Method for improving crop resistance to bacterial wilt by using tomato Holliday junction resolvase

Abstract

The present invention belongs to the field of genetic transformation technology and specifically relates to the use of tomato Holliday linker resolvase, methods for improving crop resistance to bacterial wilt, and methods for protein-based pathogen inhibition. The present invention provides a use of a tomato Holliday linker resolvase, wherein the amino acid sequence of the tomato Holliday linker resolvase SlMOC1 is shown in SEQ ID NO:2; the nucleotide sequence encoding the gene SlMOC1 is shown in SEQ ID NO:1. Results from the examples demonstrate that overexpression of the tomato Holliday linker resolvase SlMOC1 gene can improve plant resistance to bacterial wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic transformation, and particularly relates to the application of tomato Holliday junction resolvase, a method for improving crop resistance to bacterial wilt, and a method for inhibiting pathogens with proteins. Background Art

[0002] Tomato bacterial wilt, caused by the bacterium Ralstonia solanacearum, is one of the most serious bacterial diseases affecting agricultural production. The bacterium invades the plant through wounds or natural openings at the roots, colonizing the xylem and invading the vascular bundles, obstructing water transport and causing the plant to wilt and ultimately die. Ralstonia's host species include hundreds of crops, including tomatoes, tobacco, and potatoes. Its strong adaptability provides favorable selective pressure for its genetic variation.

[0003] Currently, the primary means of controlling bacterial wilt in production is still chemical control. However, the long-term use of chemical agents can easily enhance the resistance of pathogens and even threaten crop production safety. Although researchers have begun to experiment with various biological control methods in recent years, their effectiveness is still limited by region, soil, temperature and humidity, and cultivation and management measures, resulting in greater differences in control effectiveness between different regions. In contrast, breeding disease-resistant varieties is a more economical, safe, and promising strategy. Finding disease-resistant gene resources is one of the important directions of genetic breeding for resistance to bacterial wilt. There are no reports in the prior art on the application of tomato Holliday junction resolvase SlMOC1 in resistance to bacterial wilt. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of tomato Holliday junction resolvase, a method for improving crop resistance to bacterial wilt, and a method for protein inhibition of pathogens. The tomato Holliday junction resolvase can reduce the incidence of crop bacterial wilt.

[0005] In order to solve the above technical problems, the following technical solutions are proposed:

[0006] The present invention provides use of a tomato Holliday junction resolvase S1MOC1 in creating crops with resistance to bacterial wilt. The amino acid sequence of the tomato Holliday junction resolvase S1MOC1 is shown in SEQ ID NO: 2.

[0007] The present invention provides the use of a gene encoding tomato Holliday junction resolvase, SlMOC1, in creating crops with resistance to bacterial wilt. The nucleotide sequence of the gene encoding SlMOC1 is shown in SEQ ID NO: 1.

[0008] Preferably, the crops include tomatoes.

[0009] Preferably, the application comprises the following steps: transferring the coding gene SlMOC1 into crops to induce overexpression of the coding gene SlMOC1.

[0010] The present invention provides a recombinant vector, comprising a basic vector and the coding gene SlMOC1 described in the above technical solution, wherein the basic vector comprises a basic prokaryotic expression vector or a basic eukaryotic expression vector.

[0011] The present invention provides a method for improving crop resistance to bacterial wilt, comprising the following steps: introducing the recombinant vector described in the above technical solution into crops to induce overexpression of the coding gene SlMOC1; the basic vector of the recombinant vector includes a basic eukaryotic expression vector.

[0012] Preferably, the transformation method comprises Agrobacterium-mediated method.

[0013] Preferably, the basic eukaryotic expression vector includes pCAMBIA2300.

[0014] The present invention provides a method for inhibiting pathogens of vascular diseases, comprising the following steps: inhibiting the pathogens of vascular diseases by utilizing tomato Holliday junction resolvase S1MOC1.

[0015] Preferably, the method of using tomato Holliday junction resolvase S1MOC1 to inhibit pathogens includes: co-cultivation.

[0016] The present invention provides the use of a tomato Holliday junction resolvase, SlMOC1, in developing crops resistant to bacterial wilt. Based on the structural characteristics of extracellular deoxyribonucleic acid (eDNA) in Ralstonia solanacearum biofilms, the present invention screened for nucleic acid resolvases targeting extracellular Holliday junction-like structures from the tomato genome. Evolutionary analysis of Holliday junction resolvases MOC1 from plants and pathogenic bacteria revealed that MOC1 is conserved across different plants. The tomato Holliday junction resolvase SlMOC1 was expressed in Escherichia coli, and the target protein was purified and then used to treat GMI1000 biofilms. The results showed that the SlMOC1 protein significantly inhibited GMI1000 biofilm formation. The SlMOC1 gene was then overexpressed in the susceptible tomato plant Moneymaker, and the transformed plants had a certain degree of resistance to bacterial wilt, indicating that the tomato Holliday junction resolvase SlMOC1 has application prospects in disease resistance breeding by targeting Ralstonia solanacearum biofilms. In the present examples, tomato SlMOC1 was found to also inhibit biofilms of Xanthomonas oryzae pv. oryzae PXO99A, indicating that the biofilm-inhibiting ability of this resolvase is conserved across Holliday junction structures, suggesting that MOC1 may have applications in vascular diseases of different crops.

