Method for detecting SUMOylation modification of tomato protein

Abstract

The invention relates to the technical field of biology, in particular to a method for detecting SUMOylation modification of tomato protein. The method comprises the following steps: preparing-E2 and + E2 type Escherichia coli competent cells, transferring a substrate protein of a tomato to be detected containing a fusion tag into the-E2 and + E2 type competent cells, the-E2 type competent cells being a control group, and the + E2 type competent cells being an experimental group; and inducing protein expression, and comparing the signal intensity and band migration situation of Flag tag substrate protein in-E2 and + E2 sample mycoprotein to judge whether the tomato substrate protein to be detected is subjected to SUMO modification or not. The problem that substrate protein SUMO modification detection is difficult due to complex metabolism regulation in tomato cells is effectively solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting SUMOylation modification of tomato protein. Background Art

[0002] SUMO (small ubiquitin-like modifier) ​​is a class of small molecule proteins widely present in eukaryotes. SUMOylation modification is a dynamic and reversible process. SUMO molecules covalently bind to the lysine residues of substrate proteins with the participation of E1 activating enzymes, E2 conjugating enzymes and E3 ligases to regulate the structure and function of substrate proteins. SENPs specifically deSUMOylate substrate target proteins and jointly regulate the SUMOylation state of substrate proteins with SUMO molecules, thereby regulating cell functions.

[0003] At present, the methods for detecting protein SUMO modification in animals, plants and yeast mainly include in vivo and in vitro methods. In vitro SUMO modification detection has high requirements for the activity of purified proteins and the stability of buffers. So far, no universal SUMO modification reaction buffer system has been established. The SUMO reaction conditions (time, temperature, etc.) of different substrate proteins need to be explored, and the overall cost of the experiment is high.

[0004] The occurrence of SUMOylation in vivo is mainly through the co-expression of substrate protein and SUMO protein (SUMO1GG and SUMO1AA) by Agrobacterium injection of tobacco leaves, protoplast transformation, or stable genetic transgenic plants, and the SUMOylation modification is driven by plant-derived SUMOylation enzymes, and finally the detection of substrate protein SUMOylation modification is carried out by protein immunoprecipitation and methods based on tag antibodies or SUMO1 endogenous antibodies. The detection of SUMOylation in vivo is difficult due to the interference of homologous proteins, protein stability, antibody specificity, dynamic changes in SUMOylation intensity, and other factors. Moreover, although SUMOylation modification is widely present in different species, the number of SUMO and SUMOylation enzyme encoding genes in different species varies greatly, and their functions are also different. It is impossible to standardize and unify the detection of substrate proteins of different species, and it is necessary to formulate corresponding modification detection schemes for different species.

[0005] Compared with the in vivo and in vitro SUMOylation detection methods in plants and animals, the Escherichia coli prokaryotic expression detection system has obvious advantages in terms of time, economic cost and detection efficiency. So far, the prokaryotic expression detection system for protein SUMOylation has been successfully established in the model plant Arabidopsis and the important food crop corn. The Arabidopsis protein prokaryotic expression SUMOylation detection system uses the prokaryotic expression recombinant plasmids pET28-SAE1a-His-AtSAE2 (E1) and pACYCDuet-1-SCE1-SUMO1 (GG) (E2 and SUMO) to modify and detect the substrate protein fused with the Flag tag. Corn has the characteristics of complex genetic background and large amount of genetic information data. Therefore, the researchers constructed the corn cDNA library into the pCDFDuet-1 vector carrying the Myc tag and transformed it into the control group Escherichia coli competent cells pET28a-ZmSAE1-ZmSAE2a and pACYC-His 6 -FLAG-ZmSUMO1aGG(SUMO1a), pET28a-ZmSAE1-ZmSAE2a and pACYC-His 6 -FLAG-ZmSUMO1aGG-ZmSCE1b (SUMO1a+E2), protein purification by His tag and Myc tag, followed by mass spectrometry detection and SUMO modification verification, successfully identified 20 types of SUMO modified proteins and about 70 proteins that may be SUMO modified. However, there is no systematic or unified pathway for substrate protein SUMO modification, so simulating the SUMO modification pathway of other species in prokaryotes cannot be achieved by simply borrowing existing technologies.

