Application of XooRuvC, a nuclease screened from the genome of Xanthomonas oryzae, in improving the resistance of rice to bacterial blight

Abstract

The application belongs to the technical field of genetic transformation, and particularly relates to application of a lyase XooRuvC screened from a genome of Xanthomonas oryzae in improving resistance of rice to bacterial leaf blight. In the embodiment of the application, overexpression of the lyase XooRuvC in rice can significantly improve the resistance of rice to bacterial leaf blight without affecting normal growth and breeding of the crop, and is beneficial to cultivation of resistant varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic transformation, and particularly relates to application of a resolvase XooRuvC screened from a genome of Xanthomonas oryzae in improving resistance of rice to bacterial leaf blight. BACKGROUND

[0002] China is the world's major rice planting country, and the cultivation of rice and the quality of rice are closely related to the economic and social development of China, and the bacterial leaf blight of rice is a bacterial disease which has an important influence on rice production. The bacterial leaf blight is caused by Xanthomonas oryzae pv. oryzae (Xoo) infection; Xoo is a typical gram-negative bacteria, mainly infects the leaves or leaf sheaths of rice through water holes or wounds, reproduces and spreads in vascular tissues, and finally causes the rice leaves to turn yellow or even wither. If the disease occurs in the heading stage, it will form unripe or sterile grains, resulting in yield reduction or quality decline of rice.

[0003] Since Xoo mainly damages in the vascular bundle, there is currently a lack of safe and efficient systemic fungicides, and in addition, humid and high-temperature weather or rain, typhoon caused mechanical damage to rice plants all provide favorable conditions for the outbreak of the bacterial leaf blight, so the disease often has fast onset, wide spread and difficult prevention and control. At present, breeding disease-resistant varieties is still an economical and effective and targeted measure for preventing and controlling the bacterial leaf blight, and searching for related gene resources of the bacterial leaf blight resistance is one of the important directions of genetic breeding.

[0004] Previous laboratory studies found that a nuclease (CN116334036A) encoding a Holliday Junction structure DNA resolvase in Pseudomonas solanacearum can significantly improve the resistance of tomato to bacterial wilt after expressing the Holliday Junction resolvase in the susceptible tomato hairy roots. However, there is no report about exploring the potential of the Holliday Junction resolvase to improve the resistance of rice to the bacterial leaf blight. SUMMARY

[0005] The purpose of the present application is to provide application of a resolvase XooRuvC screened from a genome of Xanthomonas oryzae in improving resistance of rice to bacterial leaf blight, and the screened Holliday Junction resolvase XooRuvC can significantly improve the resistance of rice to the bacterial leaf blight.

[0006] The present application provides application of a resolvase XooRuvC screened from a genome of Xanthomonas oryzae in improving resistance of rice to bacterial leaf blight.

[0007] In specific embodiments, the amino acid sequence of the resolvase XooRuvC is shown as SEQ ID NO. 1.

[0008] The present application provides a method for improving the resistance of rice to bacterial leaf blight, comprising the following steps:

[0009] The resolvase XooRuvC screened from the genome of Xanthomonas oryzae is promoted to be overexpressed or superexpressed in target materials.

[0010] In specific embodiments, the target material comprises wild-type rice Nipponbare.

[0011] In specific embodiments, the promotion further comprises constructing an overexpression vector containing the resolvase XooRuvC.

[0012] In specific embodiments, the Xanthomonas oryzae comprises model strain PXO99A.

[0013] In specific embodiments, the basic backbone vector of the overexpression vector comprises pRHVcHA.

[0014] In specific embodiments, the construction method comprises the following steps: inserting the resolvase XooRuvC and the signal peptide OsPR1b fusion fragment into the basic backbone vector pRHVcHA.

[0015] The present application provides the application of the resolvase XooRuvC screened from the genome of Xanthomonas oryzae in constructing materials resistant to bacterial leaf blight.

[0016] The present application provides a method for constructing materials resistant to bacterial leaf blight, comprising the following steps:

[0017] The resolvase XooRuvC screened from the genome of Xanthomonas oryzae, the expression cassette or the expression vector is transformed into target species to construct the materials resistant to bacterial leaf blight.

