Construction and application method of wheat yeast two-hybrid cDNA library

Abstract

The invention provides a construction method and an application method of a wheat yeast two-hybrid cDNA library, and belongs to the technical field of biology. The invention provides a construction method of a yeast two-hybrid cDNA library, which comprises the following steps: extracting total RNA, separating mRNA, converting into cDNA, transferring into escherichia coli competent cells through a series of reactions to obtain a cDNA primary library bacterial liquid, separating primary library plasmids, and obtaining a yeast two-hybrid cDNA library bacterial liquid through an LR recombination reaction and conversion of the escherichia coli competent cells. A high-quality wheat variety Zheng wheat 158 is taken as a research material, wheat gliadin genes Tagli-gamma-1 and Tagli-gamma-11 are cloned, a corresponding bait vector is constructed, the interaction protein is screened from a cDNA library of the Zheng wheat 158 by utilizing a yeast two-hybrid technology, and a foundation is laid for carrying out interaction protein screening of genes related to wheat quality and analyzing a regulation mechanism of the interaction protein in a later period.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a construction method of a wheat yeast two-hybrid cDNA library and an application method thereof. Background Art

[0002] Yeast two-hybrid technology is a commonly used technique to study the interaction between plant proteins. At present, it is widely used in various species as a modern molecular biotechnology. In 2016, Wang Yuqiu and others successfully screened 16 rice smut fungus effector interacting proteins from rice through yeast two-hybrid technology, and analyzed the theoretical mechanism related to the infection of rice by smut fungus. In 2020, Li Jian and others identified the ZmCIPK24-2 gene involved in the regulation of salt stress from corn, and found through yeast two-hybrid technology LUC complementation imaging experiments that ZmCIPK24-2 interacts with ZmCBL1, ZmCBL4, ZmCBL8 and ZmCBL9 in the corn CBLs family, providing new experimental evidence for analyzing the function of the corn CBL-CIPK signaling pathway. In 2022, Xie Ruiying and others used yeast two-hybrid technology to explore the role of NtMYB4a in the synthesis of tobacco secondary metabolites and response to abiotic stress. They found that NtMYB4a protein may interact with vacuolar sorting proteins, B2 proteins, RALF-like 22 proteins, and stress-enhancing proteins to participate in the defense and regulation mechanism of tobacco under low temperature stress. In addition, yeast two-hybrid technology has also been widely used in cotton, bitter melon, tomato, rapeseed, etc.

[0003] This technology has also been well applied in wheat. Zhao Lei and others used forward genetics to clone the important gene TaHST1L (NAD-dependent deacetylase HST1-like gene) that controls the tillering angle of wheat, and screened the interacting proteins through the yeast library to identify a protein TaIAA17 involved in auxin signal transduction. The research found that TaHST1L may participate in the auxin signal transduction pathway by inhibiting the accumulation of TaIAA17. In 2023, Lv Guoguo and others cloned a gene TaCWI (A cell wall invertase) that can resist wheat stem base rot and sheath blight. Through yeast two-hybrid technology, it was found that the cell wall invertase protein it encodes interacts with the α-galactosidase (TaGAL) protein that can degrade cell wall components, and inhibits the expression of the TaGAL gene, offsetting the degradation of TaGAL on the cell wall and thickening the cell wall, thereby resisting the invasion of pathogens, thereby achieving wheat disease resistance. In the same year, Song Xiao et al. used yeast two-hybrid technology to screen the interacting protein SdrI-like of wheat nitrate transporter TaNRT1.1-1A, laying the foundation for further analysis of the molecular mechanism of TaNRT1.1-1A regulating wheat nitrate absorption. It can be seen that yeast two-hybrid technology has also been widely used in wheat yield, disease resistance and other aspects, but the application of this technology in wheat quality, especially in dough quality, has not been reported.

[0004] Wheat (Triticum aestivum L.) is one of the most important food crops in my country, providing 20% ​​of protein and energy for humans. While maintaining high and stable wheat yields, improving wheat quality and meeting market demand is one of the important goals that wheat researchers in my country have long pursued. Conducting research on gene functions related to wheat dough quality and exploring the regulatory mechanisms related to it can provide an important basis for the genetic improvement of wheat quality. Summary of the invention

[0005] The present invention provides a method for constructing a high-quality wheat yeast two-hybrid cDNA library and its application, which can lay a foundation for screening interacting proteins of genes related to wheat dough quality and analyzing their regulatory mechanisms.

[0006] The present invention provides a method for constructing a high-quality wheat yeast two-hybrid cDNA library, comprising the following steps: (1) isolating and purifying mRNA from total RNA of a target species, and reverse transcribing the mRNA into cDNA;

[0007] (2) transferring the cDNA described in step (1) into competent Escherichia coli cells to obtain a cDNA primary library solution;

[0008] (3) isolating the primary library plasmid from the cDNA primary library bacterial solution of step (2), and obtaining the yeast two-hybrid cDNA library bacterial solution by LR recombination reaction and transformation of Escherichia coli competent cells.

[0009] In a preferred embodiment of the present invention, the target species in step (1) include monocotyledonous plants such as high-quality wheat.