[0006] Tomato is a model plant for studying fruit development and an important fruit and vegetable economic crop, with important research value and market value. It is worth exploring whether protein SUMOylation modification is involved in the development and ripening process of tomato fruit. The main reason for the limitation of research is the limitation of SUMOylation detection method. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting SUMOylation modification of tomato protein.

[0008] According to the method for detecting SUMOylation modification of tomato protein of the present invention, the method comprises the following steps:

[0009] Constructing -E2 type Escherichia coli competent cells and +E2 type Escherichia coli competent cells, wherein the -E2 type Escherichia coli competent cells express SlSUMO1GG gene, SlSAE1a gene and SlSAE2 gene, and the +E2 type Escherichia coli competent cells express SlSUMO1GG gene, SlSCE1 gene, SlSAE1a gene and SlSAE2 gene, wherein the SlSUMO1GG gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.1, the SlSCE1 gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.2, the SlSAE1a gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.3, and the SlSAE2 gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.4;

[0010] Integrate the substrate protein to be tested containing the fusion tag into the E. coli expression vector, construct a tag fusion vector, and transfer it into the -E2 type E. coli competent cells and the +E2 type E. coli competent cells, respectively, the former being the control group and the latter being the experimental group;

[0011] Screening positive colonies on a resistance culture medium, and activating and inducing the expression of the substrate protein to be tested;

[0012] Take the induced -E2 and +E2 samples for SDS-PAGE protein electrophoresis and Western blot, and compare the signal intensity and band migration of the Flag-tagged substrate protein in the bacterial protein of -E2 and +E2 samples to determine whether the protein to be detected has undergone SUMO modification.

[0013] SlSUMO1GG, SEQ ID NO.1:

[0014] MSGVTQQEEEKKPAGDQGGHINLKVKSQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDFNSIAFLFDGRRLRAEQTPDELEMEDGDEIDAMLHQTGG

[0015] S1SCE1, SEQ ID NO.2:

[0016] MSAGIARGRLAEERKAWRRNHPHGFVAKPETLPDGSVNLMVWHCSIPGKTGTDWEGGCYPVTIHFSEDYPSKPPKCKFPQGFFHPNVYPSGTVCLSILNEDSGWRPAITVKQILVGIQDLLDQPNPADPAQTEGYHLFIQDAIEYKKRVKLQAKQYPPLV

[0017] SlSAE1a,SEQ ID NO.3:

[0018] MHKRKSRIHYSSAKFLQLFCHLRMGTEGEQLTEQETAIYDRQIRVWGVDAQRRLSKSNVFVSGLRGTSVEFCKNIVLAGVGSLTLNDDRLVTEDLLFANFLTPPAENDFRGKSVAELCCDSLKDFNPMVSVSVEKGALSIFDADFFQKFDVVVVNCCSLLTKKSVNAKCRKLPKRVAFYSVECRDSCGEIFVDLQNYSYCKKKNEETIECKLHYPSFEEAIAVPWRSLPKRMSKLYYAMRVIERFEELEGRNPGETSVDDLPNVQKLRKELCEAHSLNESQIPDSLLRRLIASISEFPPVCAIVGGILGQEVIKAISGKGDPLKNFFFFDAMDGKGIIEDISNGKS

[0019] SlSAE2,SEQ ID NO.4:

[0020] MASEEHLSAIKGAKVLMVGAGGIGCELLKTLALSGFEDIHIIDMDTIEVSNLNRQFLFRQKHVGQSKAKVARDAVLRFRPHIKITPYHANVKDPEFNVDFFKQFNVVLNGLDNLDARRHVNRLCLASGVPLVESGTTGFLGQVTVHVKGKTECYECQSKPAP KTYPVCTITSTPTKFVHCIVWAKDLLFAKLFGNKNQENDLNVRSTDASSSSEHSEDVFERRAAEDVEQYGRRIYDHVFGYNIEVALRNEETWKNRNKPRPVYSKDVLPPKPVGQNGNKDKASNTGDPSSVSAMSSLGLKNPQDLWSLRENSEVFLEALRLFFS KREKEIGQLGFDKDDQLAVEFVTSAANIRAASFGIPLHSLFEAKGIAGNIVHAVATTNAIIAGLIVLEAIKVLQNDVKSYRMTYCLEHPSRKMLLMPVEPFEPNKSCYVCSETPLTLEINTHRSKLRDFVDKIVKEKLGMSLPLMHGVALLYEVGDDLEED EVANYAANLDKVLSELPSPVTGGTILTVEDLQQELKCSINIKHREEFDEEKEPDGMVLSGWTPALAAEKTKTLDNGPSSSNASQTVPLESEDNDEIEIILKDPEILAAGKKRKSSDVSVAVDPEIFSVTGAMLTKKKVEEDDSNNDIVMVDGKLDLNKKKRVQ

[0021] According to the method for detecting SUMOylation modification of tomato protein of the present invention, the nucleotide sequence of the SlSUMO1GG gene is shown as SEQ ID NO.5, the nucleotide sequence of the SlSAE1a gene is shown as SEQ ID NO.6, the nucleotide sequence of the SlSAE2 gene is shown as SEQ ID NO.7, and the nucleotide sequence of the SlSCE1 gene is shown as SEQ ID NO.8.

[0022] The nucleotide sequence of SlSUMO1GG gene, SEQ ID NO.5:

[0023] ATGTCAGGCGTCACTCAACAGGAGGAAGAGAAGAAGCCGGCCGGCGATCAGGGAGGTCACATTAACCTCAAAGTCAAGAGCCAGGATGGAAATGAGGTGTTTTTCAGGATCAAGAGAAGCACACAGCTGAAGAAGCTCATGAATGCTTACTGTGATAGGCAATCTGTGGACTTCAACTCTATTGCCTTCTTGTTTGATGGTCGCCGTCTCAGGGCCGAGCAGACTCCTGATGAGCTCGAGATGGAAGATGGCGATGAAATTGATGCTATGCTGCATCAGACTGGTGGA。

[0024] Nucleotide sequence of SlSAE1a gene, SEQ ID NO.6:

[0025] 。

[0026] The nucleotide sequence of SlSAE2 gene, SEQ ID NO.7:

[0027]

[0028] The nucleotide sequence of S1SCE1 gene, SEQ ID NO.8:

[0029] ATGTCTGCAGGAATCGCTCGTGGCCGTCTTGCTGAAGAACGCAAAGCTTGGCGCAGGAATCATCCCCATGGGTTTGTGGCTAAACCGGAGACGCTTCCAGATGGGTCAGTGAACTTGATGGTGTGGCACTGCTCTATTCCTGGTAAAACAGGGACGGATTGGGAAGGTGGCTGTTATCCAGTTACGATTCACTTCAGTGAAGATTATCCTAGCAAACCACCAAAGTGCAAATTCCCACAAGG TTTCTTCCATCCGAATGTGTATCCATCAGGAACAGTTTGCTTGTCGATCCTCAATGAAGACAGTGGTTGGAGACCAGCCATTACAGTGAAACAGATACTCGTTGGCATCCAAGACCTGTTAGATCAGCCAAACCCTGCTGATCCTGCTCAAACTGAAGGATATCATCTCTTTATTCAGGATGCTATTGAGTACAAGAAACGGTTTAAGCTGCAGGCCAAGCAATATCCGCCTCTGGTGTAG.

[0030] According to the method for detecting SUMOylation modification of tomato protein of the present invention, the antibiotic resistance of the prokaryotic expression vector containing the substrate protein to be detected is different from that of the prokaryotic expression vector containing the SlSUMO1GG gene, the prokaryotic expression vector containing the SlSAE1a gene and the SlSAE2 gene, and the prokaryotic expression vector containing the SlSCE1 gene.