[0017] It can be seen that the tomato Holliday junction resolvase SlMOC1 synthesized from the tomato Holliday junction resolvase SlMOC1 gene of the present invention can improve the plant's resistance to bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an evolutionary analysis diagram of Holliday junction resolvase, in which MOC1 from different crops is highlighted in green, RuvC from pathogens is highlighted in blue, and Bacillus ComEA is used as an outgroup control;

[0019] Figure 2 This is the plasmid map of the prokaryotic expression pET15-D vector;

[0020] Figure 3 The results of SlMOC1 treating GMI1000 biofilm in vitro, where A is the predicted structural analysis of tomato SlMOC1, including the Chloroplast transit peptide and Holliday junction resolvase functional domains; B is SlMOC1 without the chloroplast transit peptide. △CTP Protein SDS-PAGE electrophoresis; C is different concentrations of SlMOC1 △CTPThe quantitative results of crystal violet staining of GMI1000 biofilms were processed. The staining solution was imaged in PCR tubes. N = 6. One-way ANOWA statistical analysis was used. Statistical differences are indicated by different lowercase letters in the figures.

[0021] Figure 4 This is a map of the plant overexpression vector pCAMBIA2300;

[0022] Figure 5 Figure 1 shows the results of tomato resistance to bacterial wilt after expressing secretory SlMOC1 in tomato hairy roots. A is a picture of tomato hairy root protein expression detection; B is an image of tomato disease 9 days after inoculation; C is a line graph of tomato bacterial wilt disease index; D is a line graph of tomato survival rate;

[0023] Figure 6 The results of SlMOC1 treating Xanthomonas oryzae pv. oryzae PXO99A biofilm in vitro; the staining solution was imaged in a PCR tube, N=6, and statistical analysis was performed using one-way ANOWA. Statistical differences are indicated by different lowercase letters in the figure. DETAILED DESCRIPTION

[0024] The present invention provides an application of a tomato Holliday junction resolvase S1MOC1 in creating crops with resistance to bacterial wilt. The amino acid sequence of the tomato Holliday junction resolvase S1MOC1 protein is shown in SEQ ID NO: 2.

[0025] In the present invention, the tomato Holliday junction resolvase S1MOC1 protein is overexpressed in crops to improve the resistance of crops to bacterial wilt.

[0026] In the present invention, the crop may include tomatoes; the tomatoes may include the tomato variety Moneymaker, which is a variety susceptible to bacterial wilt and commonly used in tomato bacterial wilt resistance testing.

[0027] The present invention provides a use of the gene SlMOC1 encoding a tomato Holliday junction resolvase for creating crops resistant to bacterial wilt. The nucleotide sequence of the gene encoding SlMOC1 is shown in SEQ ID NO: 1. The use of the present invention preferably comprises the following steps: introducing a recombinant vector into the crop to induce overexpression of the gene encoding SlMOC1; the recombinant vector comprises the gene encoding SlMOC1; and the base vector of the recombinant vector comprises a basic eukaryotic expression vector. The basic eukaryotic expression vector is an overexpression vector.

[0028] As one embodiment, the crop transformation method described herein includes Agrobacterium transformation. In an embodiment of the present invention, Agrobacterium rhizogenes is used to mediate plant genetic transformation. The Agrobacterium rhizogenes may be Agrobacterium rhizogenes MSU440. As one embodiment, the Agrobacterium transformation method described herein preferably employs the dip method. The present invention does not specifically limit the steps of the dip method; conventional methods can be employed.

[0029] In the present invention, the crop may include tomatoes; the tomatoes may include the tomato variety Moneymaker. As one embodiment, the present invention utilizes genetic transformation methods to transform the SlMOC1 gene shown in SEQ ID NO. 1 into tomato hairy roots. The transformed plants possess a certain degree of resistance to bacterial wilt, and the SlMOC1 gene does not affect the normal growth of the crop itself. The SlMOC1 gene has the potential to be applied to disease-resistant breeding for a wider range of crops.

[0030] The present invention provides a recombinant vector, comprising a basic vector and the coding gene SlMOC1 described in the above technical solution; the basic vector comprises a basic prokaryotic expression vector or a basic eukaryotic expression vector.

[0031] As an embodiment, the basic prokaryotic expression vector includes pET15D, and the basic eukaryotic expression vector includes pCAMBIA2300. The basic eukaryotic expression vector is an overexpression vector. The present invention does not specifically limit the preparation method of the recombinant vector, and conventional methods can be used.

[0032] The present invention provides a method for improving crop resistance to bacterial wilt, comprising the following steps: introducing the recombinant vector described in the above technical solution into the crop to induce overexpression of the gene encoding S1MOC1; the base vector of the recombinant vector comprises a basic eukaryotic expression vector. The technical features of the basic eukaryotic expression vector have been discussed above and will not be repeated here.

[0033] In the present embodiment, the gene S1MOC1 shown in SEQ ID NO. 1 was transiently expressed in the hairy roots of the tomato variety Moneymaker, which is susceptible to bacterial wilt. The transformed tomatoes had significantly improved resistance to bacterial wilt, which is beneficial for the breeding of resistant tomato varieties.