[0010] In a preferred embodiment of the present invention, the transfer in step (2) comprises transferring the cDNA into DH5α competent cells by connecting the recombination linker attB1, fractionating and performing BP recombination reaction.

[0011] The present invention also provides the use of a yeast two-hybrid cDNA library constructed by the above construction method in screening wheat dough quality related gene interacting proteins.

[0012] In a preferred embodiment of the present invention, the genes include wheat γ-gliadin 1 gene Tagli-γ-1 (TraesCS1A02G007300) and γ-gliadin 11 gene Tagli-γ-11 (TraesCS1D02G001100).

[0013] The present invention also provides a method for screening wheat dough quality-related gene interacting proteins using a yeast two-hybrid cDNA library constructed by the above construction method, comprising the following steps: (1) using cDNA obtained by reverse transcription of total RNA of a target species as a template, and performing PCR amplification using specific primers designed according to the target gene, connecting the amplified product to a T vector and then transferring it into bacteria for culturing, and extracting plasmids;

[0014] (2) using the plasmid described in step (1) to amplify the target fragment carrying two restriction sites and inserting it into the basic vector backbone to construct a bait expression vector;

[0015] (3) extracting a yeast two-hybrid library plasmid from the yeast two-hybrid cDNA library bacterial solution constructed by the above construction method, and co-transforming yeast competent cells with the yeast two-hybrid library plasmid and the bait expression vector described in step (2) to obtain transformed yeast;

[0016] (4) Spreading the bacterial solution of the transformed yeast described in step (3) on a SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficient plate, and after culturing for a period of time, picking blue colonies and continuing to culture on a SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficient plate, and screening positive clones again;

[0017] (5) Amplifying and sequencing the positive clones screened out in step (4), screening blue monoclonal colonies, and determining the candidate proteins that interact with the target gene.

[0018] In a preferred embodiment of the present invention, the target genes in step (1) include wheat dough quality-related genes Tagli-γ-1 and Tagli-γ-11.

[0019] In a preferred embodiment of the present invention, when the wheat dough quality-related gene is Tagli-γ-1, the specific primers include a primer pair having nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2;

[0020] When the wheat dough quality-related gene is Tagli-γ-1, the specific primers include a primer pair whose nucleotide sequences are shown as SEQ ID No.3 and SEQ ID No.4.

[0021] In a preferred embodiment of the present invention, the basic vector skeleton in step (2) includes a pGBKT7 vector.

[0022] In a preferred embodiment of the present invention, the yeast competent cells in step (3) include Y2H Gold competent cells.

[0023] Beneficial effects: The present invention provides a method for constructing a high-quality wheat yeast two-hybrid cDNA library, firstly extracting total RNA and separating and purifying mRNA, converting it into cDNA and then transferring it into competent Escherichia coli cells, obtaining a cDNA primary library bacterial solution by connecting a recombination linker attB1, fractionating and performing BP recombination reaction, separating the primary library plasmid, and obtaining a yeast two-hybrid cDNA library bacterial solution by LR recombination reaction and transforming competent Escherichia coli cells. The present invention uses high-quality wheat variety Zhengmai 158 as research material, clones wheat γ-alcohol-soluble protein gene Tagli-γ-1 (TraesCS1A02G007300), constructs a bait vector pGBKT7-Tagli-γ-11, uses yeast two-hybrid technology to screen candidate interacting proteins of Tagli-γ-1 from a cDNA library of Zhengmai 158, and further verifies its interaction relationship through a rotation verification test, thereby determining the candidate protein that interacts with Tagli-γ-1; uses high-quality wheat variety Zhengmai 158 as research material, clones wheat γ-alcohol-soluble protein 11 gene Tagli-γ-11 (TraesCS1D02G001100), constructs a bait vector pGBKT7-Tagli-γ-11, uses yeast two-hybrid technology to screen candidate interacting proteins of Tagli-γ-11 from a cDNA library of Zhengmai 158, and further verifies its interaction relationship through a rotation verification test, thereby determining the candidate protein that interacts with Tagli-γ-11. In the embodiment of the present invention, the grains of high-quality wheat Zhengmai 158 at different developmental stages were used as materials to construct its yeast two-hybrid cDNA library, which laid a foundation for the subsequent screening of interacting proteins of genes related to wheat quality and the analysis of their regulatory mechanisms, and provided important gene resources for the genetic improvement of wheat quality in the Huanghuaihai wheat region. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The quality detection diagram of seed RNA (A) and mRNA (B) of Zhengmai 158 at different developmental stages;

[0025] Figure 2 This is the library capacity identification diagram of Zhengmai 158 grain cRNA primary library;

[0026] Figure 3 This is the PCR detection diagram of the insert fragment of Zhengmai 158 primary library;

[0027] Figure 4 This is the PCR detection diagram of the insert fragment of the Zhengmai 158 yeast two-hybrid library;

[0028] Figure 5 This is the electrophoresis detection diagram of Tagli-γ-1-pMD18-T bacterial solution PCR product;

[0029] Figure 6This is a diagram of the digestion of pGBKT7-Tagli-γ-1 with NdeI and BamH I; M in the diagram: DL10000; 1: pGBKT7-Tagli-γ-1 bait vector; 2: fragments digested with restriction endonucleases NdeI and BamH I;