[0018] Beneficial effects: The present application provides the application of the resolvase XooRuvC screened from the genome of Xanthomonas oryzae in improving the resistance of rice to bacterial leaf blight. In the embodiments of the present application, the resolvase XooRuvC is superexpressed in rice, which can significantly improve the resistance of rice to bacterial leaf blight without affecting the normal growth and breeding of crops, and is conducive to the cultivation of resistant varieties.

[0019] The application provides a method for improving the resistance of rice to bacterial leaf blight, comprising the following steps: promoting overexpression or superexpression of resolvase XooRuvC screened from a genome of Xanthomonas oryzae in target materials.

[0020] The application provides application of resolvase XooRuvC screened from a genome of Xanthomonas oryzae to construction of materials resistant to bacterial leaf blight, and the resolvase XooRuvC is overexpressed in rice Nipponbare; after inoculation of the transformed rice with PXO99A, the disease symptom is obviously reduced, which indicates that the resolvase XooRuvC has an application prospect in disease resistance breeding by targeting the biofilm of Xanthomonas oryzae pathovar. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 XooRuvC and XooRuvX are amino acid sequence alignment results;

[0022] Figure 2 It is a prokaryotic expression pET15-D vector plasmid map;

[0023] Figure 3 XooRuvC and XooRuvX are in vitro treatment results of PXO99A biofilm, wherein A is an SDS-PAGE electrophoretogram of the purified protein; B is a crystal violet staining quantitative result of PXO99A biofilm treated by different concentrations of XooRuvX, the staining solution is imaged in a PCR tube, N=6, one-way ANOWA statistical analysis is used, and statistical differences are indicated by different lowercase letters in the figure; C is a crystal violet staining quantitative result of PXO99A biofilm treated by different concentrations of XooRuvC, the staining solution is imaged in a PCR tube, N=6, one-way ANOWA statistical analysis is used, and statistical differences are indicated by different lowercase letters in the figure; D is a confocal staining observation field diagram of PXO99A biofilm treated by XooRuvC, and the scale is 50 microns;

[0024] Figure 4 It is a plasmid map of the rice stable overexpression vector pRHVcHA;

[0025] Figure 5Figure for detection result of bacterial blight resistance of XooRuvC transgenic rice overexpressing, wherein, A: verification of XooRuvC expression in rice, RT-PCR was used to detect XooRuvC gene expression level, ACTIN1 was used as internal reference, and Image J was used to quantify relative intensity of bands, wild type Nipponbare was used as control; B: figure of 14-day lesion observation after T1 generation OxXooRuvC PR1 was inoculated with PXO99A in test field, scale bar was 10 mm; C: figure of 14-day lesion length measurement after T1 generation OxXooRuvC PR1 PXO99A inoculation, N = 20, error line indicates standard deviation;

[0026] Figure 6 Figure for part development trait of XooRuvC transgenic rice overexpressing, wherein, A: growth trait of rice plant entering heading stage under greenhouse condition; B: OxXooRuvC PR1 rice seed trait, scale bar was 10 mm; C: 100-grain weight of dried rice seed, N = 10, statistical analysis was performed by using one-way ANOWA, and statistical difference was indicated by different lowercase letters in the figure. DETAILED DESCRIPTION

[0027] The application provides application of a resolvase XooRuvC screened from a genome of Xanthomonas oryzae in improving bacterial blight resistance of rice.

[0028] In specific embodiments, the amino acid sequence of the resolvase XooRuvC is shown as SEQ NO. 1.

[0029] MTRILGIDPGSQRTGIGIIDIDEGGRSRHVHHAPLILLGEGDFSQRLKRLLHGLGELIETYRPDEVAIEKVFMGKSAASALKLGQARGAAICAVVMRDLPVHEYAATEVKLALVGKGGADKVQVQHMVGIMLNLKGKLQPDAADALAVAITHAHVRATAQCLGVNTQQAWSRKK.

