CN112048514B

CN112048514B - Phage trp574 gene and application thereof

Info

Publication number: CN112048514B
Application number: CN202010769368.XA
Authority: CN
Inventors: 刘琼光; 胡蓉花; 余成鹏; 钟敏
Original assignee: Ji'an Co Of Jiangxi Tobacco Co; South China Agricultural University
Current assignee: Ji'an Co Of Jiangxi Tobacco Co; South China Agricultural University
Priority date: 2020-08-03
Filing date: 2020-08-03
Publication date: 2022-08-23
Anticipated expiration: 2040-08-03
Also published as: CN112048514A

Abstract

The invention discloses a phage trp574 gene and application thereof, wherein the nucleotide sequence of the phage trp574 gene is shown in SEQ ID NO. 1, and the coded amino acid sequence thereof is shown in SEQ ID NO. 2. The invention discloses a transcription regulation factor trp574 gene belonging to an XRE family for the first time, the research of the invention shows that ralstonia solanacearum strain transformed with the trp574 gene almost loses pathogenicity, the trp574 gene has the comprehensive effects of obviously interfering the growth, metabolism, pathogenicity and the like of ralstonia solanacearum, and the trp574 gene can be used for preparing a ralstonia solanacearum strain without pathogenicity and has a larger application prospect in controlling the ralstonia solanacearum.

Description

Phage trp574 gene and application thereof

Technical Field

The invention relates to the technical field of ralstonia solanacearum prevention and treatment, and particularly relates to a phage trp574 gene and application thereof.

Background

Laurella solanacearum (Ralstonia solanacearum ) is a type of soil-borne gram-negative bacteria of the beta-Proteobacteria subphyla. The germs usually invade into the cortical intercellular spaces from the wound of the root or stem of the plant or from the root cap part of the non-injured secondary root, and by destroying the intercellular mesoglea layer, the cell walls are separated and deformed to form cavities, and then invade the xylem parenchyma tissue to stimulate the small cells near the duct to form an invaded body and move into the invaded body. After the infill is broken, the ralstonia solanacearum is continuously released into the conduit, and is propagated and rapidly diffused in the conduit in a large quantity, so that the plant is wilted and died. This devastating soil-borne disease is called crop bacterial wilt, and the economic losses caused by this disease are hundreds of millions each year around the world. The ralstonia solanacearum has wide hosts and can infect more than 50 crops of more than 200 families including ginger, tobacco, tomato, potato, peanut, mulberry and casuarina. The disease mainly occurs in subtropical and tropical regions and gradually spreads to temperate regions, the disease condition is aggravated year by year, and the host range is continuously enlarged. At present, the control method for the bacterial wilt of crops mainly comprises chemical control, biological control, planting of disease-resistant varieties, crop rotation and the like, and although the measures play a role in controlling the bacterial wilt to a certain degree, the disease cannot be effectively controlled.

The bacteriophage is a virus which can kill specific bacteria after being combined with specific sites on the surfaces of bacterial cells, has strong bactericidal specificity, is harmless to human and livestock,The pathogenic bacteria are used as the host to continuously proliferate by self, and the like, so the biological pesticide has the potential of developing the effective component of the novel biological pesticide. The utilization of the phage for preventing and treating the crop bacterial wilt has important practical significance. Filamentous bacteriophage

And

infection with ralstonia solanacearum can reduce the virulence of ralstonia solanacearum, and the physiology and biochemistry of ralstonia solanacearum are also changed (Addy et al, 2012), while filamentous phage are used

The infected ralstonia solanacearum is inoculated on the root of the tomato, then wild ralstonia solanacearum is inoculated, and the fluorescence detection method shows that the toxicity of the wild ralstonia solanacearum is inhibited, and the overall tomato incidence rate is reduced (Addy et al, 2012). Fujiwara et al (2011) use bacterial wilt bacteriophages to biologically control bacterial wilt, and prove that the bacteriophages have certain effects on bacterial wilt control. Because the bacteriophage does not have a complete cell structure and only contains a single nucleic acid, the bacteriophage can be regarded as an organism which preys on bacteria; although the phage genome contains many genes, all known phages utilize the ribosome of bacterial cells, various factors required for protein synthesis, various amino acids and energy generation systems to achieve their own growth and proliferation, and once they leave the host cell, the phages are neither able to grow nor replicate. Therefore, when the bacteriophage is used for controlling the bacterial diseases, the bacteriophage needs to be applied together with the host bacteria, and other risks are easily brought to the plants needing to be controlled due to the pathogenicity of the host bacteria. Therefore, it can be tried to avoid the risk to crops caused by the pathogenicity of host bacteria by culturing phage using a nonpathogenic rally bacterial wilt strain as a host of the phage and then controlling bacterial wilt of plants.