[0030] Figure 7 This is a diagram for detecting the self-activation activity of the pGBKT7-Tagli-γ-1 bait vector;

[0031] Figure 8 This is a diagram showing the screening results of Tagli-γ-1 interacting proteins;

[0032] Fig. 9 This is a rotation verification diagram of Tagli-γ-1 and some candidate interacting proteins;

[0033] Fig.10 This is the electrophoresis detection diagram of the PCR product of Tagli-γ-11-pMD18-T bacterial solution;

[0034] Fig.11 This is a diagram of the digestion of pGBKT7-Tagli-γ-11 with NdeI and BamH I, where M: DL10000; 1: pGBKT7-Tagli-γ-11 bait vector; 2: fragments digested with restriction endonucleases Nde I and BamH I;

[0035] Fig.12 This is a self-activation assay diagram of yeast transformed with the pGBKT7-Tagli-γ-11 bait vector;

[0036] Fig.13 This is a rotation verification diagram of Tagli-γ-11 and some candidate interacting proteins. DETAILED DESCRIPTION

[0037] The present invention provides a method for constructing a high-quality wheat yeast two-hybrid cDNA library, comprising the following steps: (1) isolating and purifying mRNA from total RNA of a target species, and reverse transcribing the mRNA into cDNA;

[0038] (2) transferring the cDNA described in step (1) into competent Escherichia coli cells to obtain a cDNA primary library solution;

[0039] (3) isolating the primary library plasmid from the cDNA primary library bacterial solution of step (2), and obtaining the yeast two-hybrid cDNA library bacterial solution by LR recombination reaction and transformation of Escherichia coli competent cells.

[0040] The present invention extracts total RNA from the material of the target species and separates and purifies mRNA. The extraction of the total RNA is not particularly limited. For example, in one embodiment, the CTAB method is used to extract total RNA. In the embodiment, Zhengmai 158 is taken as an example, and the CTAB method is used to extract total RNA from grains at different developmental times. The total RNA is required to be of stable and high quality. Then, the mRNA is separated and purified using conventional methods in the art. The electrophoresis band is uniformly diffused, indicating that the mRNA is not degraded and meets the requirements for constructing a library.

[0041] The present invention reverse transcribes the purified mRNA into cDNA according to the instructions of the reverse transcription kit (TaKaRa Code No.RR047A). The present invention transfers the cDNA into competent cells, such as Escherichia coli competent cells, DH5α competent cells in one embodiment, specifically, refer to GATEWAY TM The recombinant linker attB1adapter was designed by the Cloning Technology method and the cDNA was connected to it. The cDNA with attB1adapter was fractionated using a cDNA fractionation chromatography column (Invitrogen Code No.18092-015) and then purified by BP The cDNA with attB1 linker was connected to the pDONR222 vector using II enzyme mix (Invitrogen Code No. 11789-020), and the above reaction product was electrotransformed into DH5α competent cells (1500V, 200Ω, 25μF) to finally obtain the cDNA primary library bacterial solution.

[0042] The present invention extracts the primary library plasmid from the cDNA primary library bacterial solution, and then uses LR Clonase IImix (Invitrogen Code No. 11791-020) to recombinant the primary library plasmid with pGADT7-DEST through LR recombination reaction, and then electrotransforms it into DH5α (1500V, 200Ω, 25μF) to obtain the yeast two-hybrid library bacterial solution.

[0043] The yeast two-hybrid library obtained in the present invention was verified, and it was proved that the length of the inserted fragments was between 1 and 2 KB, the recombination rate was 100%, and the construction standard of the yeast two-hybrid library was met, the connection efficiency was high, and the library capacity was large.

[0044] The present invention also provides the use of a high-quality wheat yeast two-hybrid cDNA library constructed by the above construction method in screening wheat dough quality related gene interacting proteins.

[0045] In one embodiment of the present invention, the high-quality wheat variety Zhengmai 158 is used as the research material, the wheat γ-alcohol-soluble protein gene Tagli-γ-1 (TraesCS1A02G007300) is cloned, and the bait vector pGBKT7-Tagli-γ-1 is constructed. The yeast two-hybrid technology is used to screen the candidate interacting proteins of Tagli-γ-1 from the cDNA library of Zhengmai 158, and the interaction relationship is further verified by the rotation verification test, thereby determining the candidate protein that interacts with Tagli-γ-1; in another embodiment, Zhengmai 158 is used as the research material, the wheat γ-alcohol-soluble protein 11 gene Tagli-γ-11 (TraesCS1D02G001100) is cloned, and the bait vector pGBKT7-Tagli-γ-11 is constructed. The yeast two-hybrid technology is used to screen the candidate interacting proteins of Tagli-γ-11 from the cDNA library of Zhengmai 158, and the interaction relationship is further verified by the rotation verification test, thereby determining the candidate protein that interacts with Tagli-γ-11.

