGATGCTCCTTATCTGTAAACATGTACGATGGCTTCTGATATACAACTGATAGACTGTTGAACTCATCGAATATCCGATCTTTGATTTCACTGCTCTGTGTATCTGCAAATACAGGGACAGCTTGCTTGGGAGGGTTTACTACACGTTCTGCTACATTAGGACTGTATTGCAAAAGCCTATAGTAAAAAAGTGCTCTGTCATGAACATCCTGATGGGTGTCAGATAGACCAGCTGCTAATGTATCCCCTAGTGCCTTCTGTGTCTCTGGGGGTCTTTTAAAGAAACATTTCATCACTGCTGTCAGGAGATGCAAGCGAACCTCAGGAGAGTGCTCTTCATCCCAGTTTTCAACAAGACTTTCAAGGATGTATGGAGCATCATGCATGTCCTGTGAATATTCTCCCAACATCCATATGAGAGCTGCTTTTCCTTTTGGCTCCTGAATGTTTTTGCTGCTGATATTTCCCACAACTGCTATACAATCATGACCCCGGCCGTAATGGCCCATATGCAGGTCCTCCTCTGAGATCAGCTTCTGCTCCTCCATGATGGCGGCTCGCCCTATAGTGAGTCGTATTATCGAGTAGGCACACCGTACTCACTCCGTCGCGACGTCAGTGT,As shown in SEQ ID NO.145;

[0155] The sequence information of LOC-Os02g11750-999bp(-) is as follows:

[0156] GTGCGCGCGACGAGCGAGTTGGATGCTGGATGGCCATCTGCATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATTCATCTGCAGCTCGAGCTCGGGCCGAGGCGGACTTTTGTTTTTTCTTTTTTTTTTTTGGTGATACGAAACCCATTCCAAAACCAGTGGCCTATGCAGAGACACCTCTAGAAAACAAAGAGGTTCTGGACAAATTTCAAAGTCAGATAACTGCTCGAGTTGGCATGGTACGGACAGCATACACATTATCACTTGTCAAAGTCAGGCAAGAATTCCTCGGGCTAATAATCTACACCGACAAAAGTTCACACTAAACAATCTGCAGACTAATACATGGTTTCCTAGCCTTCACTTGCATGAATCACTTGTTACAGTTCTTCATGGTATATTGTACTTCTAGGAAAGTTAGCACCATGATAAACTAATAATACCGGTTTTGTGATGTGCAGATTTTCATGCTACGCCATTCTCATACATTGAACCATCTTTGGTTGTGGCTTCTTCACCAATGTCAACCTCACCCATTGCATCGTCAATAAGAGACTGGAGCTCCTTTTCAGACATCTTCACCCTGTCATTGTAGTCTTGAAGTTGCAGAAAACATTTGTACAGCTTTGTAACAACCAATTGTCGTTCTGAAGAAACTTTCCGTGCAGCAGACAATTCATCCTCTAACTTCTGGGATCTCTTTTGCAAATCTGCAATGTGTTGTTCCTTTGCTGCAACTTTATCTTTGATCTGACTAACCTCACTTTCAAGGTCCTCGCGCATTTGAATTGCTTTGTTCAACTGTCGTTTTGCATCACTGTGAAGATCATCACTGGAAACTGATAGATCCCTGCTACCCGGCCGTAATGGCCATTCATATGAGCGTAATCTGGTACGTCGTATGGGTACTCCATGGCGGCGCTCGCCCTATAGTGAGTCGTATTAGATAACTTGCACACCGTACTCACTCCGTCGCGACGTCAGTGT,As shown in SEQ ID NO.146;

[0157] The sequence information of LOC_Os07g48170-1234bp(-) is as follows:

[0158]

[0159] The sequence information of AD-LOC_Os02g58160-1724bp(+) is as follows:

[0160]

[0161] The specific sequence information of BD-LOC_Os03g22770-1100bp(-) is:

[0162]

[0163] The sequence information of AD-LOC_Os08g09250-693bp(-) is specifically as follows:

[0164] GTGAGCGTGTATGTGCGAGTTGGATGCTGGATGGTGGTCTCCATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATTCATCTGCAGCTCGAGCTCGATGGATCCCGTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTACACAAACTGGTCCTTGTTATCAACAACACTCCAACAGTCATGCTGATACAGGTAGCAATGCATAGCGGATTTACAGCCAAACCCAGACAGTACTAGCATAAGCACAACGCATGTGCCAGAATTTATTCTTGTTCATAGAGCGGAAACCGCTTCTTTATTACATTGCACTGGCATTACTACATAAGGTTTGTGCTTTGCCTCCTAGCACAGAACTTGGGCCATCTCTCATCTTCACTGGAGTTCCTTCAGGAAGTCGGCGTTATCAACCAAAACCACTTTCCAGCCATCAGGGTCAAGGAAAGAGGCAATCTTTGTGTTGAGCCCTGGTAGTGGACCCGGCCATAATGGCCACTCTGCGTTGATACCACTGCTTGGGTGGAATTCACTGGCCTCCATGGCCATATGAGCGTAATCTGGTACGTCGTATGGGTACTCCATGGCGGCGCTCGCCCTATAGTGAGTCGTATTAGGCACACAGCACACCGTACTCACTCCGTCGCGACGTCAGTGT, as shown in SEQ ID NO.150;

[0165] The sequence information of BD-LOC_Os05g22670-1859bp(+) is specifically as follows:

[0166]

[0167] The sequence information of AD-LOC_Os01g55590-1317bp(+) is as follows:

[0168]

[0169] The sequence information of BD-LOC_Os02g47870-795(+) is specifically as follows:

[0170] ATATCAGTCATGCATAGGAGTGAGTACGGTGTGCCAATGATGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGGGCCATTACGGCCGGGGGAACGCCGTCTCTCTCTGGGCCTGGGACATCGTGGTGGACAACTGCGCCACCTGCCGCAACCACATCATGGACCTTTGCATCGAGTGCCAGGCGAACCAGGCGAGCGCCACCAGCGAGGAGTGCACCGTCGCTTGGGGAATTTGCAACCATGCATTCCACTTCCACTGTATCAGCCGTTGGCTAAAGACCCGTCAAGTGTACCCTCTTGACAACAGTGAGTGGGAGTTCCAGAAGTATGGTCACTAGAGAGAATGGCTTCGGCGTGGAATTGCTTCATGTTTCTCGTCGTTTAGCAGTAGTGGTTACTGAATAGATTCTGGTGAACATGCTATGGATCTGTAACGTTTCTGAAGTTCTGCCTTACTTGAAGCTGTAGGCGAATACTCTGGCTATGCCATATTCGTCCTTCGGGGCGCTGTTGAACAGCTCTTCCTCACATGAAGGTGTAGACGAATTACTCAGTTTGCAGAAATTAATTGGTAATTGTGGGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAGAAAAAACAAAAGGCCGCCTCGGCCGGATCCGTCGACCTGCAGCGGCCGCATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGCATCAGGTCCATCCAGCATCCAACTCTGTCACTCAT, as shown in SEQ ID NO.153;

[0171] The sequence information of AD-LOC_Os12g01190-928bp(-) is specifically as follows:

[0172] ACAGACGCATATGTACGAGTTGGATGCTGGATGGGCTGATCGATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATTCATCTGCAGCTCGAGCTCGGGCCGAGGCGGCCTTTTGTTTTTTCTTTGGTGATGTCGTTGACTTCGATGTAAGATGCCCTGATTCAGCTGGCTTTGAAGAAACCGAACATTCAAACTTTCCATTGTCATTTACAACCTGCTTCTCGTCACTTCCTTTTGACATGCCATCTGATGTGACATCATCATCGTTCCAAGCTGAAGGTATGGTAGAGAAGTCTATGTCAAGCGATGACTCCAGTGATGTTACAGCTTGGGTTTCCTTTGCAATACCAGGCTTATATGGCTTCAAGGTTTTAGACAAACTATCTCGTCCTTCCGTCATTTTCGGAACTGTGCTTGTGTCATCATTCCATGAAGAAGGTAGTTTTGTACTTGAAACAGTCGAAGAACTCGAGAATGAATTACTTTGAGGATCTGCTTTTGATTTAGTAAGAGATTGTGACGATGCCACCCCAGTTGCTGTTGTCCCGGTATTCAAATCGCGATGCCCCCTGCTCATACCAAAAAAATTTTAGCAACAAATTCACATTTTAAAACATATCATGAGACCCTAAGCAGCATCATACCATGAAGCAGCTGGTGGAAGTGTGGATCTCCCAGAACTACTGTTTGGAGGCGAAAGCCTGGATTGATTTGCAGTATTCTGTACAAAATAAGACCAGTTAATGTAGCAGCATGAAATCAAACAGAAATGAACATGTCCAGTGTCCCCCCGGCCGTAATGGCCACATATGAGCGTAATCTGGTACGTCGTATGGGTACTCCATGGCGGCGCTCGCCCTATAGTGAGTCGTATTAGATAACTTGCACACCGTACTCACTCCGTCGCGACGTCAGTGT, as shown in SEQ ID NO.154;