[0030] In specific embodiments, the present application screens the related genes from the genome of Xanthomonas oryzae pv. oryzae PXO99A which are highly conserved with known Holliday Junction resolvase sequences, and obtains XooRuvC. In the embodiments of the present application, the information of Xanthomonas oryzae pv. oryzae pathogenic genes used is from the KEGG (https: / / www.genome.jp / kegg / ) genome database; the amino acid sequences used for sequence alignment are from the UniPort (https: / / www.uniprot.org / ) or NCBI (https: / / www.ncbi.nlm.nih.gov / ) database. In specific embodiments, the present application first uses GeneDoc to perform alignment analysis on the Holliday Junction resolvase amino acid sequences of different pathogenic bacteria, mainly including Pseudomonas aeruginosa RuvC (PaRuvC), the sequence number of which in NCBI is PaRuvC (NP_249656.1); Pseudomonas syringae pv. Tomato RuvC (PstRuvC), the sequence number of which in NCBI is PstRuvC (WP_011104812.1); Escherichia coli RuvC (EcRuvC), the sequence number of which in NCBI is EcRuvC (NP_416377.1); Ralstonia solanacearum RuvC (RsRuvC), the sequence number of which in NCBI is RsRuvC (WP_013206954.1); Xanthomonas oryzae pv. oryzae RuvC (XooRuvC), the sequence number of which in NCBI is XooRuvC (WP_014504180.1); Bacillus subtilis RuvC (BsRuvC), the sequence number of which in NCBI is BsRuvC (WP_146876833.1); and Xanthomonas oryzae pv. oryzae RuvX (XooRuvX), the sequence number of which in NCBI is XooRuvX (WP_014502544.1).

[0031] The present application provides a method for improving the resistance of rice to bacterial leaf blight, comprising the following steps:

[0032] Promote the overexpression or superexpression of the resolvase XooRuvC screened from Xanthomonas oryzae in the target material.

[0033] In specific embodiments, the application first constructs an overexpression vector containing the resolvase XooRuvC, and then promotes overexpression or superexpression of the resolvase XooRuvC in target materials. The application performs prokaryotic expression of the candidate gene, and after in vitro protein purification, the biofilm of the model strain PXO99A is treated, and it is found that the biofilm can be significantly inhibited after the addition of the resolvase XooRuvC in vitro. The resolvase XooRuvC is expressed in rice, and after inoculation of the model strain PXO99A, the occurrence of lesions is significantly reduced. The gene is the Holliday Junction resolvase XooRuvC against bacterial leaf blight.

[0034] In specific embodiments, the prokaryotic expression vector of Escherichia coli used in the application is pET15-D (see Figure 2 ), which carries a 6xHis tag at the N' terminal. In the embodiments of the application, the pET15-D vector is first linearized by double digestion with NdeI / XhoI, and the full-length sequence of the candidate gene is amplified using the genomic DNA of Xanthomonas oryzae pv. oryzae PXO99A as a template, and the full-length sequence of the candidate gene is connected to the linearized pET15-D to form the prokaryotic expression vector pET15-XooRuvC.

[0035] In specific embodiments, the pET15-XooRuvC is transformed into DE3 competent cells by plasmid heat shock transformation, and after incubation at 37℃, single colonies are picked for induction expression. After incubation at 37℃ at 190rpm to OD 600 = 0.5, 0.25mM IPTG is added for induction at 16℃ for at least 12h, and after induction expression, protein extraction is performed. The purity of the extracted protein product is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis.

[0036] In specific embodiments, after determining the purity of the resolvase XooRuvC protein, the in vitro purified protein is mixed with the biofilm of the model strain PXO99A to verify whether the Holliday Junction resolvase XooRuvC has inhibitory activity on the biofilm (see Figure 3 ). If the protein has an inhibitory effect on the biofilm in vitro, it can be used to continuously express in host plants such as rice, and it is verified whether the plant after expression has the ability to resist vascular diseases such as bacterial leaf blight.

[0037] The application also provides the use of the resolvase XooRuvC screened from the genome of Xanthomonas oryzae for constructing materials against bacterial leaf blight.

[0038] The present invention provides a method for constructing a bacterial blight-resistant material, comprising the following steps: transforming a resolvase XooRuvC, obtained from the genome of Xanthomonas oryzae, and an expression cassette or expression vector into a target species to construct the bacterial blight-resistant material. In an embodiment of the present invention, genetic transformation is used to construct the bacterial blight-resistant material. The present invention does not particularly limit the specific method of genetic transformation; genetic transformation can be performed using conventional methods in the art to obtain a transgenic line that stably expresses the gene.