[0046] The present invention also provides a method for screening wheat dough quality-related gene interacting proteins using a high-quality wheat yeast two-hybrid cDNA library constructed by the above construction method, comprising the following steps: (1) using cDNA obtained by reverse transcription of total RNA of a target species as a template, and performing PCR amplification according to specific primers designed according to the target gene, connecting the amplified product to a T vector and then transferring it into bacteria for culturing, and extracting plasmids;

[0047] (2) using the plasmid described in step (1) to amplify the target fragment carrying two restriction sites and inserting it into the basic vector backbone to construct a bait expression vector;

[0048] (3) extracting a yeast two-hybrid library plasmid from the high-quality wheat yeast two-hybrid cDNA library bacterial solution constructed by the above construction method, and co-transforming yeast competent cells with the yeast two-hybrid library plasmid and the bait expression vector described in step (2) to obtain transformed yeast;

[0049] (4) Spreading the bacterial solution of the transformed yeast described in step (3) on a SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficient plate, and after culturing for a period of time, picking blue colonies and continuing to culture on a SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficient plate, and screening positive clones again;

[0050] (5) Amplifying and sequencing the positive clones screened out in step (4), screening blue monoclonal colonies, and determining the candidate proteins that interact with the target gene.

[0051] The present invention first clones the target gene, and uses the cDNA obtained by reverse transcription of total RNA as a template during cloning, and amplifies it through specific primers, connects the amplified product to the pMD18-T vector, selects the positive single clone with correct sequencing for bacterial culture, and extracts the plasmid. In the embodiment of the present invention, when the target gene is Tagli-γ-1 gene, the specific primer sequence is as follows:

[0052] F1(SEQ ID No.1):5'-ATGAAGACCTTACTCATCCTG-3';

[0053] R1(SEQ ID No.2):5'-TTATTGGCCACCAATGTCG-3';

[0054] When the target gene is Tagli-γ-11 gene, the specific primer sequences are as follows:

[0055] F11(SEQ ID No.3):5'-ATGAAGACCTTACTCATCCTAACA-3';

[0056] R11 (SEQ ID No. 4): 5'-TTATTGGCCACCAATGCCG-3'.

[0057] The present invention uses the extracted plasmid as a template, designs primer sequences with restriction sites at both ends, inserts the target cDNA into the basic backbone vector, and constructs a bait expression vector. In the embodiment of the present invention, the Tagli-γ-1 gene and the Tagli-γ-11 gene are respectively inserted between the NdeI and BamHI restriction sites of the basic backbone vector, thereby constructing two bait expression vectors: pGBKT7-Tagli-γ-1 and pGBKT7-Tagli-γ-11.

[0058] The present invention transforms the bait expression vector into a yeast competent cell, and the yeast competent cell can be a Y2H Gold competent cell, and a negative control and a positive control are set at the same time. After the transformation, the present invention also includes performing a self-activation test, specifically, after the bait expression vector + pGADT7, the positive control pGBKT7-p53 + pGADT7-T and the negative control pGBKT7-Lam + pGADT7-T are respectively transferred into the Y2H Gold recipient bacteria, they are coated on SD-Trp / -Leu two-deficiency plates, SD / -Trp / -Leu / -His / X-α-gal / AbA three-deficiency plates, and SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA four-deficiency plates, and the growth of yeast on these three nutritional deficiency plates is observed to determine whether the vector has successfully transformed the yeast strain, and to verify whether the bait expression vector has self-activation activity.

[0059] The present invention performs a subsequent screening process for interacting proteins on the correctly verified transformed yeast, including extracting a yeast two-hybrid library plasmid from the above-constructed high-quality wheat yeast two-hybrid cDNA library bacterial solution, co-transforming the yeast two-hybrid library plasmid and the bait expression vector into yeast competent cells, and then applying the transformed yeast liquid on a SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficient plate for inverted culture, culturing to a colony size of 2 mm, picking blue colonies to continue growing on a SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficient plate, and screening positive clones again. At the same time, positive and negative controls are set, and repeated 3 times.

[0060] Then, the screened positive clones were identified by PCR and sequenced. If the target gene is Tagli-γ-1 gene or Tagli-γ-11 gene, the specific primer sequences used are as follows:

[0061] DF(SEQ ID No.5):5'-CGGCTAGTAAAATTGATGATGGTAATAATTCA-3';

[0062] AD-R (SEQ ID No. 6): 5'-CACAGTTGAAGTGAACTTGCGG-3'.

[0063] After sequencing, the sequencing results were compared and analyzed using the NCBI BLAST database. After two rounds of screening, 10 candidate proteins that interact with Tagli-γ-1 and are involved in the synthesis and degradation of starch and storage proteins in wheat seeds were identified; 8 candidate proteins that interact with Tagli-γ-11 and are involved in the synthesis and degradation of starch and storage proteins or cysteine ​​in wheat grains and other reproductive growth were identified.

[0064] To further illustrate the present invention, a high-quality wheat yeast two-hybrid cDNA library and a method for screening interacting proteins provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0065] During PCR amplification in the embodiment of the present invention, the PCR reaction system was prepared according to the method of Wang Shasha et al. (Wang Shasha, Huang Shaomin, Song Xiao, et al. Analysis of the relationship between allelic variation of wheat nitrogen utilization efficiency gene TaARE1-A and yield-related traits [J]. Henan Agricultural Science, 2023, 52(12): 14-21.), and the PCR amplification program was set. The reaction system was placed in a PCR amplifier (BIO-RADS T100 TM PCR amplification reaction was carried out in a Thermal Cycler.