[0031] In view of the above-mentioned defects and shortcomings and in view of the fact that SUMOylation modification may not be completely dependent on E3 in vitro, the technical solution of the present application uses tomatoes as experimental materials, clones the coding genes of SUMO, activating enzyme E1, and conjugating enzyme E2 in tomatoes and constructs recombinant plasmids, and builds a prokaryotic detection system for SUMOylation modification of tomato proteins in Escherichia coli cells. Through protein induced expression and protein immunoblotting technology, rapid and efficient SUMOylation modification detection of any intended protein in tomatoes can be carried out, helping to break through the bottleneck of tomato SUMOylation modification research.

[0032] Beneficial technical effects

[0033] The method for detecting SUMOylation modification of tomato protein according to the present invention effectively simulates the SUMOylation modification reaction mechanism in tomato cells in Escherichia coli cells, and effectively reduces the difficulty of detecting SUMOylation of substrate proteins caused by complex metabolic regulation in plant cells such as tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Show the principle of the detection method of the present application;

[0035] Figure 2 The component identification of the tomato protein SUMO modification detection system according to the specific embodiment of the present application is shown, A: PCR identification of recombinant plasmids in -E2 and +E2 type bacterial liquids; B: Protein immunoblotting detection of each protein factor after induced expression of recombinant plasmids in -E2 and +E2 type bacterial liquids;

[0036] Figure 3 The results of detecting SUMOylation modification of tomato proteins using the method of the present application are shown: A: SUMOylation modification detection of AtADA2b; B: SUMOylation modification detection of SlHsfA1; C: SUMOylation modification detection of SlMMS21; D: SUMOylation modification detection of SlH2B. DETAILED DESCRIPTION

[0037] The tomato protein SUMO modification detection system of the present invention is established based on the occurrence pathway of SUMO modification in tomatoes and the fact that SUMO modification in vitro may not be completely dependent on SUMO E3 ligase. Figure 1 As shown, the specific experimental principle is to clone and embed the coding genes of SUMO1 and E1 (-E2 type) and SUMO1 / E2 and E1 (+E2 type) in tomatoes into prokaryotic expression vectors of different resistances, and co-express SUMO1 and E1, SUMO1 / E2 and E1 proteins in BL21 (DE3) Escherichia coli cells through prokaryotic induced expression. On this basis, the two co-expression colonies are used to prepare the corresponding -E2 and +E2 type competent cells, which are used to accept the introduction of the substrate protein coding gene fusion plasmid, and co-express the substrate protein and the protein factors of the SUMO modification pathway in the corresponding competent cells. Finally, the substrate protein in the -E2 and +E2 type competent cells is detected by immunoblotting with a specific tag antibody, and the number and molecular weight differences of the specific bands in the -E2 and +E2 protein samples are compared and analyzed to determine whether the substrate protein can be modified by SUMO.

[0038] The plasmids used in the following examples are pACYCDuet-1, pET28a(+), and pCDFDuet-1 prokaryotic expression vectors. Among them, pACYCDuet-1 carries a 6his tag and a chloramphenicol resistance marker gene (Cm); pET28a(+) carries a 6his tag and a kanamycin resistance marker gene (Kan); and pCDFDuet-1 carries a 6his tag and a streptomycin resistance marker gene (Str).

[0039] In the following examples, pACYCDuet-1 is mainly responsible for the embedding of the nucleotide sequence of the gene encoding SlSUMO1GG and SlSUMO1GG-SlSCE1 fusion protein and the induced expression of the corresponding protein; pET28a(+) is mainly responsible for the embedding of the nucleotide sequence of the gene encoding SlSAE1a-SlSAE2 fusion protein and the induced expression of the corresponding protein; pCDFDuet-1 is mainly responsible for the embedding of the gene encoding the tomato substrate protein to be detected and the induced expression of the corresponding protein. There are no special requirements for the compatible use of the three plasmids, and the stability of the three antibiotics added needs to be ensured when using them.