[0039] In the embodiment of the present invention, the basic backbone vector of the overexpression vector used is the pRHVcHA plasmid (see Figure 4 ), for cloning the target gene, the vector pRHVcHA was first linearized by double enzyme digestion with BamHI / KpnI. Then, the sequence of the rice signal peptide OsPR1 was predicted using SignaIP 6.0 (SignalP-6.0-Services-DTU HealthTech). The gene sequence of the signal peptide OsPR1 and the full-length gene sequence of the resolvase XooRuvC were ligated to the linearized pRHVcHA to construct the overexpression vector pRHVcHA-35s:OsPR1-XooRuvC-HA. The correctly constructed plasmid was then transformed into Agrobacterium tumefaciens EHA105 by electroporation for use.

[0040] In a specific embodiment, the bacterial blight-resistant material described herein is derived from the wild-type rice Nipponbare, subspecies japonica of the genus Oryza in the Poaceae family. In this embodiment, the gene for the resolvase XooRuvC was transformed into Nipponbare. The resulting material possesses a certain degree of resistance to bacterial blight without affecting the normal growth of the crop itself, potentially enabling application in disease-resistant breeding for a wider range of crops.

[0041] The present invention also provides a method for constructing a species resistant to bacterial blight, comprising the following steps: transforming the resolvase XooRuvC, expression cassette or expression vector screened from the genome of Xanthomonas oryzae into the target species to construct the bacterial blight-resistant material.

[0042] The present invention does not particularly limit the conversion method, and conventional methods in the art can be used.

[0043] For example, in the embodiment of the present invention, a His (histidine) tag affinity protein purification method is used to extract and purify the protein. Specifically, the His (histidine) tag protein extraction and purification method preferably includes the following steps:

[0044] (1) Induction of expression: pET15-XooRuvC and pET15-XooRuvX were transformed into DE3 competent cells by heat shock transformation, and after 37°C culture, single colonies were picked for induction of expression, 37°C, 190 rpm, until OD 600 = 0.5, 16°C induction for at least 12h after adding IPTG with a final concentration of 0.25mM, 4°C, 4000rpm, centrifugation for 15min, discard the supernatant, and collect the bacterial cells.

[0045] (2) Protein crude extraction: (the following process is carried out on ice)

[0046] 1) Add 30mL His extraction buffer to the precipitate, resuspend the precipitate with a vortex, and then add 1mM PMSF;

[0047] 2) Transfer to a 50mL round-bottom tube (for ultrasonic and centrifugation), and the cell resuspension is a brownish yellowish white viscous substance;

[0048] 3) Add lysozyme with a final concentration of 100μg / mL and react on ice for 15min;

[0049] 4) Place the resuspension on ice, and use an ultrasonic instrument to break the cells (Xinzhi JY99-IIDN, 6mm amplitude rod, output power: 300W), break for 30s, and then break again after 5min, and use ultrasonic treatment until the solution is clear and no longer viscous, 4°C, 12000rpm, centrifugation for 15min, and transfer the supernatant to a new 15mL centrifuge tube;

[0050] (3) Protein adsorption: after transferring the supernatant, add Triton-100 with a final concentration of 1%, mix, and then add 200μL His-tag Ni-NTA agarose affinity resin that has been previously washed with His extraction buffer, and rotate incubate at 4°C for more than 3h;

[0051] (4) Protein purification: after adsorption, centrifuge at 1000rpm for 1min using a horizontal centrifuge, and use a vacuum pump to dry the supernatant, leaving only the Ni-NTA affinity resin at the bottom of the tube, wash the precipitate with His washing buffer containing 20mM imidazole three times, and use a vacuum pump to slowly absorb the supernatant, transfer the Ni-NTA affinity resin to a new 1.5mL centrifuge tube, add His elution buffer containing 250-500mM imidazole after washing, and rotate incubate for more than 15min, and the supernatant collected by horizontal centrifugation is the protein;

[0052] (5) Protein concentration and purity determination: Measure protein concentration (unit: mg / mL) using a microspectrophotometer; perform sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) analysis; and store the protein product at low temperature by adding 80% glycerol at a volume ratio of 1:1.

[0053] For example, in the embodiment of the present invention, Agrobacterium-mediated rice genetic transformation is used, and the specific rice genetic transformation method preferably includes the following steps:

[0054] (1) Induction: Select rice seeds without mold spots and with normal buds, disinfect them with 75% alcohol for 1 min, rinse with sterile water for 1 min each time, disinfect them with 15% sodium hypochlorite for 20 min, and rinse with sterile water three times for 1 min each time. The disinfected seeds are inoculated into induction medium and cultured under light at 26°C for 20 days.