[0066] Example 1 Construction of yeast two-hybrid cDNA library of seeds of high-quality wheat variety Zhengmai 158 at different developmental stages and its quality identification:

[0067] 1. Total RNA extraction and mRNA detection: The CTAB method was used to extract total RNA from grains of Zhengmai 158 at different developmental times, such as 7, 14, 21, 28, 35 and 42 DPA after flowering. The electrophoresis results showed that the 28S and 18S bands were clearly visible in the lanes, indicating that the total RNA was stable and of high quality ( Figure 1 After separation and purification of mRNA, the electrophoresis band is uniformly diffuse ( Figure 1 Middle B), indicating that the mRNA was not degraded and met the requirements for library construction.

[0068] 2. Construction and identification of the primary library: The purified Zhengmai 158 mRNA was reverse transcribed into cDNA, and the cDNA was electroporated into DH5α competent cells through ligation of the recombination linker attB1, fractionation and BP recombination reaction, and finally the Zhengmai 158 cDNA primary library was obtained.

[0069] After diluting the above bacterial solution 1000 times, take 50 μL and spread it on the LB plate containing Kan (containing Kan resistance), and record the number of colonies on the plate as 202 ( Figure 2), the primary library titer was calculated as 4.0×10 6 CFU / mL. The volume of the transformed bacterial solution is 2 mL, so the bacterial solution storage capacity of the E. coli library is 4.0×10 6 CFU / mL×2mL=8.0×10 6 CFU. Then 24 single clones were selected for PCR detection. The results showed that all 24 clones had insert fragments, of which 4 were less than 1KB in length, 1 was greater than 2KB in length, and the rest were between 1 and 2KB in length on average. The recombination rate was 100%, which met the quality standards of the primary library ( Figure 3 ).

[0070] 3. Construction and quality identification of yeast two-hybrid library

[0071] The primary library plasmid was transformed into DH5α by LR recombination reaction and electroporation to obtain yeast two-hybrid library. It was diluted 1000 times and then spread on LB (containing Kan resistance) plate. The number of colonies on the plate was recorded as 237, and the titer of yeast two-hybrid library was 4.7×10 6 CFU / mL. Yeast two-hybrid library capacity is 9.4×10 6 CFU( Figure 4 ). Then 24 single clones were selected for PCR detection, and the results showed that all 24 clones contained inserts, of which 8 were less than 1KB, 5 were greater than 2KB, and the remaining were between 1 and 2KB. The recombination rate was 100%, meeting the construction standard of yeast two-hybrid library. In summary, the Zhengmai 158 cDNA library constructed by BP recombination reaction and LR recombination reaction had high connection efficiency and large library capacity.

[0072] Example 2 Cloning of wheat γ-gliadin gene Tagli-γ-1 and screening of its interacting proteins

[0073] The high-quality wheat variety Zhengmai 158 was used as the research material, the wheat γ-alcohol-soluble protein 1 gene Tagli-γ-1 (TraesCS1A02G007300) was cloned, and the bait vector pGBKT7-γ-alcohol-soluble protein 1 (pGBKT7-Tagli-γ-1) was constructed. The yeast two-hybrid technique was used to screen the candidate interacting proteins of γ-alcohol-soluble protein 1 from the cDNA library of Zhengmai 158, and the interaction relationship was further verified by the rotation verification experiment.

[0074] 2.1 Tagli-γ-1 gene cloning: Total RNA from Zhengmai 158 seeds was extracted using the TRIzol method and reverse transcribed to obtain cDNA according to the instructions of the reverse transcription kit (TaKaRa Code No. RR047A) and stored at -20°C.

[0075] According to the cDNA sequence of Tagli-γ-1 gene, specific primers (SEQ ID No.1 and SEQ ID No.2) were designed, and PCR amplification was performed using Zhengmai 158 cDNA as template. The PCR product was detected by 1.0% agarose gel electrophoresis, recovered, and connected to the pMD18-T cloning vector. After bacterial liquid PCR detection and sequencing, the wheat Tagli-γ-1 gene cDNA sequence with a length of 1020 bp was finally obtained ( Figure 5 ).

[0076] 2.2 Construction of Tagli-γ-1 bait vector and yeast transformation: Based on the wheat gene Tagli-γ-1 cDNA sequence and the pGBKT7 vector sequence, specific primers carrying Ndel and BamHⅠ restriction sites were designed using Primer premier 3.0 online software:

[0077] F2 (SEQ ID No. 7):5'-GGTACCATGAAGACCTTACTCATCC-3';

[0078] R2 (SEQ ID No. 8): 5'-TCTAGATTATTGGCCACCAATGTC-3'.

