Application of tomato Halide linker dissociation enzyme, method for improving crop bacterial wilt resistance and method for inhibiting pathogenic bacteria by protein

Abstract

The invention belongs to the technical field of genetic transformation, and particularly relates to application of tomato Halide linker dissociation enzyme, a method for improving crop bacterial wilt resistance and a method for inhibiting pathogenic bacteria through protein. The invention provides application of a tomato Halide linker dissociation enzyme. The amino acid sequence of the tomato Halide linker dissociation enzyme SlMOC1 is as shown in SEQ ID NO: 2. The nucleotide sequence of the coding gene SlMOC1 is as shown in SEQ ID NO: 1. The result of the embodiment shows that the bacterial wilt resistance of the plant can be improved by overexpression of the tomato Halide linker dissociation enzyme SlMOC1 gene.

Description

Technical Field

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

[0002] Tomato bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum, and is one of the most serious bacterial diseases that harm agricultural production. Ralstonia invades the roots through plant wounds or natural openings, colonizes in the xylem, invades the vascular bundle, and blocks the transport of water, causing the plant to wilt and eventually die. Ralstonia hosts hundreds of crops including tomatoes, tobacco, potatoes, etc., and Ralstonia has a strong adaptability, which provides suitable selection pressure for the genetic variation of Ralstonia.

[0003] At present, the main means of prevention and control of bacterial wilt in production is still chemical control, but the long-term use of chemical agents can easily enhance the resistance of pathogens and even threaten the safety of crop production; although researchers have begun to try a variety of biological control methods in recent years, the effect is still limited by the region, soil, temperature and humidity, cultivation and management measures, etc., resulting in greater differences in control effects between different regions. In contrast, breeding disease-resistant varieties is a more economical, safe and considerable strategy, and finding disease-resistant related gene resources is one of the important directions of genetic breeding for resistance to bacterial wilt. There is no report on the application of tomato Holliday junction resolvase SlMOC1 in resistance to bacterial wilt in the prior art. 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 application of tomato Holliday junction resolvase S1MOC1 in creating crops with resistance to bacterial wilt. The amino acid sequence of tomato Holliday junction resolvase S1MOC1 is shown in SEQ ID NO:2.

[0007] The present invention provides an application of a gene S1MOC1 encoding tomato Holliday junction resolvase in creating crops with resistance to bacterial wilt. The nucleotide sequence of the gene S1MOC1 encoding the gene is shown in SEQ ID NO:1.

[0008] Preferably, the crop comprises 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 comprises a basic eukaryotic expression vector.

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

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

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

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

[0016] Beneficial effects of the present invention: The present invention provides the use of tomato Holliday junction resolvase SlMOC1 in creating crops with resistance to bacterial wilt. The present invention first screened a nucleic acid resolvase targeting its extracellular Holliday junction structure from the tomato genome based on the structural characteristics of the extracellular deoxyribonucleic acid (eDNA) of the Ralstonia solanacearum biofilm, and conducted an evolutionary analysis of the Holliday junction resolvase MOC1 of plants and pathogenic bacteria, and found that MOC1 is conservatively present in different plants. The tomato Holliday junction resolvase SlMOC1 was expressed in Escherichia coli prokaryotically, and the target protein was purified and then treated with GMI1000 biofilm. The results showed that the SlMOC1 protein could significantly inhibit the formation of GMI1000 biofilm; the SlMOC1 gene was then overexpressed in the susceptible tomato Moneymaker, and the transformed plants had a certain resistance to bacterial wilt, indicating that the tomato Holliday junction resolvase SlMOC1 has an application prospect in disease-resistant breeding through bacterial wilt biofilm. In the examples of the present invention, it was found that tomato SlMOC1 also had an inhibitory effect on the biofilm of Xanthomonas oryzae pv. oryzae PXO99A, indicating that the ability of this resolvase to inhibit biofilm is conservative for the effect of the Holliday junction structure, which means that MOC1 may have the ability to be applied to vascular diseases of different crops.