[0055] (2) Agrobacterium infection: Pick Agrobacterium into the infection solution and prepare OD 600 = 0.2 Agrobacterium resuspension, pick callus into a triangular flask, add Agrobacterium resuspension, infect for 10-15 minutes, discard the bacterial solution, inoculate callus into co-cultivation medium, and co-cultivate at 20℃ for 48-2 hours;

[0056] (3) Callus screening: The co-cultured callus tissue was inoculated into the screening medium and cultured in the dark at 26°C for 20-30 days; the positive callus was inoculated into the secondary screening medium. During the callus selection process, single clone callus must be selected and cultured in the dark at 26°C for 7-10 days;

[0057] (4) Differentiation and rooting: The positive callus was inoculated into differentiation medium and cultured at 25-27°C for 15-20 days. After the buds of 2-5 cm were differentiated, they were inoculated into rooting medium and cultured at 30°C for 7-10 days.

[0058] (5) Positive seedling detection: Extract total RNA from rice, amplify the target gene fragment after reverse transcription, and perform PCR detection.

[0059] To further illustrate the present invention, the application of the resolvase XooRuvC screened from Xanthomonas oryzae provided by the present invention in improving the resistance of rice to bacterial blight is described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0060] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0061] 1. Plant materials

[0062] Rice (Oryza sativa), mainly using the japonica variety Nipponbare (wild rice);

[0063] The rice genetic transformation culture platform is provided by Wuhan Boyuan Biotechnology Co., Ltd.

[0064] 2. Vector and strain

[0065] The stable overexpression vector used for constructing the rice transgenic plant is based on the backbone vector pRHVcHA, and the E. coli expression vector pET15-D is used for protein purification.

[0066] DE3 (Escherichia coli) for prokaryotic expression, MC1061 (Escherichia coli) for vector construction purchased from Wuhan Bio Basic & Greenray Biotech Co., Ltd., EHA105 for rice gene expression from Wuhan Boyuan Biotech Co., Ltd., model strain PXO99A (Xanthomonas oryzae pv. oryzae, Xoo) for biofilm culture and pathogenicity inoculation from the laboratories of Prof. Li Guotian and Prof. Yuan Meng, respectively, of Hubei Hongshan Laboratory. (See: [1]. Xu Z, Xu X, Li Y, Liu L, Wang Q, Wang Y, Wang Y, Yan J, Cheng G, Zou L, Zhu B, Chen G. Tal6b / AvrXa27A, a hidden TALE targeting the susceptibility gene OsSWEET11a and the resistance gene Xa27 in rice. Plant Commun. 2024 Feb 12;5(2):100721. doi: 10.1016 / j.xplc.2023.100721. Epub 2023 Sep 20. PMID: 37735868; PMCID: PMC10873877. [2]. Sha G, Sun P, Kong X, Han X, Sun Q, Fouillen L, Zhao J, Li Y, Yang L, Wang Y, Gong Q, Zhou Y, Zhou W, Jain R, Gao J, Huang R, Chen X, Zheng L, Zhang W, Qin Z, Zhou Q, Zeng Q, Xie K, Xu J, Chiu TY, Guo L, Mortimer JC, Boutté Y, Li Q, Kang Z, Ronald PC, Li G. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance. Nature. 2023 Jun;618(7967):1017-1023. doi: 10.1038 / s41586-023-06205-2. Epub 2023 Jun 14. PMID: 37316672.)

[0067] 3. Main reagents:

[0068] Gold medal Mix TSE101 was purchased from GenScript Biotech Corporation; restriction endonuclease and T4 ligase were purchased from Takara Bio Inc.; columnar DNA recovery kit was purchased from Shanghai Biotech Engineering Corporation; isopropyl alcohol, anhydrous alcohol, chloroform and other inorganic salts and organic solvents were purchased from China National Pharmaceutical Group Corporation; RNA extraction related reagents and reverse transcription kit were purchased from Novozyme Biological Technology Co., Ltd.; protein peptone, beef infusion powder and sodium chloride in the culture medium were purchased from OXOID Company; live / dead bacterial staining kit was purchased from Yikesheng Biological Technology Co., Ltd.