[0079] The target fragment was obtained by PCR amplification using wheat Tagli-γ-1-pMD18-T plasmid DNA as a template and the designed specific primers F2 / R2. The target fragment was digested with NdeI and BamHⅠ and ligated with T4 DNA ligase to construct the bait expression vector pGBKT7-Tagli-γ-1. The target fragment was digested with NdeI and BamHⅠ and the target fragment was ligated with pGBKT7 vector to construct the bait expression vector pGBKT7-Tagli-γ-1. Figure 6 ) and sequencing, it was found that the pGBKT7-Tagli-γ-1 bait vector was successfully constructed.

[0080] pGBKT7-Tagli-γ-1 was transformed into Y2H Gold competent cells using the PEG / LiAC-mediated yeast transformation method, and a negative control (pGBKT7-Lam) and a positive control (pGBKT7-p53) were set up at the same time (CHEN J, ZHOU JH, SANDERS CK, et al. A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping [J]. Analytical Biochemistry, 2009, 390 (1): 29-37.).

[0081] 2.3 Self-activation detection of bait expression vector pGBKT7-Tagli-γ-1: pGBKT7-Tagli-γ-1+pGADT7, positive control pGBKT7-p53+pGADT7-T and negative control pGBKT7-Lam+pGADT7-T were respectively transferred into Y2HGold recipient bacteria, and then spread on SD-Trp / -Leu double-deficiency plates, SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficiency plates, and SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficiency plates, and the growth of yeast on these three nutritional deficiency plates was observed to determine whether the vector was successfully transformed into the yeast strain and to verify whether pGBKT7-Tagli-γ-1 has self-activation activity.

[0082] The results are as follows Figure 7 As shown, yeast transformed with the three plasmids can grow on SD / -Leu / -Trp double-deficiency plates. The positive control can grow on SD / -Trp / -Leu / -His / X-α-Gal triple-deficiency plates and SD / -Trp / -Leu / -His / -Ade / X-α-Gal quadruple-deficiency plates. The yeast containing pGBKT7-Tagli-γ-1 and the negative control cannot grow normally on SD-Trp / -Leu / -His / X-α-Gal triple-deficiency plates and SD-Trp / -Leu / -His / -Ade / X-α-Gal quadruple-deficiency plates, indicating that the three plasmids have been successfully transformed into yeast and the protein encoded by Tagli-γ-1 does not have self-activation activity.

[0083] 2.4 Screening of Tagli-γ-1 interacting proteins

[0084] In order to screen Tagli-γ-1 interacting proteins, the constructed Zhengmai 158 yeast two-hybrid library plasmid and the bait vector pGBKT7-Tagli-γ-1 plasmid were first co-transformed into Y2H Gold yeast competent cells. The transformed yeast solution was then spread on the SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficient plate and inverted at 30°C for 3 to 5 days. When the colony grows to 2 mm in size, pick the blue colony and continue to grow on the SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficient plate, inverted at 30°C for 3 to 5 days, and screen the positive clones again. Set up positive and negative controls and repeat 3 times.

[0085] The positive clones were identified by PCR and sequenced using specific primers SEQ ID No.5 and SEQ ID No.6. The sequencing results were analyzed for homology using the BLAST database of NCBI. After two rounds of screening, a total of 117 blue monoclonal colonies were obtained ( Figure 8 ), and identified 10 candidate proteins interacting with Tagli-γ-1, which were cysteine-rich and transmembrane domain protein 1 (XM_044546254.1), α-amylase inhibitor (XM_044509330.1), specific endo-1,3:1,4-β-D-glucanase 2 (XM_044550285.1, XM_044594213.1), sucrose 1-fructosyltransferase (XM_044554537.1), pullulanase 1 (XM_0445546254.1), and α-amylase inhibitor (XM_044509330.1). M_044577151.1) and cysteine ​​protease B-like (XM_044577994.1), as well as cell number regulatory factors CNR8-like (XM_044483337.1, XM_044491902.1) and CNR2-like (XM_044468782.1) related to plant reproductive growth, especially fruit or grain growth, and ubiquitin domain proteins DSK2b-like (Dominant suppressor of KAR 2b like, XM_044546840.1) and CIP73 (CCa MK-interacting protein of 73kμ, XM_044554473.1) involved in protein modification (Table 1). These proteins are mainly involved in the synthesis and degradation of starch and storage proteins or cysteine ​​in wheat seeds.

[0086] Table 1 Proteins interacting with Tagli-γ-1 screened by yeast two-hybrid system

[0087] Serial number Accession Number Notes 1 XM_044509330.1 Alpha-amylase inhibitors 2 XM_044546254.1 Cysteine-rich and transmembrane domain protein 1 3 XM_044546840.1 Ubiquitin domain protein DSK2b-like 4 XM_044550285.1, XM_044594213.1 Specific endo-1,3:1,4-β-D-glucanase 2 5 XM_044554473.1 Ubiquitin domain protein CIP73 6 XM_044554537.1 Sucrose 1-fructosyltransferase 7 XM_044577151.1 Pullulanase 1 8 XM_044577994.1 Cysteine ​​protease B-like 9 XM_044483337.1, XM_044491902.1 Cell number regulator CNR8-like 10 XM_044490442.1 Cell number regulator CNR2-like