[0173] The specific sequence information of BD-LOC_Os03g44484-1297bp(+) is:

[0174]

[0175] In order to further optimize the above technical solution, step 4) is specifically to use the PacBio PE150 sequencing platform to sequence the purified PCR product, with a sample sequencing depth of 5G to obtain high-throughput sequencing results.

[0176] In order to further optimize the above technical solution, the gene information of the interacting proteins in step 4) is shown in Table 5.

[0177] Table 5

[0178] AD B.D. LOC_Os01g43520 LOC_Os06g01450 LOC_Os01g39000 LOC_Os02g43330 LOC_Os05g39110 LOC_Os04g53350 LOC_Os06g05100 LOC_Os03g61620 LOC_Os01g60830 LOC_Os04g51120 LOC_Os08g04500 LOC_Os01g63160 LOC_Os03g11580 LOC_Os01g14950 LOC_Os10g10130 LOC_Os05g05950 LOC_Os02g04325 LOC_Os01g58700 LOC_Os08g04840 LOC_Os01g14950 LOC_Os10g02930 LOC_Os03g22740 LOC_Os08g27010 LOC_Os01g43630 LOC_Os02g11750 LOC_Os07g48170 LOC_Os02g58160 LOC_Os03g22770 LOC_Os08g09250 LOC_Os05g22670 LOC_Os01g55590 LOC_Os02g47870 LOC_Os12g01190 LOC_Os03g44484

[0179] At the same time, the present invention uses the DNA gel recovery kit (product model) of Yisheng Biology to recover the target fragment; the concentration and purity of the recovered product are detected using the Agilent 2010 kit (product model), and it is stored in a -20°C refrigerator for standby use; the model of the kit is Gel Extraction Kit (19101ES50) Agilent 2010 kit (5067-1508).