[0069] 4. Main culture medium:

[0070] NB (Nutrient Broth) culture medium: protein peptone 10 g / L, beef infusion powder 3 g / L, sodium chloride 5 g / L, dissolved in 1 L ddH2O, sterilized at 121℃ for 20 min. Solid culture medium needs to add 15 g agar powder.

[0071] 5. Main instruments:

[0072] PCR instrument (Mastercycler nexus, Germany Eppendorf), centrifuge (5810R, Germany Eppendorf), shaking incubator (MQL-61R, Mingquan), plant growth chamber (HP1500GS-B, Ruishua), electrophoresis instrument (EPS600, Shanghai Tianni), balance (JY5002, Shanghai Shunyu Hengping), super-clean workbench (DL-CJ-2NDI, Beijing Donglian Haer), water bath (XMTD6000, Beijing Changfeng), gel imaging instrument (ChemiDoc XRS+, USA BIO-RAD), single-lens reflex camera (B700, Japan Nikon), ultramicro spectrophotometer (DS-11, Beijing Bei Hui), enzyme label instrument (SPARK, Switzerland TECAN), ultraviolet-visible spectrophotometer (UV1300, Shanghai Meizan Instrument), laser confocal microscope (Leica, SP8).

[0073] Example 1 Holliday Junction resolvase amino acid sequence alignment

[0074] First, GeneDoc was used to analyze and compare the Holliday Junction resolvase amino acid sequences of different pathogenic bacteria, and the results were as follows: Figure 1From top to bottom, they represent Pseudomonas aeruginosa (PaRuvC), Pseudomonas syringae pv. Tomato (PstRuvC), Escherichia coli (EcRuvC), Ralstonia solanacearum (RsRuvC), Xanthomonas oryzae (XooRuvC), Bacillus subtilis (BsRuvC), Xanthomonas oryzae (XooRuvX). Figure 1 The different degrees of gray background correspond to the similar amino acid sequences between different pathogens, and in Figure 1 In the example of the pathogen, the higher the repetition of amino acids at this position between pathogen sequences, the darker the gray; Figure 1 As can be seen in the example of the pathogen, there are many similar or repeated amino acids between XooRuvC and other pathogen examples, indicating that the sequence is highly conserved. In sharp contrast to the sequence of Bacillus subtilis BsRuvC, the only Gram-positive bacterium among the pathogen examples (other examples are all Gram-negative bacteria), the gray marked amino acids of BsRuvC are much less, indicating that many of its amino acid codes are different from the RuvC of Gram-negative bacteria, and it is considered to be low in conservation. Through amino acid sequence alignment, it is found that XooRuvC has high sequence conservation compared with other pathogens, and has multiple conserved Holliday Junction resolvase enzyme active sites predicted by UniPort, and XooRuvX has relatively low sequence conservation.

[0075] Example 2 Effect of Holliday Junction resolvase on biofilm in vitro

[0076] The full-length genes of XooRuvX and XooRuvC were respectively connected to the pET15-D vector, and after enzyme digestion and sequencing verification, the plasmid was transformed into Escherichia coli DE3, and then induced and expressed. After 37°C culture to OD 600 = 0.5, 0.25 mM IPTG was added, and 16°C low-temperature induction was performed. After induction, the bacterial solution was clarified by ultrasonic disruption, and the supernatant was obtained by centrifugation. The supernatant was adsorbed by Ni-NTA agarose affinity resin at low temperature, and then eluted with His elution buffer containing 400 mM imidazole. The extracted quality of the protein was detected by SDS-PAGE electrophoresis after protein elution, and the results are shown in FIG. 1A. Figure 3 ​

[0077] Wild type PXO99A strain was activated on NB plate, and a single colony was picked and cultured in 2 mL NB liquid medium at 28°C, 190 rpm for 14-16 h. The cultured bacteria were collected by centrifugation, resuspended in fresh NB medium, and adjusted to OD600=0.01. The bacteria were treated with different concentrations of XooRuvC and XooRuvX in a 96-well cell culture plate, respectively. The concentration gradient (relative to the medium) was set as 0, 50 ng / μL, 100 ng / μL, 150 ng / μL, and 200 ng / μL, respectively. The plate was sealed with a gas-permeable membrane and incubated at 28°C for 2 d. 600