[0017] It can be seen that the tomato Holliday junction resolvase SlMOC1 synthesized by 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 the evolution analysis diagram of Holliday junction resolvase, in which MOC1 of different crops is highlighted in green, pathogenic bacteria RuvC 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 dissociation enzyme domains; B is SlMOC1 without chloroplast transit peptide △CTP Protein SDS-PAGE electrophoresis; C is different concentrations of SlMOC1 △CTPThe quantitative results of crystal violet staining of GMI1000 biofilm 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 figure.

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

[0022] Figure 5 The results of tomato resistance to bacterial wilt after secretory SlMOC1 was expressed in tomato hairy roots, where 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 one-way ANOWA statistical analysis was used. Statistical differences are represented 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 detection.

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

[0028] As an embodiment, the method of transferring to crops described in the present invention includes Agrobacterium transformation; in the embodiment of the present invention, Agrobacterium rhizogenes is used to mediate plant genetic transformation. The Agrobacterium rhizogenes may be Agrobacterium rhizogenes MSU440. As an embodiment, the Agrobacterium transformation method described in the present invention preferably adopts the dipping method. The present invention has no special limitation on the operation steps of the dipping method, and conventional methods can be used.

[0029] In the present invention, the crop may include tomatoes; the tomatoes may include the tomato variety Moneymaker. As an embodiment, the present invention uses a genetic transformation method to transform the SlMOC1 gene shown in SEQ ID NO.1 into tomato hairy roots, and the transformed plants have a certain 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 of more 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 has no special limitation on the preparation method of the recombinant vector, and a conventional method 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 coding gene S1MOC1; the basic 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 embodiment of the present invention, the gene SlMOC1 shown in SEQ ID NO.1 is transiently expressed in the hairy roots of Moneymaker, a tomato variety susceptible to bacterial wilt, and the resistance of the transformed tomatoes to bacterial wilt is significantly improved, which is beneficial to the breeding of resistant tomato varieties.

[0034] The present invention provides a method for inhibiting pathogens of vascular diseases, comprising the following steps: using tomato Holliday junction dissociation enzyme SlMOC1 to inhibit pathogens of vascular diseases. As an embodiment, the method of inhibiting pathogens by using tomato Holliday junction dissociation enzyme SlMOC1 in the present invention includes: co-cultivation. During co-cultivation, tomato Holliday junction dissociation enzyme SlMOC1 inhibits the formation of pathogen biofilm, indicating that tomato Holliday junction dissociation enzyme SlMOC1 has the effect of inhibiting the activity of pathogens of vascular diseases. As an embodiment, the co-cultivation time can be 2d, and the co-cultivation temperature can be 28°C. As an embodiment, the pathogens of vascular diseases in the present invention can be rice bacterial blight pathogens and / or solanacearum pathogens. In a specific embodiment, the strain of rice bacterial blight pathogens can be Xanthomonas oryzae pv. oryzae PXO99A; the solanacearum pathogen can be the solanacearum pathogen GMI1000. The present invention performs in vitro functional detection on the Holliday junction resolvase SlMOC1, and the results show that SlMOC1 has the ability to inhibit the biofilm formation of Ralstonia solanacearum GMI1000; it is 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] As an embodiment, the preparation method of the tomato Holliday junction resolvase SlMOC1 protein of the present invention comprises: constructing a prokaryotic expression vector containing the gene SlMOC1, transferring the prokaryotic expression vector into Escherichia coli, and constructing a recombinant Escherichia coli; culturing, inducing and purifying the recombinant Escherichia coli to obtain the tomato Holliday junction resolvase SlMOC1 protein. The prokaryotic expression vector of the present invention comprises a basic prokaryotic expression vector and the encoding gene SlMOC1 described in the above technical scheme; as an embodiment, the basic prokaryotic expression vector can be pET15D. In an embodiment of the present invention, the gene SlMOC1 is prokaryotically expressed, and the SlMOC1 protein is purified and then treated with GMI1000. It is found that the addition of SlMOC1 in vitro can significantly inhibit the formation of GMI1000 biofilm.