[0034] The present invention provides a method for inhibiting pathogens of vascular diseases, comprising the following steps: using the tomato Holliday junction resolvase S1MOC1 to inhibit pathogens of vascular diseases. As an example, the method of using the tomato Holliday junction resolvase S1MOC1 to inhibit pathogens includes co-cultivation. During co-cultivation, the tomato Holliday junction resolvase S1MOC1 inhibits pathogen biofilm formation, demonstrating that the tomato Holliday junction resolvase S1MOC1 has the activity to inhibit pathogens of vascular diseases. In one embodiment, the co-cultivation period can be 2 days, and the co-cultivation temperature can be 28°C. In one embodiment, the pathogens of vascular diseases described herein can be Xanthomonas oryzae pv. oryzae and / or Ralstonia solanacearum. In a specific embodiment, the strain of Xanthomonas oryzae pv. oryzae PXO99A can be used; and the Ralstonia solanacearum can be used. The present invention conducted in vitro functional testing on the Holliday junction resolvase SlMOC1, and the results showed that SlMOC1 has the ability to inhibit the biofilm formation of Ralstonia solanacearum GMI1000; it was also found that the SlMOC1 protein can also inhibit the biofilm formation of the vascular disease Xanthomonas oryzae pv. oryzae PXO99A, indicating that the gene SlMOC1 has the potential to be applied to disease resistance breeding of more crops.

[0035] In one embodiment, the method for preparing the tomato Holliday junction resolvase SlMOC1 protein described herein includes: constructing a prokaryotic expression vector containing the SlMOC1 gene, transferring the prokaryotic expression vector into Escherichia coli to construct recombinant E. coli; and culturing, inducing, and purifying the recombinant E. coli to obtain the tomato Holliday junction resolvase SlMOC1 protein. The prokaryotic expression vector described herein includes a basic prokaryotic expression vector and the SlMOC1 encoding gene described in the above technical solution; in one embodiment, the basic prokaryotic expression vector can be pET15D. In an example of the present invention, the SlMOC1 gene was prokaryotically expressed, the purified SlMOC1 protein was then treated with GMI1000, and it was found that the addition of SlMOC1 significantly inhibited GMI1000 biofilm formation in vitro.

[0036] When the basic vector of the recombinant vector includes a basic prokaryotic expression vector, the present invention preferably transfers the recombinant vector described in the aforementioned technical solution into Escherichia coli, cultures, induces and purifies the recombinant Escherichia coli to obtain the tomato Holliday junction resolvase SlMOC1 protein, and more preferably cultures, induces, crudely extracts, adsorbs and purifies the recombinant Escherichia coli to obtain the tomato Holliday junction resolvase SlMOC1 protein. The method of transferring the recombinant vector into Escherichia coli in the present invention is preferably a plasmid heat shock transformation method. There is no special limitation on the steps of the plasmid heat shock transformation method, and conventional methods can be used. The culture temperature can be 33 to 40°C, and the culture is preferably carried out until the OD of the culture solution reaches 0. 600 The induction method is preferably IPTG induction. There is no particular limitation on the parameters of the IPTG induction method, and conventional methods can be used. The crude extraction, adsorption and purification methods can adopt the His (histidine) tag affinity protein purification method.

[0037] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] In the examples of the present invention, the test materials used are all common commercial products in the field unless otherwise specified.

[0039] 1. Plant materials

[0040] The tomato (Solanum lycopersicum) used was the susceptible tomato variety Moneymaker, which is a susceptible variety commonly used in tomato bacterial wilt resistance testing.

[0041] 2. Vectors and strains

[0042] The plant expression vector pCAMBIA2300 was used for the construction of tomato transgenic plants; the E. coli expression vector pET15-D was used for protein purification;

[0043] DE3 (Escherichia coli) for prokaryotic expression and MC1061 (Escherichia coli) for vector construction were purchased from Wuhan Bovino Biotechnology Co., Ltd., MSU440 (Agrobacterium tumefaciens) for tomato hairy root gene expression came from the Alberto Macho laboratory of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, and the pathogenic bacterial wilt GMI1000 (Ralstonia solanacearum) was from the laboratory of Professor Chen Huilan of the College of Horticulture and Forestry, Huazhong Agricultural University.

[0044] 3. Main reagents

[0045] Gold Mix TSE101 was purchased from Beijing Qingke Biotechnology Co., Ltd.; restriction endonucleases and T4 ligase were purchased from Takara Biotechnology Co., Ltd.; column-based DNA recovery kits were purchased from Shanghai Sangon Biotechnology Co., Ltd.; inorganic salts and organic solvents such as isopropanol, anhydrous ethanol, and chloroform were purchased from Sinopharm Group Co., Ltd.; peptone and casein hydrolysate in the culture medium were purchased from Thermo Fisher Scientific (China) Co., Ltd.; and antibiotics were purchased from Anhui White Shark Biotechnology Co., Ltd.

[0046] 4. Main culture medium

[0047] CPG liquid medium is composed of: 10g bacterial peptone (Bacteriological peptone No. 2), 1g casein hydrolyzate acid, and 5g glucose, dissolved in 1L ddH2O and sterilized at 121°C for 20 minutes. Solid medium requires the addition of 15g / L agar powder.

[0048] NB (Nutrient Broth) medium is composed of 10 g / L peptone, 3 g / L beef extract powder, and 5 g / L sodium chloride dissolved in 1 L ddH2O and sterilized at 121°C for 20 min. For solid culture media, add 15 g / L agar powder.

[0049] 1 / 2MS medium is composed of 2.15 g MS basal salt mixture and 0.5 g MES dissolved in 800 mL ddH2O, adjusted to pH 5.8 with 5 M KOH, and 5.0 g sucrose, dilute to 1 L. For solid medium, add 8 g / L agar powder.