[0078] After incubation, the culture solution was aspirated along the wall of the hole, and the hole plate was washed twice with 100 μL of sterile water and dried. Each hole was fixed with 100 μL of methanol for 10 min, and then the excess methanol was aspirated and air-dried at room temperature. The hole plate was dyed with 200 μL of 0.1% (W / V) crystal violet at room temperature for 15-20 min, and then the excess crystal violet was aspirated and the hole plate was washed with 200 μL of sterile water. The attached crystal violet was dissolved in 200 μL of 95% ethanol at 37°C for 30 min, and the absorbance at 590 nm was measured using an enzyme-labeled instrument. The measurement results are shown in Tables 1, 2, and 3. Figure 3 The observation and quantitative statistics of crystal violet staining showed that the co-culture of XooRuvX and PXO99A did not affect the production and accumulation of biofilm, while XooRuvC could significantly inhibit the biofilm of PXO99A, indicating that XooRuvC had an in-vitro biofilm inhibition activity, while XooRuvX did not have the inhibition function.

[0079] Table 1 Quantitative results of crystal violet staining of PXO99A biofilm treated with different concentrations of XooRuvX

[0080]

[0081]

[0082] Table 2 Quantitative results of crystal violet staining of PXO99A biofilm treated with different concentrations of XooRuvC

[0083]

[0084] ​Since previous studies have found that Holliday Junction resolvase can mediate the dissociation of biofilm structure by targeting biofilm eDNA, PXO99A biofilms were cultured statically on cell culture slides and 100 ng / μL XooRuvC (the final concentration added to the culture system) was added. After static co-culture at 28°C for 2 days, the cells were stained with a live / dead bacteria staining kit diluted with 0.85% (w / v) NaCl solution at room temperature in the dark for 15 minutes. After staining, the excess dye was washed off, and the cells were covered with a coverslip and observed under a confocal microscope. The green fluorescent channel is DMAO staining, representing the total amount of DNA in living cells and biofilms. The red fluorescent channel is EthD-III staining, which does not have cell membrane permeability and represents extracellular eDNA in the biofilm. The imaging results are shown in Figure 2. Figure 3 As shown in Figure D, the untreated imaging sample served as the control group (Mock), that is, the biofilm naturally formed by the strain. The fluorescence intensity of the biofilm and eDNA in the sample treated with XooRuvC was significantly reduced compared with the untreated control group, indicating that XooRuvC may have inhibited the biofilm by targeting the eDNA of PXO99A.

[0085] Example 3OxXooRuvC PR1 Resistance detection to PXO99A

[0086] Replace T1 with OxXooRuvC PR1 (Transgenic rice was provided by Wuhan Boyuan Biotechnology Co., Ltd.) Rice leaves were quick-frozen in liquid nitrogen and then total plant RNA was obtained. The extracted RNA was reverse transcribed, and the cDNA concentration was uniformly controlled at around 200 ng / μL. Primers for the full-length XooRuvC and the rice internal reference gene OsACTIN1 were designed.

[0087] PCR identification reaction system: 1 μL rice cDNA, 8 μL gold medal mix, 0.5 μL each primer (primer sequence information is shown in Table 3, i.e., SEQ NO. 2 to SEQ NO. 5); PCR identification reaction procedure: 98 ° C pre-denaturation for 3 min; 98 ° C denaturation for 30 s; 55 ° C annealing for 30 s; 72 ° C extension for 45 s; a total of 35 cycles; 72 ° C final extension for 10 min; 12 ° C storage for 10 min; PCR products were subjected to agarose gel electrophoresis, and the electrophoresis results were as follows: Figure 5 As shown in Figure A. L refers to different transgenic individual lines. Since the offspring seeds are harvested individually during the transgenic rice propagation process, the different lines are numbered. The values ​​of 0.1.2 to 4.2 represent the relative intensity of the target gene XooRuvC band identified in the DNA of different lines. A larger value indicates a brighter band, indicating a higher copy number of the gene in the rice.