[0088] 2.5 Verification of Tagli-γ-1 interacting proteins

[0089] In order to further verify the interaction relationship between Tagli-γ-1 and candidate proteins (XM_044509330.1, XM_044550285.1, XM_044577994.1, XM_044546254.1), the constructed pGBKT7-Tagli-γ-1 vector plasmid and pGADT7-candidate interaction protein vector plasmid were transformed into Y2H Gold yeast competent cells, and then spread on SD / -Trp / -Leu / -His / X-α-gal / AbA and SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA four-deficient plates in turn and cultured for 3 to 5 days. If blue colonies grow on the four-deficient plates, it means that there is an interaction relationship between Tagli-γ-1 and the candidate protein. Otherwise, it means that there is no interaction relationship between Tagli-γ-1 and the candidate protein. The results are as follows Fig. 9 As shown, blue colonies grew on the four-deficient plates, suggesting that the four positive clones obtained through screening may interact with Tagli-γ-1.

[0090] Example 3 Cloning of wheat γ-gliadin gene Tagli-γ-11 and screening of its interacting proteins

[0091] Using Zhengmai 158 as the research material, the wheat γ-alcohol-soluble protein gene Tagli-γ-11 was cloned, and the bait vector pGBKT7-γ-alcohol-soluble protein 11 (pGBKT7-Tagli-γ-11) was constructed. The yeast two-hybrid technique was used to screen the candidate interacting proteins of Tagli-γ-11 from the cDNA library of Zhengmai 158, and the interaction relationship was further verified by the rotation verification experiment.

[0092] 3.1 Tagli-γ-11 gene cloning:

[0093] The total RNA of seeds of Zhengmai 158 at different developmental stages was extracted using the TRIzol method. The cDNA was obtained by reverse transcription according to the instructions of the reverse transcription kit (TaKaRa Code No.RR047A) and stored at -20°C. Specific primers SEQ ID No.3 and SEQ ID No.4 were designed for PCR amplification reaction. The PCR product was detected by 1.0% agarose gel electrophoresis, recovered, ligated to the pMD18-T cloning vector, and sequenced. Finally, the cDNA sequence of the wheat Tagli-γ-11 gene with a length of 880 bp was obtained ( Fig.10 ).

[0094] 3.2 Construction of Tagli-γ-11 bait vector and yeast transformation:

[0095] According to the cDNA sequence of wheat Tagli-γ-11 gene and the pGBKT7 vector sequence, specific primers carrying two restriction enzyme sites, Ndel and BamHI, were designed using Primer premier3.0 online software:

[0096] F2 (SEQ ID No. 9):5'-CATATGATGAAGACCTTACTCATCC-3';

[0097] R2 (SEQ ID No. 10): 5'-GGATCCTTATTGGCCACCAAT-3'.

[0098] Wheat Tagli-γ-11-pMD18-T plasmid DNA was used as a template and PCR amplification was performed using the designed specific primers F2 / R2. The target fragment was digested with the pGBKT7 vector using Nde I and BamH I endonucleases and ligated with T4 DNA ligase to construct the bait expression vector pGBKT7-Tagli-γ-11. The double digestion of Nde I and BamH I was detected ( Fig.11 ) and sequencing, the pGBKT7-Tagli-γ-11 bait vector was successfully constructed. Using the PEG / LiAC-mediated yeast transformation method, pGBKT7-Tagli-γ-11 was transformed into the Y2H Gold yeast strain, and negative and positive controls were set up at the same time.

[0099] 3.3 Self-activation detection of bait expression vector pGBKT7-Tagli-γ-11:

[0100] According to step 3.2, pGBKT7-Tagli-γ-11+pGADT7, positive control pGBKT7-p53+pGADT7-T and negative control pGBKT7-Lam+pGADT7-T were respectively transferred into Y2HGold yeast, and then spread on SD-Trp / -Leu double deficiency plate, SD / -Trp / -Leu / -His / X-α-gal / AbA triple deficiency plate, and SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple deficiency plate in turn, and the growth of yeast on these three nutritional deficiency plates was observed to determine whether the vector was successfully transformed into the yeast strain and to verify whether pGBKT7-Tagli-γ-11 has self-activation activity.

[0101] The results are as follows Fig.12As shown, yeast transformed with the three plasmids can grow colonies on the SD-Trp / -Leu double-deficiency plate. The positive control can grow colonies on the SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficiency plate and the SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficiency plate. The yeast containing the pGBKT7-Tagli-γ-11 recombinant vector and the negative control cannot grow colonies on the SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficiency plate and the SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficiency plate, indicating that the three plasmids have been successfully transferred into the yeast strain, and the protein encoded by the Tagli-γ-11 gene does not have self-activation activity.

[0102] 3.4 Screening of Tagli-γ-11 interacting proteins: Using the same method as in Example 2, 8 candidate interacting proteins with Tagli-γ-11 were identified, namely, cysteine ​​protease (XM_044577944.1), fructose bisphosphate aldolase (XM_044518121.1), cysteine-rich and transmembrane domain protein 1 (XM_044546254.1) and (1,3:1,4)-β-D-glucanase (XM_044550279.1); The auxin response factor ARF17-like (XM_044586904.1) and cell number regulatory factor CNR8-like (XM_044499954.1) related to reproductive growth (especially pollen and grain growth and development) and the ubiquitin domain protein DSK2b (XM_044546840.1, XM_044474147.1) and transcription factor PIF1-like (XM_044597895.1) related to plant stress response (Table 2) were mainly involved in the synthesis and degradation of starch and storage protein or cysteine ​​in wheat grains and other aspects of reproductive growth.