[0050] Example 1 Evolutionary Analysis of Holliday Junction Resolvases from Different Species

[0051] 1. Acquisition of the Tomato SlMOC1 Gene

[0052] For the Arabidopsis thaliana Holliday junction resolvases AtMOC1 gene, see “Yusuke Kobayashi, Osami Misumi, Masaki Odahara, Kota Ishibashi, Masafumi Hirono, Kumi Hidaka, Masayuki Endo, Hiroshi Sugiyama, Hiroshi Iwasaki, Tsuneyoshi Kuroiwa, Toshiharu Shikanai, Yoshiki Nishimura. Holliday junction resolvases mediate chloroplast nucleoid segregation [J]. Science, 2017, 356(6338): 631-634.” published in Science magazine in 2017.

[0053] The tomato genome was obtained from the Solanaceae Genomics Network (https: / / www.solgenomics.net / ), version 2.40 of the tomato genome database. The tomato genome was screened for candidate proteins with functional and structural similarities to the reported Arabidopsis Holliday junction resolvase AtMOC1 gene. The corresponding SlMOC1 gene was identified in the tomato genome database. The nucleotide sequence of the SlMOC1 gene is shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by the SlMOC1 gene is shown in SEQ ID NO: 2.

[0054] SEQ ID NO: 1:

[0055] >SlMOC1(Solyc01g008700)CDS sequence

[0056] ATGGAATCAATTATCTTACAAACTCAACCATTACAATTCATGAACCCAATTACTTCAAAATTCATTCCCACAATTCGATTTACTGCTTCCTCTGTAGCAGCATTCAAGCTCTTCTCATCTTCATCTTCAGCAGCCATTACCACAGATTCCCCTGTCATAGAGCAAAACCCATCTCCTAAACCAGCTATTTCTCCGAGAAATGGGGTTGTAAAGGCTAGGGTTAGTAAAGGCATTTTGGAAGCTCAGCTGAAAATGGACTGGTTGGAGTCTTTGTCTTGCCCATTTCCATGTACAAAGCCTATGGACAGTGGTTGGGTTATTGGGGTTGACCCAGATACTTCTGGTGCTTTGGCACTCTTGAAACCAAATCAAACTCCTCAGGTGTTTGATTCTCCTCACTTGAAGGTACTTGTTGGAAAAGGAGTGCGGAAACGCTTAGATGCAAAAGCTATTGTTCAGTTGCTTCAAAGTTTTGAAGCTCCGTTAGGAACAACTGTGTATATTGAACAATCAACTCCGTATCCACAAGATGGTAAGCAGGGATGGTGGAGTGGAGGATTCGGCTATGGACTATGGATTGGTTTGTTAGTTGCATCCGGATTTTCCGTTACTCCTGTGCCATCAAGTGCATGGAAGAGTGAGTTTCGACTTACAAGAGAACGCTCAAACAAGGATTATAGCAGGGAACTAGCATCTTCATTGTTTCCCTCTTTGAGTTCTTCATTGAAAAGGAAGAAAGATCATGGTCGAGCTGAGGCTCTTCTCATTGCTGCTTATGGCAAAGGCATCAAGATAAATTCTGATTCTCCTTGCGCCGTGGAAAATTTAGATGCTTTAGCAACCGGAGAAAAGCTAGTCAATGAATTCCATCTTTTATCAACTCCAGCGCTTAACAACTAG。

[0057] SEQ ID NO:2:

[0058] >SlMOC1(Solyc01g008700)protein sequence

[0059] MESIILQTQPLQFMNPITSKFIPTIRFTASSVAAFKLFSSSSSAAITTDSPVIEQNPSPKPAISPRNGVVKARVSKGILEAQLKMDWLESLSCPFPCTKPMDSGWVIGVDPDTSGALALLKPNQTPQVFDSPHLKVLVGKGVRKRLDAKA IVQLLQSFEAPLGTTVYIEQSTPYPQDGKQGWWSGGFGYGLWIGLLVASGFSVTPVPSSAWKSEFRLTRERSNKDYSRELASSLFPSLSSSLKRKKDHGRAEALLIAAYGKGIKINSDSPCAVENLDALATGEKLVNEFHLLSTPALNN.

[0060] 2. Construction of evolutionary tree

[0061] In the prior art published by Yoshiki Nishimura's laboratory in 2017 (see "Yusuke Kobayashi, Osami Misumi, Masaki Odahara, Kota Ishibashi, Masafumi Hirono, Kumi Hidaka, Masayuki Endo, Hiroshi Sugiyama, Hiroshi Iwasaki, Tsuneyoshi Kuroiwa, Toshiharu Shikanai, Yoshiki Nishimura. Holliday junction resolvases mediate chloroplast nucleoid segregation[J]. Science, 2017, 356(6338): 631-634"), by performing homology modeling on the tertiary structures of MOC1 and RuvC, it was found that the structures of the two are very similar.