[0040] Example 1 Construction of a tomato protein SUMO modification detection system

[0041] 1. Gene cloning and recombinant plasmid construction

[0042] (1) Using the cDNA of the whole MT tomato plant as a template, specific amplification primers were designed and high-fidelity enzymes were used to amplify the genes encoding the activated form of SlSUMO1, SlSUMO1GG, SlSAE1a, SlSAE2, and SlSCE1;

[0043] (2) After gel recovery, the recombinant plasmids pACYCDuet-Myc-SlSUMO1GG, pACYCDuet-Myc-SlSUMO1GG-His-SlSCE1 and pET28a-His-SlSAE1a-HA-SlSAE2 were sequenced correctly using homologous recombination technology and used for the preparation of competent cells. Different tags were added to the coding genes required for the SUMOylation pathway in order to detect whether SlSUMO1GG, SlSCE1, SlSAE1a and SlSAE2 were normally expressed in -E2 and +E2 Escherichia coli competent cells by Western blot to ensure the feasibility of competent cells.

[0044] 2. Preparation of Competent Cells for Tomato Protein SUMOylation Detection System

[0045] (1) Heat shock method: pACYCDuet-Myc-SlSUMO1GG and pET28a-His-SlSAE1a-HA-SlSAE2 were co-transformed into E. coli BL21 competent cells, and pACYCDuet-Myc-SlSUMO1GG-His-SlSCE1 and pET28a-His-SlSAE1a-HA-SlSAE2 were co-transformed into E. coli BL21 competent cells. + and Cm + LB solid screening medium was cultured at 37°C for 12-16h;

[0046] (2) Pick a single clone and transfer it to a centrifuge tube containing 2 mL of liquid LB medium and expand the culture at 37°C / 220 rpm / 2 h;

[0047] (3) The cultured monoclonal bacterial solution was used as the PCR template DNA, and specific amplification primers were used to perform molecular identification of SlSUMO1GG, SlSAE1a, SlSAE2 and SlSCE1. The bacterial solution containing pACYCDuet-Myc-SlSUMO1GG and pET28a-His-SlSAE1a-HA-SlSAE2 plasmids was named -E2 bacterial solution, and the competent cells prepared from it were named -E2 competent cells. The bacterial solution containing pACYCDuet-Myc-SlSUMO1GG-His-SlSCE1 and pET28a-His-SlSAE1a-HA-SlSAE2 plasmids was named +E2 bacterial solution, and the competent cells prepared from it were named +E2 competent cells. Take 50 μL of -E2 and +E2 bacterial solutions respectively and add them to 5 mL of Kan. + and Cm + LB liquid medium was cultured at 37°C for 12-16h;

[0048] (4) Take appropriate amount (1.0-1.5 mL) of newly revived -E2 and +E2 bacterial solution to 100 mL containing Kan + and Cm + LB liquid medium was placed in a conical flask and its OD 600 =0.1, shake at 37°C-180rpm / min for 2-3h until OD 600 =0.5~0.6;

[0049] (5) Place the -E2 and +E2 E. coli cultures on ice for 30 min, divide into 50 mL centrifuge tubes, centrifuge at 4°C-4000 rpm / min-10 min, discard the supernatant, and collect the cells.

[0050] (6) Add 10 mL of pre-cooled Inove transformation solution (2.176 g MnCl 2 .4H 2 O,0.44gCaCl 2 .2H 2 O, 3.73g KCl and 3.02g PIPES to 200ml deionized water, filter and sterilize before use) Resuspend the cells and gently mix to prevent cell death. Centrifuge at 4℃-4000rpm / min-10min and discard the supernatant.

[0051] (7) Take 6 mL of pre-cooled Inove transformation solution to collect -E2 and +E2 type bacteria, gently invert and mix until the bacteria are completely suspended, add 1.5 mL of DMSO to each of the -E2 and +E2 cell suspensions, gently mix, and place on ice for 10 min.

[0052] (8) Dispense into sterile 1.5 mL tubes at a rate of 100 uL / tube, quickly freeze in liquid nitrogen, and store at -80°C to obtain -E2 and +E2 competent cells for detecting SUMOylation of tomato proteins.