[0103] Table 2 Proteins interacting with Tagli-γ-11 screened by yeast two-hybrid system

[0104] Serial number Accession Number Notes 1 XM_044577944.1 Cysteine ​​proteases 2 XM_044518121.1 Fructose bisphosphate aldolase 3 XM_044546254.1 Cysteine-rich and transmembrane domain protein 1 4 XM_044550279.1 (1,3:1,4)-β-D-glucanase 5 XM_044499954.1 Cell number regulator CNR8-like 6 XM_044586904.1 Auxin response factor ARF17-like 7 XM_044546840.1, XM_044474147.1 Ubiquitin domain protein DSK2b 8 XM_044597895.1 Transcription factor PIF1-like

[0105] 3.5 Reciprocal verification of Tagli-γ-11 interacting proteins and identification of candidate proteins interacting with Tagli-γ-11

[0106] In order to further verify the interaction between Tagli-γ-11 and candidate proteins (XM_044518121.1, XM_044546254.1 and XM_044550279.1), the constructed pGBKT7-Tagli-γ-11 vector plasmid and pGADT7-candidate interaction protein vector plasmid were transformed into Y2H Gold yeast competent cells, and then coated on SD / -Trp / -Leu / -His / X-α-gal / AbA and SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA four-deficient plates in turn and cultured for 3 to 5 days. If blue colonies grow on the four-deficient plates, it means that there is an interaction between Tagli-γ-11 and the candidate protein, otherwise, it means that there is no interaction between Tagli-γ-11 and the candidate protein.

[0107] The results are as follows Fig.13 As shown, blue colonies grew on the four-deficient plates, and it was speculated that the three positive clones obtained by screening may have an interaction relationship with Tagli-γ-11.

[0108] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a yeast two-hybrid cDNA library, characterized in that: The following steps are involved: (1) isolating and purifying mRNA from total RNA of the target species, and reverse transcribing the mRNA into cDNA; (2) transferring the cDNA described in step (1) into competent Escherichia coli cells to obtain a cDNA primary library solution; (3) isolating the primary library plasmid from the cDNA primary library bacterial solution of step (2), and obtaining the yeast two-hybrid cDNA library bacterial solution by LR recombination reaction and transformation of Escherichia coli competent cells.

2. The construction method according to claim 1, characterized in that: The target species in step (1) includes wheat.

3. The construction method according to claim 1, characterized in that: The transfer in step (2) includes transferring the cDNA into DH5α competent cells by connecting the recombination linker attB1, fractionating and performing BP recombination reaction.

4. Use of a yeast two-hybrid cDNA library constructed by the construction method according to any one of claims 1 to 3 in screening for interacting proteins of genes related to wheat quality.

5. The application according to claim 4, characterized in that: The genes include wheat γ-gliadin 1 gene and wheat γ-gliadin 11 gene.

6. A method for screening wheat quality-related gene interacting proteins using a yeast two-hybrid cDNA library constructed by the construction method according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Using the cDNA obtained by reverse transcription of the total RNA of the target species as a template, PCR amplification was performed with specific primers designed based on wheat quality-related genes, the amplified product was connected to a T vector and then transferred into bacteria for culture, and the plasmid was extracted; (2) using the plasmid described in step (1) to amplify the target fragment carrying two restriction sites and inserting it into the basic vector backbone to construct a bait expression vector; (3) extracting a yeast two-hybrid library plasmid from the yeast two-hybrid cDNA library bacterial solution constructed by the construction method according to any one of claims 1 to 3, and co-transforming yeast competent cells with the yeast two-hybrid library plasmid and the bait expression vector described in step (2) to obtain transformed yeast; (4) Spreading the bacterial solution of the transformed yeast described in step (3) on a SD / -Trp / -Leu / -His / X-α-gal / AbA triple-deficient plate, and after culturing for a period of time, picking blue colonies and continuing to culture on a SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA quadruple-deficient plate, and screening positive clones again; (5) Amplifying and sequencing the positive clones screened out in step (4), screening blue monoclonal colonies, and determining the candidate proteins that interact with the target gene.

7. The method according to claim 6, characterized in that: The wheat quality-related genes in step (1) include wheat γ-gliadin 1 gene and wheat γ-gliadin 11 gene.

8. The method according to claim 7, characterized in that: When the wheat dough quality-related gene is the wheat γ-gliadin 1 gene, the specific primers include a primer pair having nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2; When the wheat dough quality-related gene is the wheat γ-gliadin 11 gene, the specific primers include a primer pair whose nucleotide sequences are shown as SEQ ID No. 3 and SEQ ID No.

4.

9. The method according to claim 6, characterized in that: The basic vector skeleton in step (2) includes the pGBKT7 vector.

10. The method according to claim 6, characterized in that: The yeast competent cells in step (3) include Y2H Gold competent cells.

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