[0062] The present invention first uses MEGA software to perform evolutionary analysis on the amino acid sequences of Holliday junction resolvase of plants and pathogenic bacteria, wherein the plants are Arabidopsis thaliana, cotton (Gossypium raimondii), tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), soybean (Glycine max), wheat (Triticum aestivum), foxtail grass (Setaria italica), wild rice (Oryza brachyantha), Asian cultivated rice (Oryza sativa), and African cultivated rice (Oryza glaberrima); and the pathogens are Ralstonia solanacearum, Xanthomonas oryzae, Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas syringae. syringaepv.Tomato); the sequence numbers of MOCI of various plants and RuvC of pathogenic bacteria in NCBI are as follows Figure 1 As shown in the figure, the amino acid sequence accession numbers of the Holliday junction resolvase of plant MOC1 and pathogen RuvC used in the construction of the phylogenetic tree are shown in Table 1. The phylogenetic tree was constructed using the Neighbor-Joining algorithm, with MOC1 of different crops highlighted in green and RuvC of different pathogens highlighted in blue. Bacillus ComEA was used as an outgroup control. The constructed Holliday junction resolvase evolutionary analysis diagram is shown in the figure. Figure 1 As shown, according to Figure 1 It can be seen that MOC1 is conserved across different plants, and RuvC is conserved across pathogens. This suggests that the function and protein structure of the Holliday junction resolvase MOC1 should be conserved across species. MOC1 is not a gene specific to tomatoes, the subject of this study, but rather widespread across species. This widespread presence suggests that functional analysis of MOC1 in tomatoes could be extended to other crops, allowing for broader research and utilization of this resource.

[0063] The gene accession numbers used to construct the phylogenetic tree are shown in Table 1 below:

[0064] Table 1 Gene information for constructing phylogenetic tree

[0065] Gene name Accession number ArabidopsisthalianaMOC1 AT2G26840 GlycinemaxMOC1 LOC100775189 GossypiumraimondiiMOC1 LOC105768794 NicotianatabacumMOC1 LOC107809858 OryzasativaMOC1 Os01g16340 OryzabrachyanthaMOC1 LOC102700669 OryzaglaberrimaMOC1 LOC127759809 Triticum aestivum MOC1 LOC123069662 SetariaitalicaMOC1 LOC101784623 RalstoniasolanacearumRuvC WP_013206954.1 XanthomonasoryzaeRuvC WP_014504180.1 EscherichiacoliRuvC NP_416377.1 PseudomonasaeruginosaRuvC NP_249656.1 Pseudomonassyringaepv.TomatoRuvC WP_011104812.1 XanthomonasoryzaeRuvX WP_014502544.1 BacillusatrophaeusComEA WP_004398514.1

[0066] The sequences of amplification primers used in the construction of eukaryotic and prokaryotic vectors are shown in Table 2.

[0067] Table 2 Nucleotide sequence information of primers used

[0068] Primer name Primer sequences <![CDATA[SlMOC1 △CTP -SpeI-FP]]> CGGACTAGTGCCATTACCACAGATTCCC(SEQ ID NO:3) SlMOC1-StuI-RP AAAAGGCCTGTTGTTAAGCGCTGGAGTTG(SEQ ID NO:4) <![CDATA[SlMOC1 △CTP -I-FP]]> CGCCATATGGCCATTACCACAGATTCCC(SEQ ID NO:5) <![CDATA[SlMOC1 △CTP -XhoI-RP]]> CCGCTCGAGGTTGTTAAGCGCTGGAGTTG(SEQ ID NO:6)

[0069] Example 2 Effect of Holliday junction resolvase S1MOC1 on Ralstonia solanacearum biofilm in vitro

[0070] 1. Prokaryotic expression vector

[0071] The tomato protein domain in tomato Holliday junction resolvase was predicted using the InterPro website (https: / / www.ebi.ac.uk / interpro / ).

[0072] The predicted results of the nucleotide sequence of chloroplast transit peptide (CTP) and the nucleotide sequence of Holliday junction resolvase are as follows: Figure 3 As shown in A. After removing the nucleotide sequence encoding the chloroplast transit peptide from the nucleotide sequence of the SlMOC1 gene, primer sequences were designed for its Holliday Junction resolvase functional domain. The target sequence product was obtained by PCR amplification using Gold Mix and then connected to the pET15-D vector. After successful restriction enzyme digestion and sequencing of the pET15-D vector, the plasmid was transformed into Escherichia coli DE3, induced for expression, and cultured at 37°C until OD 600 =0.72, add IPTG to a final concentration of 0.25mM, and induce at 16°C. After induction, sonicate until the bacterial solution becomes clear. Centrifuge the solution and retain the supernatant, then adsorb it onto Ni-NTA nickel-containing affinity resin at low temperature. After adsorption, elute with His elution buffer containing 400mM imidazole. The specific process is as follows:

[0073] (1) Construct the vector.

[0074] The E. coli prokaryotic expression vector used was pET15-D, and the vector map is as follows: Figure 2As shown, its N-terminus carries a 6xHis (histidine) tag. First, the pET15-D vector was double-digested and linearized using restriction endonucleases NdeI and XhoI. The nucleotide sequence of the Holliday junction resolvase functional domain of SlMOC1 was amplified using the cDNA of the tomato variety Moneymaker susceptible to bacterial wilt as a template. The DNA polymerase used for amplification was Gold Mix. The amplified fragment was recovered and purified using a column DNA recovery kit. The recovered product and the pET15-D original plasmid were digested with restriction endonucleases NdeI and XhoI, and then ligated to the linearized plasmid using T4 ligase at 16°C. On pET15-D, the ligation product was transformed into Escherichia coli MC1061 strain to screen plasmid transformants, and single transformant colonies were picked and placed in 2 mL of LB liquid culture medium containing 50 μg / mL kanamycin. The culture was shaken at 37°C for 10 hours, and the bacterial liquid was transferred to a 1.5 mL centrifuge tube. Centrifuged at room temperature for 5 minutes, the supernatant was discarded, and the bacteria were collected for plasmid extraction. The plasmid DNA product was obtained after removing the protein by chloroform extraction and precipitating the plasmid DNA with isopropanol. The product was dissolved in sterile water and then digested with restriction endonucleases NdeI and XhoI for enzyme verification. The verified correct plasmid was the prokaryotic expression vector pET15-SlMOC1.