On the other hand, the use of a. nondisease bacterial strain for controlling crop bacterial wilt is one of the major research points for biological control of plant diseases at present and has been successfully applied (Dongchun, Zengxian, Liu Jong. Currently, ralstonia solanacearum strains without pathogenicity are mainly obtained by primary research on the disease control effect of ralstonia solanacearum strains on tobacco bacterial wilt through separation and screening from plant tissues (Xiaotian, Xiaochonggang, Zhouyang, etc.. ralstonia solanacearum strains [ J ]. plant protection, 2008,34(002):79-82.), or are obtained through artificial selective culture so that pathogenic strains are converted into non-pathogenic strains, or are obtained through an ultraviolet light induction method (Chenqinghe, Weng inspire, Hufanping, the control effect of ralstonia solanacearum strains on tomato bacterial wilt [ J ]. Chinese biological control, 2004,20(001):42-44.), or are obtained through knocking out virulence factors related to pathogenicity in ralstonia solanacearum strains, and no reports related to the ralstonia solanacearum strains by introducing genes into the ralearia solanacearum strains are found.

Disclosure of Invention

The present invention is directed to overcoming the above-mentioned deficiencies of the prior art and providing a phage trp574 gene.

The second object of the present invention is to provide a protein encoded by the phage trp574 gene.

The third objective of the invention is to provide a primer pair for amplifying and detecting the phage trp574 gene.

The fourth purpose of the invention is to provide a recombinant expression vector, a host bacterium or a cell line containing the phage trp574 gene.

The fifth purpose of the invention is to provide the application of the phage trp574 gene in constructing nonpathogenic ralstonia solanacearum strains.

The above purpose of the invention is realized by the following technical scheme:

a phage trp574 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

According to the invention, a brand-new transcription regulatory factor orf30 is discovered by performing genome sequencing on a lytic bacteriophage P574, then a primer is designed according to a bacteriophage genome sequence and PCR amplification is performed to obtain a target fragment of about 688bp, the target fragment is sequenced to discover that the orf30 sequence is 555bp, the nucleotide sequence is shown as SEQ ID NO. 1, and the coded amino acid sequence is shown as SEQ ID NO. 2; the BALST alignment search found that orf30 has 82% homology with Ralstonia phase GP4, and the protein search results showed that it has a conserved sequence of transcription regulatory factor (transcription regulator), and the amino acid sequence homology was not higher than 70%. Therefore, rf30 was named as trp574 gene, where T stands for Transcriptional regulator, R stands for Ralstonia (Ralstonia), and P574 stands for this gene from bacteriophage P574.

The amino acid sequence of the protein coded by the phage trp574 gene is shown in SEQ ID NO. 2.

The invention also provides a primer pair for amplifying and detecting the phage trp574 gene, which is characterized in that the sequence of the primer pair is shown as SEQ ID NO. 3-4.

The invention also provides a recombinant expression vector containing the phage trp574 gene.

Preferably, the expression vector is pBBR1MCS 4.

The invention also provides a host bacterium containing the recombinant expression vector.

Preferably, the host bacterium is escherichia coli or ralstonia solanacearum; the ralstonia solanacearum is ralstonia solanacearum Tb15 or Tb 1546.

The invention also provides a cell line containing the recombinant expression vector.

The research of the invention shows that the ralstonia solanacearum strain transformed with the trp574 bacteriophage gene almost loses pathogenicity. The growth speed of the transformed strain in LB is accelerated, the colony on the TTC plate is dry and flat, and is deep red, the yield of extracellular polysaccharide is reduced, and the activity of cellulase is reduced. The qRT-PCR result shows that the trp574 gene can regulate and control phc systems and a plurality of two-component systems, so that pathogenicity of the ralstonia solanacearum is influenced, and the trp574 gene has comprehensive effects of obviously interfering growth, metabolism, pathogenicity and the like of the ralstonia solanacearum. the trp574 gene can be used for preparing pathogenic-free ralstonia solanacearum strains, so that the bacterial wilt can be prevented and treated.

Therefore, the invention requests to protect the application of the phage trp574 gene in preparing a pathopoiesia ralstonia solanacearum strain.