[0180] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0181] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for constructing high-throughput interaction protein analysis, characterized in that: Including plate barcode primers and block barcode primers; The plate barcode primer consists of three parts: a vector backbone sequence, an 8 bp barcode sequence, and an 18 bp bridge sequence; The vector backbone sequence includes: backbone-T7-F:TAATACGACTCACTATAGG, as shown in SEQ ID NO.1; backbone-AD-R: ATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.2; backbone-BD-R: CTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO. 3; The 8 bp barcode sequence includes: barcode -F1:CGCTGCTC; barcode -F2:CTACTCGA; barcode -F3:TGTGTGCC; barcode -F4:AAGTTATC; barcode -F5:CAATGATG; barcode -F6:GTCGCTGT; barcode -F7: GATGCCGG; barcode -F8:AGCAGAGA; barcode -F9:GGTGTCCG; barcode -F10:CCGTGCCA; barcode -F11:TTGTAGAT; barcode -F12:TAATAGGA; barcode-R1:GATCTGCC; barcode-R2: GTTCATCT; barcode-R3:TGACCGAG; barcode-R4:GATTCACA; barcode-R5: CGTCCGTG; barcode-R6: CTCTAGTG; barcode-R7: CCATCTGC; barcode-R8:ACCTGATG; barcode-R9:TGGTCTCC; barcode-R10: GCTGATCG; The 18 bp bridge sequence includes: Bridge-F: GGAGTGAGTACGGTGTGC, as shown in SEQ ID NO. 4; Bridge-R: GAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 5; The block barcode primer consists of two parts: an 18 bp bridge sequence and a 16 bp barcode sequence; Wherein, the 18 bp bridge sequence is shown in SEQ ID NO.4 and SEQ ID NO.5; The 16bp barcode sequence includes: Barcode-F1: TCAGACGATGCGTCAT, as shown in SEQ ID NO.6; Barcode-F2: CTATACATGACTCTGC, as shown in SEQ ID NO.7; Barcode-F3: TACTAGAGTAGCACTC, as shown in SEQ ID NO. 8; Barcode-F4: TGTGTATCAGTACATG, as shown in SEQ ID NO.9; Barcode-F5: ACACGCATGACACACT, as shown in SEQ ID NO.10; Barcode-F6: GATCTCTACTATATGC, as shown in SEQ ID NO.11; Barcode-F7: ACAGTCTATACTGCTG, as shown in SEQ ID NO. 12; Barcode-F8: ATGATGTGCTACATCT, as shown in SEQ ID NO. 13; Barcode-F9: CTGCGTGCTCTACGAC, as shown in SEQ ID NO. 14; Barcode-F10: GCGCGATACGATGACT, as shown in SEQ ID NO.15; Barcode-F11: CGCGCTCAGCTGATCG, as shown in SEQ ID NO. 16; Barcode-F12: GCGCACGCACTACAGA, as shown in SEQ ID NO. 17; Barcode-F13: ACACTGACGTCGCGAC, as shown in SEQ ID NO.18; Barcode-F14: CGTCTATATACGTATA, as shown in SEQ ID NO. 19; Barcode-F15: ATATCAGTCATGCATA, as shown in SEQ ID NO. 20; Barcode-F16:TAGATGCGAGAGTAGA, as shown in SEQ ID NO.21; Barcode-F17: CATAGCGACTATCGTG, as shown in SEQ ID NO. 22; Barcode-F18: CATCACTACGCTAGAT, as shown in SEQ ID NO. 23; Barcode-F19: CGCATCTGTGCATGCA, as shown in SEQ ID NO. 24; Barcode-F20: TATGTGATCGTCTCTC, as shown in SEQ ID NO.25; Barcode-R1:TAGTCACAGCGTGTAC, as shown in SEQ ID NO.26; Barcode-R2: AGATATGCGCGACACG, as shown in SEQ ID NO. 27; Barcode-R3: TATGCATGACTGATAT, as shown in SEQ ID NO. 28; Barcode-R4: CGAGACTGTCGATCTC, as shown in SEQ ID NO. 29; Barcode-R5: CGCGCGTGTGTGCGTG, as shown in SEQ ID NO.30; Barcode-R6: CACACGCGCGTGCTCG, as shown in SEQ ID NO.31; Barcode-R7: ATCTGTGCGAGACTAC, as shown in SEQ ID NO.32; Barcode-R8: CACTATCTCTAGTCTC, as shown in SEQ ID NO. 33; Barcode-R9: TGTCGCGCGACGAGC, as shown in SEQ ID NO.34; Barcode-R10: GTCATCACACATCTCT, as shown in SEQ ID NO.35; Barcode-R11: CTGATATGTAGTCGTA, as shown in SEQ ID NO. 36; Barcode-R12: TGTCGCGCGACGAGC, as shown in SEQ ID NO.37; Barcode-R13: GCGCATGTCTCTCTCT, as shown in SEQ ID NO.38; Barcode-R14: TCTACAGAGCGAGAGT, as shown in SEQ ID NO. 39; Barcode-R15: ACAGACGCATATGTAC, as shown in SEQ ID NO.40; Barcode-R16: CGCATGAGATATACGC, as shown in SEQ ID NO.41; Barcode-R17: GTAGCGACATACGCAC, as shown in SEQ ID NO.42; Barcode-R18: TGTGATACTGCGAGCA, as shown in SEQ ID NO.43; Barcode-R19: ACTCAGATGAGTGACA, as shown in SEQ ID NO.44; Barcode-R20: GTGAGCGTGTATGTGC, as shown in SEQ ID NO.45; The plate barcode primer specifically includes the following sequence: plate-T7F1: GGAGTGAGTACGGTGTGCCGCTGCTCTAATACGACTCACTATAGG, as shown in SEQ ID NO.46; plate-T7F2: GGAGTGAGTACGGTGTGCCTACTCGATAATACGACTCACTATAGG, as shown in SEQ ID NO.47; plate-T7F3: GGAGTGAGTACGGTGTGCTGTGTGCCTAATACGACTCACTATAGG, as shown in SEQ ID NO.48; plate-T7F4: GGAGTGAGTACGGTGTGCAAGTTATCTAATACGACTCACTATAGG, as shown in SEQ ID NO. 49; plate-T7F5: GGAGTGAGTACGGTGTGCCAATGATGTAATACGACTCACTATAGG, as shown in SEQ ID NO.50; plate-T7F6: GGAGTGAGTACGGTGTGCGTCGCTGTTAATACGACTCACTATAGG, as shown in SEQ ID NO.51; plate-T7F7: GGAGTGAGTACGGTGTGCGATGCCGGTAATACGACTCACTATAGG, as shown in SEQ ID NO.52; plate-T7F8: GGAGTGAGTACGGTGTGCAGCAGAGATAATACGACTCACTATAGG, as shown in SEQ ID NO.53; plate-T7F9: GGAGTGAGTACGGTGTGCGGTGTCCGTAATACGACTCACTATAGG, as shown in SEQ ID NO.54; plate-T7F10: GGAGTGAGTACGGTGTGCCCGTGCCATAATACGACTCACTATAGG, as shown in SEQ ID NO.55; plate-T7F11: GGAGTGAGTACGGTGTGCTTGTAGATTAATACGACTCACTATAGG, as shown in SEQ ID NO.56; plate-T7F12: GGAGTGAGTACGGTGTGCTAATAGGATAATACGACTCACTATAGG, as shown in SEQ ID NO.57; plate-ADR1: GAGTTGGATGCTGGATGGGATCTGCCATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.58; plate-ADR2: GAGTTGGATGCTGGATGGGTTCATCTATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.59; plate-ADR3: GAGTTGGATGCTGGATGGTGACCGAGATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.60; plate-ADR4: GAGTTGGATGCTGGATGGGATTCACAATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.61; plate-ADR5: GAGTTGGATGCTGGATGGCGTCCGTGATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO. 62; plate-ADR6:GAGTTGGATGCTGGATGGCTCTAGTGATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.63; plate-ADR7: GAGTTGGATGCTGGATGGCCATCTGCATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.64; plate-ADR8: GAGTTGGATGCTGGATGGACCTGATGATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.65; plate-ADR9: GAGTTGGATGCTGGATGGTGGTCTCCATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.66; plate-ADR10: GAGTTGGATGCTGGATGGGCTGATCGATGCACAGTTGAAGTGAAC, as shown in SEQ ID NO.67; plate-BDR1:GAGTTGGATGCTGGATGGGATCTGCCCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.68; plate-BDR2:GAGTTGGATGCTGGATGGGTTCATCTCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.69; plate-BDR3:GAGTTGGATGCTGGATGGTGACCGAGCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.70; plate-BDR4: GAGTTGGATGCTGGATGGGATTCACACTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.71; plate-BDR5: GAGTTGGATGCTGGATGGCGTCCGTGCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO. 72; plate-BDR6:GAGTTGGATGCTGGATGGCTCTAGTGCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.73; plate-BDR7: GAGTTGGATGCTGGATGGCCATCTGCCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO. 74; plate-BDR8:GAGTTGGATGCTGGATGGACCTGATGCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.75; plate-BDR9:GAGTTGGATGCTGGATGGTGGTCTCCCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO.76; plate-BDR10: GAGTTGGATGCTGGATGGGCTGATCGCTCAAGACCCGTTTAGAGG, as shown in SEQ ID NO. 77; The block barcode primer specifically includes the following sequence: Block-F1: TCAGACGATGCGTCATGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.78; Block-F2: CTATACATGACTCTGCGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.79; Block-F3: TACTAGAGTAGCACTCGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.80; Block-F4: TGTGTATCAGTACATGGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.81; Block-F5: ACACGCATGACACACTGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.82; Block-F6: GATCTCTACTATATGCGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.83; Block-F7: ACAGTCTATACTGCTGGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.84; Block-F8: ATGATGTGCTACATCTGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.85; Block-F9: CTGCGTGCTCTACGACGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.86; Block-F10: GCGCGATACGATGACTGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.87; Block-F11: CGCGCTCAGCTGATCGGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.88; Block-F12: GCGCACGCACTACAGAGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.89; Block-F13: ACACTGACGTCGCGACGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.90; Block-F14: CGTCTATATACGTATAGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.91; Block-F15: ATATCAGTCATGCATAGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.92; Block-F16: TAGATGCGAGAGTAGAGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.93; Block-F17: CATAGCGACTATCGTGGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.94; Block-F18: CATCACTACGCTAGATGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.95; Block-F19: CGCATCTGTGCATGCAGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.96; Block-F20: TATGTGATCGTCTCTCGGAGTGAGTACGGTGTGC, as shown in SEQ ID NO.97; Block-R1: TAGTCACAGCGTGTACGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.98; Block-R2: AGATATGCGCGACACGGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.99; Block-R3: TATGCATGACTGATATGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 100; Block-R4: CGAGACTGTCGATCTCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.101; Block-R5: CGCGCGTGTGTGCGTGGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.102; Block-R6: CACACGCGCGTGCTCGGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 103; Block-R7: ATCTGTGCGAGACTACGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 104; Block-R8: CACTATCTCTAGTCTCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 105; Block-R9: TGTCGCGCGACGAGCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 106; Block-R10: GTCATCACACATCTCTGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 107; Block-R11: CTGATATGTAGTCGTAGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.108; Block-R12: TGTCGCGCGACGAGCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.109; Block-R13: GCGCATGTCTCTCTCTGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.110; Block-R14: TCTACAGAGCGAGAGTGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.111; Block-R15: ACAGACGCATATGTACGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 112; Block-R16: CGCATGAGATATACGCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO. 113; Block-R17: GTAGCGACATACGCACGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.114; Block-R18: TGTGATACTGCGAGCAGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.115; Block-R19: ACTCAGATGAGTGACAGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.116; Block-R20: GTGAGCGTGTATGTGCGAGTTGGATGCTGGATGG, as shown in SEQ ID NO.