[0075] (2) Induced expression. pET15-SlMOC1 was transformed into DE3 competent cells by heat shock transformation. After culturing at 37°C, a single colony was picked for induced expression. The cells were cultured at 37°C and 190 rpm until OD 600 =0.72, add IPTG with a final concentration of 0.25 mM, induce at 16°C for at least 12 h, centrifuge at 4°C, 4000 rpm for 15 min, discard the supernatant, and collect the bacteria.

[0076] (3) Protein crude extraction: The protein crude extraction process was carried out on ice.

[0077] Add 30 mL of His tag resuspension solution to the bacterial pellet, resuspend the pellet using a vortexer, and then add 1 mM benzylsulfonamide (PMSF) to obtain a resuspension solution;

[0078] The resuspension was transferred to a 50 mL round-bottom tube (for ultrasound and centrifugation). The cell resuspension was brownish-yellow and viscous. Lysozyme with a final concentration of 100 g / mL was added and reacted on ice for 15 minutes. The resuspension was then placed on ice and the cells were disrupted with an ultrasonic instrument (parameters of the ultrasonic instrument were: Xinzhi JY99-IIDN, 6 mm amplitude rod, output power: 300 W). The cells were disrupted for 30 seconds with an interval of 5 minutes, which was recorded as one cycle. The maximum disruption time in this cycle was 45 minutes until the solution was clear and no longer viscous. The solution was then centrifuged at 4°C, 12,000 rpm for 15 minutes, and the supernatant was transferred to a new 15 mL centrifuge tube.

[0079] Protein adsorption: After transferring the supernatant, add Triton-100 to a final concentration of 1% to the centrifuge tube, mix well, add 200 μL of His-tag Ni-NTA nickel-containing affinity resin (the His-tag Ni-NTA nickel-containing affinity resin has been washed with His tag resuspension solution), and then incubate with rotation at 4°C for at least 3 hours;

[0080] Protein purification: After adsorption, centrifuge at 1000 rpm for 1 min. Drain the supernatant with a vacuum pump, leaving only the Ni-NTA nickel-containing affinity resin at the bottom of the tube. Wash the precipitate three times with His-tag wash buffer containing 20 mM imidazole. Slowly drain the supernatant by rotating the vacuum pump. Transfer the Ni-NTA nickel-containing affinity resin to a new 1.5 mL centrifuge tube. After washing, add His-tag eluent containing 400 mM imidazole. Incubate with rotation for more than 15 min. The supernatant collected by horizontal centrifugation is the SlMOC1 protein.

[0081] Protein concentration and purity determination: The concentration of the obtained SlMOC1 protein was measured by microspectrophotometer and was 3.9 mg / mL. The purity of the obtained SlMOC1 protein was detected by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After protein elution, the amount of extracted protein was detected by SDS-PAGE electrophoresis. The results were as follows: Figure 3 As shown in B, it can be seen that the SlMOC1 protein was purified.

[0082] The obtained SlMOC1 protein product was stored at low temperature by adding glycerol with a volume concentration of 80%, and the volume ratio of 80% glycerol to the obtained SlMOC1 protein product was 1:1.

[0083] After testing the SlMOC1 protein concentration at 3.9 mg / mL, the present invention mixed the in vitro purified SlMOC1 protein with cells of the model strain GMI1000 of Ralstonia solanacearum to verify that the Holliday junction resolvase has inhibitory activity against biofilm formation of the model strain GMI1000. If the protein inhibits biofilm formation in vitro, the SlMOC1 protein's ability to target biofilm degradation could be exploited to express it in host plants to verify whether the expressed plants possess resistance to the vascular disease bacterial wilt.

[0084] 2. The wild-type GMI1000 strain was activated on a CPG plate. A single colony was picked and cultured overnight in 2 mL of CPG liquid medium at 28°C and 190 rpm for 14 to 16 hours. The cultured bacteria were collected by centrifugation and resuspended in fresh CPG medium to adjust the OD 600to 0.1 to obtain a resuspension; the resuspension was placed in a 96-well cell culture plate, and then different concentrations of SlMOC1 △CTP Protein (SlMOC1 △CTP The protein is the MOC1 protein that removes the chloroplast transit peptide) for co-culture, and a 96-well cell culture plate is set up with SlMOC1 △CTP The protein concentration gradient was 0, 50, 100, 150, and 200 ng / μL, and the co-culture was sealed with a breathable membrane and incubated at 28°C for 2 days.

[0085] After co-cultivation, the culture medium was aspirated along the wall of the well, and the well plate was washed twice with 100 μL of sterile water, and the well plate was dried. Each well in the well plate was fixed with 100 μL of methanol for 10 minutes, and then the excess methanol was aspirated and air-dried at room temperature. Stain with 200 μL of 0.1% (W / V) crystal violet for 15 to 20 minutes at room temperature, and the excess crystal violet was aspirated, and the well plate was rinsed with 200 μL of sterile water. The attached crystal violet was dissolved in 200 μL of 95% ethanol by volume, dissolved at 37°C for 30 minutes, and the absorbance at 590 nm was measured using an enzyme reader. The measurement results are shown in Table 3 and Figure 3 As shown in C, different letters represent significant differences at the P < 0.05 level. Crystal violet staining observation and quantitative statistics showed that SlMOC1 △CTP It can significantly inhibit the biofilm formation of strain GMI10000, indicating that SlMOC1 has the activity of inhibiting biofilm in vitro.