[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 of the present invention is preferably a plasmid heat shock transformation method, and the steps of the plasmid heat shock transformation method are not particularly limited, 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 0.1. 600 The induction method is preferably IPTG induction method; there is no special 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 in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope 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 tomato susceptible 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 (Escherichiacoli) for vector construction were purchased from Wuhan Bovino Biotechnology Co., Ltd., MSU440 (Agrobacterium tumefaciens) for tomato hairy root gene expression came from Alberto Macho's laboratory, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, and GMI1000 (Ralstonia solanacearum) pathogenic Ralstonia solanacearum came from the laboratory of Professor Chen Huilan, 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 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.; antibiotics were purchased from Anhui White Shark Biotechnology Co., Ltd.

[0046] 4. Main culture medium

[0047] The composition of CPG liquid medium is: 10g bacterial peptone (Bacteriological peptone No. 2), 1g casein hydrolyzate acid and 5g glucose, dissolved in 1L ddH2O, sterilized at 121℃ for 20min. Solid medium needs to add 15g / L agar powder.

[0048] The composition of NB (Nutrient Broth) medium is: 10g / L peptone, 3g / L beef extract powder and 5g / L sodium chloride, dissolved in 1L ddH2O, sterilized at 121℃ for 20min. Solid medium needs to add 15g / L agar powder.

[0049] The composition of 1 / 2MS medium is: 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, 5.0 g sucrose, and fixed to 1 L. Solid medium needs to 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 resolvase 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 / ) website, the tomato genome ITAG 2.40 version. Candidate proteins with similar functions and structures to the Arabidopsis Holliday junction resolvase AtMOC1 gene reported in the literature were screened from the tomato genome, and the corresponding SlMOC1 gene was determined 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 resolution mediates 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 invention 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, Gossypium raimondii, Nicotiana tabacum, Solanum lycopersicum, Glycine max, Triticum aestivum, Setaria italica, Oryza brachyantha, Oryza sativa and 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 The amino acid sequence accession numbers of the Holliday junction resolvase of plant MOC1 and pathogen RuvC used in constructing the evolutionary tree are shown in Table 1. The phylogenetic tree was constructed using the Neighbor-Joining algorithm. MOC1 of different crops was highlighted in green, RuvC of different pathogens was highlighted in blue, and Bacillus ComEA was used as an outgroup control. The evolutionary analysis diagram of the constructed Holliday junction resolvase is shown in Figure 1 As shown, according to Figure 1 It can be seen that MOC1 is conservatively present in different plants, and RuvC is conservatively present in pathogens. It can be inferred that the function and protein structure of the Holliday junction resolvase MOC1 should be conservative in different species. MOC1 is not a unique gene in the tomato, which is the research subject of the present invention. It is widely present in different species. Widespread presence means that the functional analysis of MOC1 in tomatoes can be extended to other crops, and such resources can be studied or used more widely.

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

[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 Pseudomonas aeruginosaRuvC NP_249656.1 Pseudomonassyringaepv.TomatoRuvC WP_011104812.1 XanthomonasoryzaeRuvX WP_014502544.1 Bacillus atrophaeusComEA 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 nucleotide sequence prediction results of chloroplast transit peptide (CTP) and chloroplast 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, the primer sequence was designed for its Holliday Junction resolvase functional domain, and the target sequence product was obtained by PCR amplification using Gold Mix and then connected to the pET15-D vector. After the pET15-D vector was successfully digested and sequenced, the plasmid was transferred into Escherichia coli DE3, induced for expression, and cultured at 37°C until OD 600 =0.72, add IPTG with a final concentration of 0.25mM, induce at low temperature at 16℃; after induction, ultrasonically disrupt the bacterial solution until it becomes clear, centrifuge the bacterial solution after disruption, retain the supernatant, adsorb it with Ni-NTA nickel-containing affinity resin at low temperature, and elute with His elution buffer containing 400mM imidazole after adsorption. The specific process is as follows:

[0073] (1) Construct 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 linearized by double restriction endonucleases NdeI and XhoI, and 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, and 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 colonies of transformants 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 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 plasmid 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 0.25mM, induce at 16℃ for at least 12h, centrifuge at 4℃, 4000rpm for 15min, 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 with 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 min. The resuspension was placed on ice and the cells were disrupted with an ultrasonic device (parameters of the ultrasonic device were: Xinzhi JY99-IIDN, 6 mm amplitude transformer, output power: 300 W). The cells were disrupted for 30 s with an interval of 5 min, which was counted as one cycle. The maximum disruption time in this cycle was 45 min until the solution was clear and no longer viscous. Centrifuge at 4°C, 12,000 rpm for 15 min, and the supernatant was transferred to a new 15 mL centrifuge tube.

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

[0080] Protein purification: After adsorption, centrifuge at 1000 rpm for 1 min in a horizontal centrifuge, 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 washing 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, add His-tag elution buffer containing 400 mM imidazole after washing, and rotate and incubate 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 a micro-spectrophotometer and was 3.9 mg / mL. The purity of the obtained SlMOC1 protein was detected by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After the protein was eluted, the amount of extracted protein was detected by SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown in B, it can be seen that the SlMOC1 protein was purified.

[0082] Glycerol with a volume concentration of 80% was added to cryopreserve the obtained SlMOC1 protein product, and the volume ratio of 80% glycerol to the obtained SlMOC1 protein product was 1:1.

[0083] After the concentration of SlMOC1 protein is detected to be 3.9 mg / mL, the in vitro purified SlMOC1 protein is mixed with the bacterial body of the model strain GMI1000 of Ralstonia solanacearum to verify that the Holliday junction resolvase has inhibitory activity on the formation of biofilm of the model strain GMI1000 of Ralstonia solanacearum. If the protein has an inhibitory effect on the formation of biofilm in vitro, the characteristic of SlMOC1 protein in targeted degradation of biofilm can be utilized to express it in the host plant to verify whether the expressed plant has the ability to resist the vascular disease bacterial wilt.

[0084] 2. The wild-type GMI1000 strain was activated on a CPG plate, and a single colony was picked and cultured overnight in 2 mL CPG liquid medium at 28°C and 190 rpm for 14 to 16 hours. The cultured bacterial cells were collected by centrifugation, resuspended in fresh CPG medium, and the OD was adjusted. 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, respectively. The co-culture was sealed with a breathable membrane and incubated at 28°C for 2 days.

[0085] After co-culture, the culture medium was aspirated along the well wall, 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, 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 Tables 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 for 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 of the pCAMBIA2300-35s:MCS-HA vector in the pCAMBIA2300·terminator reading frame. Experimental group: First, the vector was linearized by double restriction digestion with BamHI and StuI based on pCAMBIA2300-35s:MCS-HA, and then the signal peptide sequence of tomato pathogenesis-related protein SlPR1 was predicted through 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 connected 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 transferred into Agrobacterium rhizogenes (MSU440) by Agrobacterium electroporation. The obtained Agrobacterium rhizogenes was recorded 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. The seeds were washed with 75% alcohol (mixed and diluted with anhydrous ethanol and sterile water in a volume ratio of 3:1) for 5 min. The alcohol was discarded. The seeds were washed three times with sterile water. The seeds were then washed with 50% sodium hypochlorite solution (mixed with 84 disinfectant and distilled water in a volume ratio of 1:1 to obtain a 50% sodium hypochlorite solution) for 5 min. This process should be carried out in a clean bench. The sodium hypochlorite solution was discarded and the seeds were washed three 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 roots of the tomatoes, 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 tomatoes back on the 1 / 2MS plate, and cover the base of the hypocotyl with a layer of moist filter paper; after 1 to 2 weeks, the tomatoes grow new first-generation hairy roots, use a scalpel to cut off the first-generation hairy roots (the hairy roots at this time are false positives), place the tomatoes back on the 1 / 2MS 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 of 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 being that Agrobacterium 1 in step 1 was replaced by Agrobacterium 2 in step 1.