[0053] -E2 and +E2 competent cells were cultured in a culture medium containing Kan + and Cm + LB solid screening medium was used for streaking activation culture (37°C constant temperature inverted culture for 12-16 hours). Two single clones were picked and inoculated into 2mL liquid LB medium centrifuge tubes respectively, and expanded culture was carried out at 37°C / 220rpm / 2h. The cultured single clone bacterial liquid was used as PCR template DNA, and the PCR reaction products were detected by agarose gel electrophoresis. Figure 2As shown in Figure A, gene fragments of SlSUMO1GG, SlSAE1a and SlSAE2 can be detected in two single colonies of -E2 type, and gene fragments of SlSUMO1GG, SlSCE1, SlSAE1a and SlSAE2 can be detected in two single colonies of +E2 type, indicating that -E2 and +E2 type competent cells contain plasmid combinations of pACYCDuet-Myc-SlSUMO1GG and pET28a(+)-His-SlSAE1a-HA-SlSAE2, pACYCDuet-Myc-SlSUMO1GG-His-SlSCE1 and pET28a(+)-His-SlSAE1a-HA-SlSAE2, respectively. The occurrence of SUMO modification mainly depends on the efficient translation of the SUMO pathway protein factor encoding gene carried by the plasmid in -E2 and +E2 type competent cells. Therefore, the activated -E2 and +E2 monoclonal bacterial cultures were expanded and induced with IPTG, and the induced protein samples were subjected to SDS-PAGE electrophoresis, and then Western blot detection was performed using different types of tag antibodies (Anti-His, Anti-HA and Anti-Myc). Figure 2 As shown in Figure B, based on the location and intensity of the immune signal, it was found that the signal bands of Myc-SlSUMO1GG, HA-SlSAE1a and His-SlSAE2 could be detected simultaneously in the -E2 sample; the signal bands of Myc-SlSUMO1GG, His-SlSCE1, HA-SlSAE1a and His-SlSAE2 could be detected simultaneously in the +E2 sample, and the positions of the bands were basically consistent with the calculated molecular weight based on the amino acid coding sequence of each protein, indicating that -E2 and +E2 type competent cells can co-express SUMOylation pathway protein factor encoding genes carried by different plasmid combinations.

[0054] Example 2 Using the tomato protein SUMO modification detection system to detect whether the protein is SUMO modified

[0055] The tomato protein SUMOylation detection system constructed in the above embodiment was used to detect the SUMOylation of the following proteins: AtADA2b, SlHsfA1, SlMMS21, and SlH2B. AtADA2b is a known SUMOylation-modified protein in Arabidopsis, SlHsfA1 is a SUMOylation-modified protein that has been reported in tomato, and SlMMS21 and SlH2B are tomato proteins, but they have not been proven to be SUMOylation-modified. Bioinformatics prediction shows that the two have potential SUMOylation sites, so this system is used for verification.

[0056] Protein prokaryotic expression and SUMO modification detection

[0057] (1) The protein to be tested containing the fusion tag was integrated into the pCDFDuet-1 vector to construct a tag fusion vector, and then transformed into -E2 competent cells and +E2 competent cells respectively, the former being the control group and the latter being the experimental group. + , Cm + and Str + The solid LB medium plates were inverted and cultured at 37°C overnight.

[0058] (2) Select positive colonies for activation and culture at 37°C with horizontal shaking at 220 rpm / min for more than 12 h.

[0059] (3) Xiang Han Kan + , Cm + and Str + Add an appropriate amount of activated bacterial solution to 50 mL of LB liquid medium to make OD 600 =0.1-0.15.

[0060] (4) Transfer the conical flask containing liquid LB to a 37°C constant temperature shaker and culture at 220 rpm until the OD 600 =0.4-0.6. 1 mL of -E2 and +E2 bacterial liquid in this state can be taken and centrifuged at 25°C-12000rpm / min for 1 min to collect the bacterial cells as the pre-induction sample.

[0061] (5) Add 250 μL of 0.1 M IPTG (working concentration 0.5 mM / L) to 50 mL of bacterial solution, incubate at 25°C (set in the range of 18°C-25°C depending on the expression of the protein) and 200 rpm / min for 16-18 h to induce protein expression.

[0062] (6) Measure the OD of the bacterial solution 600 (The normal value is about 2.0). 600 OD after induction 600 ×1mL was used to take the sample after induction to ensure that the number of bacteria in the samples before and after induction was roughly the same. Centrifuge at 25℃-12000rpm / min for 1min, discard the supernatant, and freeze the bacteria in liquid nitrogen and store at -80℃.