[0088] Table 3 Primer sequence information

[0089] Primer Primer name Primer sequence SEQ NO. 2 XooRuvC-FLFP-SpeI 5'-CGGACTAGTATGACCCGCATCCTGGGC-3' SEQ NO. 3 XooRuvC-FLRP-SmaI 5'-TCCCCCGGGTTTCTTGCGGCTCCAGGC-3' SEQ NO. 4 OsACTINl-FP: 5'-CCCTCAAGTACCCCATCGAG-3' SEQ NO. 5 OsACTINl-RP: 5'-TCCTAATATCCACGTCGCACT-3'

[0090] Wild type (WT) strain PXO99A was activated in NB medium, cultured at 28°C for 14-16h, and then centrifuged to collect the bacterial cells. After resuspension, the OD of the bacterial cells was adjusted to 1.0, and the bacterial cells were diluted to 0.5. The bacterial liquid was used to cut off the leaf tips of about 2-3 cm in length with scissors. One piece of leaf was cut off every time the bacterial liquid was dipped. The leaves were left in the natural environment to cause disease, and the length of the disease spots was measured 14 days after inoculation. The disease conditions were as shown in Table 1 and Table 2. 600 Diluted to 0.5, using scissors dipped in bacterial liquid, parallel cut off 2-3 cm or so leaf tip, every cut a piece of leaf dipped in bacterial liquid, natural environment to make it sick, 14 days after inoculation measure the length of the disease spot, the disease conditions as shown in Figure 5 Table 4 and Table 5. Figure 5 Table 4 and Table 5.

[0091] Table 4 T1 OxXooRuvC PR1 The length of the disease spot was measured 14 days after inoculation with PXO99A

[0092]

[0093]

[0094] The results show that OxXooRuvC PR1 The length of the disease spot was measured 14 days after inoculation with PXO99A

[0095] Example 4 OxXooRuvC PR1 Growth and development related trait detection

[0096] Greenhouse planting of wild type and T1 OxXooRuvC PR1 L2, L3, L2 and L3 are the seeds planted by the offspring of two independent strains of T0 transgenic rice, and the development traits of rice in the whole growth period are observed, Figure 6 Table 4 and Table 5. PR1 L2 and L3 can grow and develop normally.

[0097] Greenhouse planting of wild type and T1 OxXooRuvC PR1 L2, L3, the seed phenotype is observed after the seeds are collected, and the seed traits are as shown in Figure 6 Table 4 and Table 5. PR1 The size and traits of the rice seeds are not significantly different from the wild type; at the same time, the hundred-grain weight of the dried seeds is weighed, and the results show that OxXooRuvC PR1 The hundred-grain weight is slightly heavier than the wild type, as shown in Table 5 and Table 6. Figure 6 Table 4 and Table 5.

[0098] Table 5. 100-seed weight of rice seeds after drying

[0099]

[0100]

[0101] It can be seen that the lyase XooRuvC screened from Xanthomonas oryzae can significantly improve the resistance of rice to bacterial leaf blight.

[0102] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of the resolvase XooRuvC screened from the genome of Xanthomonas oryzae in improving the resistance of rice to bacterial blight, characterized in that: The resolvase XooRuvC is overexpressed in rice, and the amino acid sequence of the resolvase XooRuvC is shown in SEQ NO.

1.

2. A method for improving rice resistance to bacterial blight, characterized in that: The following steps are involved: The overexpression of the resolvase XooRuvC screened from the Xanthomonas oryzae genome in rice is promoted; the amino acid sequence of the resolvase XooRuvC is shown in SEQ NO.

1.

3. The method according to claim 2, characterized in that The method further comprises: constructing an overexpression vector containing the resolvase XooRuvC.

4. The method according to claim 3, characterized in that The Xanthomonas oryzae includes: a model strain PXO99A.

5. The method according to claim 3, characterized in that The basic backbone vector of the overexpression vector includes pRHVcHA.

6. The method according to claim 3, characterized in that The method for constructing an overexpression vector containing the resolvase XooRuvC comprises the following steps: inserting a fusion fragment of the resolvase XooRuvC and the signal peptide OsPR1b into a basic skeleton vector pRHVcHA.

7. Screening the bacterial blight-resistant resolvase XooRuvC from the Xanthomonas oryzae genome and its application in constructing bacterial blight-resistant rice, characterized in that: The amino acid sequence of the resolvase XooRuvC is shown in SEQ NO.

1.

8. A method for constructing rice resistant to bacterial blight, characterized in that: The following steps are involved: The expression cassette or expression vector containing the resolvase XooRuvC is transformed into rice to construct the rice resistant to bacterial blight; the amino acid sequence of the resolvase XooRuvC is shown in SEQ NO.1.

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