[0086] Table 3 Different concentrations of SlMOC1 △CTP OD value measurement results at 590nm of GMI1000 biofilm treatment

[0087]

[0088] Example 3 Transient expression of OxS1MOC1 in tomato PR1 Resistance detection to Ralstonia solanacearum

[0089] 1. Construction of plant overexpression vector

[0090] The plant overexpression vector backbone used was pCAMBIA2300 (see Figure 4), the laboratory previously obtained the cloning of the target gene SlMOC1 gene in the pCAMBIA2300-35s:MCS-HA vector in the pCAMBIA2300·terminator reading frame. Experimental group: First, the vector pCAMBIA2300-35s:MCS-HA was linearized by double enzyme digestion with BamHI and StuI. Then, the signal peptide sequence of the tomato pathogenesis-related protein SlPR1 was predicted using the website SignaIP 5.0 (SignalP-5.0-Services-DTU Health Tech). The signal peptide sequence and the Holliday junction resolvase functional domain of the candidate gene SlMOC1 were ligated to the linearized pCAMBIA2300-35s:MCS-HA to construct the overexpression vector pCAMBIA2300-35s:SlPR1-SlMOC1-HA. The correctly constructed pCAMBIA2300-35s:SlPR1-SlMOC1-HA plasmid was transformed into Agrobacterium rhizogenes (MSU440) by Agrobacterium electroporation. The obtained Agrobacterium rhizogenes was designated as Agrobacterium 1. The obtained Agrobacterium 1 mediated tomato hairy root transformation to obtain transformed seedlings.

[0091] pCAMBIA2300-35s:MCS-HA was transformed into Agrobacterium rhizogenes, and the resulting Agrobacterium rhizogenes was designated as Agrobacterium 2. The resulting Agrobacterium 2-mediated tomato hairy root transformation was used as a control (EV).

[0092] 2. Tomato hairy root transformation mediated by Agrobacterium rhizogenes. The genetic transformation steps are as follows:

[0093] Experimental group: (1) About 100 plump and healthy Moneymaker seeds were selected and poured into a 50 mL centrifuge tube. They were washed with 75% alcohol (diluted by mixing anhydrous ethanol and sterile water in a volume ratio of 3:1) for 5 min. The alcohol was discarded. The seeds were washed 3 times with sterile water. Then, the seeds were washed with 50% sodium hypochlorite solution (mixed by mixing 84 disinfectant and distilled water in a volume ratio of 1:1) for 5 min. This process should be carried out in a clean bench. The sodium hypochlorite solution was discarded and the seeds were washed 3 times with sterile water. The washed seeds were placed on the surface of 1 / 2 MS culture medium and cultured vertically on the culture medium plate in the tissue culture room for about 14 days until the first true leaf began to grow.

[0094] (2) Cut off the tomato roots, use a sterilized pipette tip to pick up a single colony of Agrobacterium 1 in step 1 that expresses the target gene (pCAMBIA2300-35s: SlPR1-SlMOC1-HA), place the tomato back on the 1 / 2 MS plate, and cover the base of the hypocotyl with a layer of moist filter paper; after 1 to 2 weeks, the tomato grows new first-generation hairy roots, cut off the first-generation hairy roots with a scalpel (the hairy roots at this time are false positives), place the tomato back on the 1 / 2 MS plate, and place a layer of moist filter paper at the base of the hypocotyl to continue rooting.

[0095] (3) After cutting off the first generation of hairy roots, place the seedlings on a slant 1 / 2 MS solid culture medium containing 50 μg / mL kanamycin (a layer of filter paper is laid on the culture medium in advance), and cover the wound on the hypocotyl with a layer of moist filter paper. Generally, the second generation of hairy roots can be seen growing after 14 to 18 days.

[0096] Control group: Same as (1) to (3) of the experimental group, the only difference is that Agrobacterium 1 in step 1 is replaced by Agrobacterium 2 in step 1.

[0097] After the second generation of hairy roots grew out in the experimental and control groups, the tomato seedlings were transplanted into Jiffypot nutrient soil. Appropriate amount of roots from the experimental group were taken for Western Blot detection of protein expression (see Figure 5 (A) The experimental group was selected from the group with the correct expression of SlPR1-SlMOC1 PR1 The positive plants of the gene were cultured until the four true leaves of the tomato grew out and then inoculated into the fresh bacterial solution of GMI1000. The OD of the fresh bacterial solution of GMI1000 was 600 Dilute to 0.01.

[0098] After the second generation of hairy roots grew out, the tomato seedlings of the control group were transferred to Jiffypot nutrient soil and cultured until the four true leaves of the tomato grew out. Then they were inoculated with fresh GMI1000 bacterial solution. The OD of the fresh GMI1000 bacterial solution was 600 Diluted to 0.01. The disease index is a comprehensive indicator that takes into account the incidence and severity. The more severe the disease, the higher the disease index. The disease severity is divided into 0, 1, 2, 3, and 4 levels, among which, level 0, no wilting symptoms; level 1, 1% to 25% of the leaves wilt; level 2, 26% to 50% of the leaves wilt; level 3, 51% to 75% of the leaves wilt; level 4, 76% to 100% of the leaves wilt. In the control group, pCAMBIA2300-35s:MCS-HA was transformed with a blank vector (EV), and in the experimental group, pCAMBIA2300-35s:SlPR1-SlMOC1-HA was a vector connected with the target gene. After transforming tomatoes, OxSlMOC1 was obtained. PR1 , observe the incidence of the experimental group and the control group on a daily basis (see Figure 5B) and disease index (see Figure 5 C and Table 4), the results showed that OxSlMOC1 PR1 The disease index was significantly lower than that of the blank vector EV (see Figure 5 D and Table 5), indicating that expressing secretory MOC1 in tomato (due to the presence of the PR1 front signal peptide sequence) can reduce the severity of tomato disease. Each control and treatment group had 14 independent plants.