[0063] (7) Take protein samples before and after induction and perform SDS-PAGE protein electrophoresis and Western blot to detect whether the target protein is expressed.

[0064] (8) On the premise of confirming that the protein to be detected is expressed normally, the induced -E2 and +E2 samples are taken for SDS-PAGE protein electrophoresis and Western blot. By comparing the signal intensity and band migration of the Flag-tagged substrate protein in the bacterial protein of -E2 and +E2 samples, it can be determined whether the protein to be detected has undergone SUMO modification.

[0065] Into -E2 and +E2 type competent cells, recombinant plasmids pCDFDuet-1-Flag-AtADA2b, pCDFDuet-1-Flag-SlHsfA1, pCDFDuet-1-Flag-SlMMS21 and pCDFDuet-1-Flag-SlH2B were introduced respectively, and protein expression was induced. If the substrate protein to be detected can undergo SUMO modification, SUMO will bind to a single or multiple specific lysine residues of the substrate protein, resulting in an increase in the molecular weight of the substrate protein and a decrease in the molecular mobility. Figure 3 As shown, the Flag tag antibody was used to perform Western blot detection on the samples after induction of each substrate protein. In addition to detecting the Flag-AtADA2b, Flag-SlHsfA1, Flag-SlMMS21 and Flag-SlH2B band signals, compared with the signal bands in -E2, different intensities and numbers of signal bands (* marks) were detected above the substrate protein in the +E2 sample, thereby proving that Flag-AtADA2b, Flag-SlHsfA1, Flag-SlMMS21 and Flag-SlH2B can undergo SUMOylation modification, thereby illustrating the feasibility of the technical method of the present patent application.

[0066] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A method for detecting SUMOylation modification of tomato protein, characterized in that: The method comprises the following steps: Preparing -E2 type and +E2 type Escherichia coli competent cells, wherein the -E2 type Escherichia coli competent cells express SlSUMO1GG gene, SlSAE1a gene and SlSAE2 gene, and the +E2 type Escherichia coli competent cells express SlSUMO1GG gene, SlSCE1 gene, SlSAE1a gene and SlSAE2 gene, wherein the SlSUMO1GG gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.1, the SlSCE1 gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.2, the SlSAE1a gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.3, and the SlSAE2 gene encodes a protein having an amino acid sequence as shown in SEQ ID NO.4; Integrate the base sequence of the Flag tag and the gene encoding the tomato substrate protein to be tested into a prokaryotic expression vector, and transfer them into the -E2 type Escherichia coli competent cells and the +E2 type Escherichia coli competent cells respectively, the cells transferred into the -E2 type Escherichia coli competent cells are the control group, and the cells transferred into the +E2 type Escherichia coli competent cells are the experimental group; Screening positive colonies on a resistance culture medium, activating and inducing the expression of the substrate protein of the tomato to be tested, and detecting the expression of the substrate protein; The samples of the control group and the test group after induction were respectively taken for Western blot. If no signal band was detected in the control group, but a signal band was detected in the test group, it indicated that the tomato substrate protein to be tested was SUMOylated.

2. The method for detecting SUMOylation modification of tomato protein according to claim 1, characterized in that: The nucleotide sequence of the SlSUMO1GG gene is shown in SEQ ID NO.5, the nucleotide sequence of the SlSAE1a gene is shown in SEQ ID NO.6, the nucleotide sequence of the SlSAE2 gene is shown in SEQ ID NO.7, and the nucleotide sequence of the SlSCE1 gene is shown in SEQ ID NO.

8.

3. The method for detecting SUMOylation modification of tomato protein according to claim 1, characterized in that: The antibiotic resistance of the prokaryotic expression vector containing the substrate protein to be tested is different from that of the prokaryotic expression vector containing the SlSUMO1GG gene, the prokaryotic expression vector containing the SlSAE1a gene and the SlSAE2 gene, and the prokaryotic expression vector containing the SlSCE1 gene.

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