[0097] After the second generation of hairy roots grew out, the tomato seedlings in the experimental and control groups were moved to Jiffypot nutrient soil, and 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 correct expression of SlPR1-SlMOC1 PR1 The positive plants of the gene were cultured until the tomato had four true leaves 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 in the control group were transferred to the Jiffypot nutrient soil and cultured until the four true leaves of the tomato grew out. Then they were inoculated with the fresh bacterial solution of GMI1000. The OD of the fresh bacterial solution of GMI1000 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 , daily statistical observation of the incidence of the experimental group and the control group (see Figure 5B) and disease index (see Figure 5 C and Table 4), the results showed that OxS1MOC1 PR1 The disease index was significantly lower than that of the blank vector EV (see Figure 5 D and Table 5), indicating that the expression of secretory MOC1 in tomato (because of the front signal peptide sequence of PR1) can reduce the severity of tomato disease. The control group and the treatment group were repeated in 14 independent plants.

[0099] Table 4 Disease index data of tomato bacterial wilt in the control group and 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 document "A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition" published in 2020, written by Liu Xian, Gang Yu, Yali Wei, Hao Xue, Rafael J. L. 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 the NB plate, and a single colony was picked and cultured overnight in 2 mL NB liquid medium at 28°C and 190 rpm for 14 h to 16 h. The cultured bacterial cells were collected by centrifugation and resuspended in fresh NB medium to adjust the OD 600 to 0.01, and the obtained 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 in 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, 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-labeled instrument. 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 for cross-use 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 determination 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 a tomato variety with resistance to bacterial wilt. SlMOC1 is derived from tomatoes themselves, and its expression in the hairy roots of the target tomato variety can improve the resistance of tomatoes to bacterial wilt. It has the potential to be conveniently, safely and effectively used in genetic breeding.

[0111] 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. Application of tomato Holliday junction resolvase S1MOC1 in creating crops with resistance to bacterial wilt, wherein the amino acid sequence of tomato Holliday junction resolvase S1MOC1 is shown in SEQ ID NO:

2.

2. Application of the gene SlMOC1 encoding tomato Holliday junction resolvase in creating crops with resistance to bacterial wilt, the nucleotide sequence of the gene SlMOC1 being shown in SEQ ID NO:

1.

3. The use according to claim 1 or 2, characterized in that: The crops include tomatoes.

4. The use according to claim 2, characterized in that: The application comprises the following steps: transferring the coding gene SlMOC1 into crops to induce overexpression of the coding gene SlMOC1.

5. A recombinant vector, characterized in that: It comprises a basic vector and the coding gene S1MOC1 according to claim 2, wherein the basic vector comprises a basic prokaryotic expression vector or a basic eukaryotic expression vector.

6. A method for improving crop resistance to bacterial wilt, characterized in that: The method comprises the following steps: introducing the recombinant vector according to claim 5 into crops to induce overexpression of the coding gene SlMOC1; the basic vector of the recombinant vector comprises a basic eukaryotic expression vector.

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

8. The method according to claim 6, characterized in that: The basic eukaryotic expression vector includes pCAMBIA2300.

9. A method for inhibiting pathogenic bacteria of vascular diseases, characterized in that: The following steps are involved: Using the tomato Holliday junction resolvase SlMOC1 to inhibit pathogens of vascular diseases.

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

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