[0099] Table 4 Tomato bacterial wilt disease index data for the control group and the treatment group

[0100]

[0101]

[0102] Table 5 Tomato survival rate data of control group and treatment group

[0103]

[0104] Note: Plants with a disease index <2 at each time point were recorded as "0 (survival)"; plants with a disease index ≥2 were recorded as "1"; this standard refers to the paper "A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition" published in 2020, by Liu Xian, Gang Yu, Yali Wei, Hao Xue, Rafael JL Morcillo, Alberto P. Macho, etc.

[0105] Example 5 Effect of S1MOC1 on Xanthomonas oryzae pv. oryzae PXO99A Biofilm in Vitro

[0106] The wild-type PXO99A strain was activated on NB plates, and a single colony was picked and cultured in 2 mL NB liquid medium at 28°C and 190 rpm overnight for 14 to 16 hours. The cultured bacteria were collected by centrifugation and resuspended in fresh NB medium to adjust the OD 600 to 0.01, and the resulting resuspension was placed in a 96-well cell culture plate, and different concentrations of SlMOC1 were added to the 96-well cell culture plate. △CTP Co-culture and set up SlMOC1 △CTP The concentration gradient was 0, 50, 100, 150, and 200 ng / μL, respectively. The co-culture was sealed with a breathable membrane and incubated at 28°C for 2 days.

[0107] After co-culture, the culture medium was aspirated along the wall of the well, and the well plate was washed twice with 100 μL of sterile water and air-dried. Each well of the cell culture plate was fixed with 100 μL of methanol for 10 minutes, and then the excess methanol was aspirated and air-dried at room temperature. Stain with 200 μL of 0.1% (W / V) crystal violet for 15 to 20 minutes at room temperature, and the excess crystal violet was aspirated, and the well plate was rinsed with 200 μL of sterile water. The attached crystal violet was dissolved in 200 μL of 95% ethanol and dissolved at 37°C for 30 minutes, and the absorbance at 590 nm was measured using an enzyme-linked microplate reader. The measurement results are as follows. Figure 6 As shown in Table 6, different letters represent significant differences at the P < 0.05 level. Crystal violet staining observation and quantitative statistics showed that SlMOC1 △CTP It can significantly inhibit PXO99A biofilm, indicating that Holliday junction resolvase has the potential to be cross-used in different vascular diseases. The species origin may not be the determining factor, but depends on its characteristics of targeting structures such as Holliday junction.

[0108] Table 6 OD value measurement results of S1MOC1 in vitro treatment of PXO99A biofilm at 590nm

[0109]

[0110] In summary, the tomato gene SlMOC1 encoding Holliday junction resolvase provided by the present invention is used to construct tomato varieties with resistance to bacterial wilt. SlMOC1 is derived from tomatoes themselves. Its expression in the hairy roots of target tomato varieties can improve the resistance of tomatoes to bacterial wilt, and has the potential to be used conveniently, safely and effectively in genetic breeding.

[0111] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Use of tomato Holliday junction resolvase S1MOC1 in creating tomatoes with resistance to bacterial wilt, the amino acid sequence of the tomato Holliday junction resolvase S1MOC1 being shown in SEQ ID NO:

2.

2. Tomato Holliday junction resolvase encoding gene SlMOC1 Application of the encoding gene in creating a tomato with resistance to bacterial wilt SlMOC1 The nucleotide sequence is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that The application comprises the following steps: SlMOC1 into tomato, inducing the coding gene SlMOC1 Overexpression of 。 4. A recombinant vector, characterized in that Including basic vector and coding gene SlMOC1 The nucleotide sequence encoding the gene SlMOC1 The nucleotide sequence is shown in SEQ ID NO: 1; the basic vector includes a basic prokaryotic expression vector or a basic eukaryotic expression vector.

5. A method for improving resistance to bacterial wilt in tomatoes, characterized in that: The invention comprises the following steps: introducing the recombinant vector of claim 4 into tomatoes, inducing the coding gene SlMOC1 Overexpression; the basic vector of the recombinant vector includes a basic eukaryotic expression vector.

6. The method according to claim 5, characterized in that The transfer method includes Agrobacterium-mediated method.

7. The method according to claim 5, characterized in that The basic eukaryotic expression vector includes pCAMBIA2300.

8. A method for inhibiting pathogens of vascular diseases, characterized in that: The method comprises the following steps: using the tomato Holliday junction resolvase S1MOC1 shown in SEQ ID NO: 2 to inhibit the pathogen of vascular disease; the pathogen is Ralstonia solanacearum ( Ralstonia solanacearum ) or Xanthomonas oryzae pv. oryzae ( Xanthomonas oryzae pv. oryzae ).

9. The method according to claim 8, characterized in that Methods for using tomato Holliday junction resolvase SlMOC1 to inhibit pathogens include: co-cultivation.

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