117.

2. A primer set for constructing high-throughput interaction protein analysis according to claim 1, characterized in that: The concentration ratio of the plate barcode primer to the block barcode primer is 1:

25.

3. A method for constructing a high-throughput analysis of interacting proteins, characterized in that: include: 1) Screening yeast interacting cloned proteins by constructing DNA libraries; 2) preparing yeast interactive cloning protein bacterial lysis products based on the yeast interactive cloning protein, and performing a first round of PCR amplification using the yeast interactive cloning protein bacterial lysis products as a template to obtain a PCR amplification product, i.e., a mixture of target gene and bait gene fragments; 3) Using the product of the first round of PCR amplification as a template, adding the primer set of claim 1 or 2, and performing a second round of Barcode-PCR amplification to obtain a tag sequence; 4) Mix, purify, sequence and analyze the PCR products with barcodes in the tag sequences to obtain the genetic information of the interacting proteins.

4. A method for constructing a high-throughput analysis of interacting proteins according to claim 3, characterized in that: Step 1) The specific operations are: 11) Using the yeast two-hybrid library-to-library strategy, 0.8 mL of the AD library and BD library were incubated in 2×YPDA medium containing kanamycin; 12) After the incubation is completed, observe the yeast strain under a microscope to see whether the conjugation is successful. If the conjugation is successful, proceed to step 13). If the conjugation is not successful, continue to incubate for 4 hours and observe again. 13) Centrifuge at 1000 g for 10 min, then suspend the cells in 50 mL of 0.5×YPDA medium containing kanamycin; centrifuge at 1000 g for 10 min again, and finally suspend the culture in 5 mL of 0.5×YPDA medium containing kanamycin to obtain bacterial suspension; 14) Dilute the bacterial solution 50 times, take 200 μL of the bacterial solution and spread it on SD / -Leu-Trp-His-Ade / +5mM 3-AT medium, and culture it in a 30°C incubator for 5-7 days; 15) After the culture is completed, screening is performed to obtain yeast-interacting cloned proteins; Wherein, in step 11), the concentration of kanamycin is 50 µg / mL; the volume of the 2×YPDA medium is 50 mL; the incubation conditions are 30°C, 30-50 rpm, and incubation for 20 h; Step 13) The concentration of kanamycin is 50 µg / mL; Step 15) The diameter of the yeast interactive cloned protein is 0.2-1 mm.

5. A method for constructing a high-throughput analysis of interacting proteins according to claim 3, characterized in that: Step 2) The specific preparation process of yeast interaction clone protein bacterial lysis products is as follows: 17 yeast interaction clone proteins with a diameter of 0.2-1 mm are placed in a 200 μL PCR tube, 10 μL of 1x yeast lysis buffer is added, mixed and treated with a PCR instrument at 100°C for 5 minutes; The first round of PCR amplification is specifically as follows: using the yeast interactive cloning protein bacterial lysis product as a template, adding the front-end and back-end common primers of the AD and BD vectors, and amplifying the bait and target gene fragments at the same time; wherein the common primers are: AD-F: CGATGATGAAGATACCCCACC, as shown in SEQ ID NO.118; AD-R:GAGATGGTGCACGATGCACAG, as shown in SEQ ID NO.119; BD-F: GCGACATCATCATCGGAAGAG, as shown in SEQ ID NO. 120; BD-R: GAAAGCAACCTGACCTACAGG, as shown in SEQ ID NO.121.

Images