Specifically, the application is to convert a recombinant expression vector containing a phage trp574 gene into wild type ralstonia solanacearum to obtain a non-pathogenic ralstonia solanacearum strain. The ralstonia solanacearum without pathogenicity can be used as a host of the phage, the phage is cultured in a large quantity, then the bacterial wilt of the plant is prevented and controlled, and the influence brought by the pathogenicity of host bacteria can be avoided. Meanwhile, the ralstonia solanacearum strain without pathogenicity has a certain biological control effect on bacterial wilt.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers and clones a brand-new bacteriophage trp574 gene, and the nucleotide sequence and the amino acid sequence of the coded protein are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2. The bacteriophage trp574 gene has the comprehensive effects of obviously interfering the growth, metabolism, pathogenicity and the like of the ralstonia solanacearum, and the ralstonia solanacearum strain transformed by the bacteriophage trp574 gene almost loses pathogenicity, so that the ralstonia solanacearum is used as a culture host bacterium of the bacteriophage or is used for controlling the ralstonia solanacearum; the invention obtains the ralstonia solanacearum without pathogenicity by transferring the bacteriophage exogenous gene trp574 into the ralstonia solanacearum strain for the first time, and has a larger application prospect in controlling the bacterial wilt.

Drawings

FIG. 1 shows the alignment of orf30 with the amino acid sequences of the kindred proteins; wherein YP _007236754.1, WP _066491583.1, WP _034473760 and WP _090835493 are NCBI protein sequence numbers corresponding to transcription regulatory or [ Burkholderia virus Bcmp ], XRE family translational regulator [ Burkholderia sp.BDU8], XRE family translational regulator [ Cabalbrenia fire ] and XRE family translational regulator [ Paraburkholderia hospita ], respectively.

FIG. 2 is the orf30 protein structure prediction based on SWISS-MODEL; in the figure, a1 and a2, b1 and b2, and c1 and c2 are the front and back sides of the same protein respectively.

FIG. 3 shows the results of orf30 gene cloning and enzyme digestion. M1 is DL2000Marker, M2 is DL5000 Marker; a is orf30 fragment clone; b is PCR verification of the recombinant plasmid, wherein 2 is the recombinant plasmid, and 3 is pBBR1MCS4 plasmid; c is orf30 recombinant plasmid and empty plasmid transformed into Tb1546, wherein 4 is orf30-Tb1546, 5 is pBBR-Tb1546, 6 is pBBR1MCS4 plasmid, and 7 is negative control; d is orf30 recombinant plasmid and empty plasmid transformed into Tb15, wherein 8 is negative control, 9 is orf30-Tb15, 10 is pBBR-Tb15, and 11 is pBBR1MCS4 plasmid; e is a restriction enzyme verification diagram of the recombinant plasmid, wherein 12 is a blank control, 13 is a pBBR-orf30 recombinant plasmid, and 14 is a pBBR1MCS4 plasmid.

FIG. 4 is the growth curves of Tb1546, Tb15 and its trans-orf 30 gene strains in different media; wherein A, B is LB liquid culture medium; C. d is NA liquid culture medium.

FIG. 5 shows the colony morphology differences of Tb1546, Tb15 and its trans-orf 30 gene strains. A. B, C, D, E, F are Tb1546, orf30-Tb1546, pBBR-Tb1546, Tb15, orf30-Tb15 and pBBR-Tb15, respectively.

FIG. 6 is EPS production assay of Tb1546, Tb15 and its trans-orf 30 gene strains. The LSD method performed multiple comparisons (p ═ 0.05).

FIG. 7 is the determination of the hydrolysis diameter of Tb1546, Tb15 and its trans-orf 30 gene strains on cellulase detection plates. CK uses NA culture medium as blank control; A. c is the diameter of the hydrolysis ring of the supernatant extracts of Tb1546, orf30-Tb1546 and pBBR-Tb1546 on the cellulase detection plate; B. d is the hydrolysis ring diameter of Tb15, orf30-Tb15 and pBBR-Tb15 supernatant extracts on the cellulase detection plate. The LSD method performed multiple comparisons (p ═ 0.05).

FIG. 8 is the competitive growth of Tb1546 with orf30-Tb1546 in LB.

FIG. 9 shows the index change of tobacco bacterial wilt disease after inoculation of Tb1546 and orf30-Tb1546 by needle punching and irrigation. Note: a and B are the morbidity after Tb1546 and orf30-Tb1546 are inoculated by a needle punching method, and E is the disease index statistics; c and D are the disease conditions after Tb1546 and orf30-Tb1546 are inoculated by irrigation, and F is the statistics of disease index.

FIG. 10 shows the relative expression levels of Tb1546 and orf30-Tb 1546.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

1. Test strains

The strains, plasmids and phages described in the examples of the invention are detailed in table 1. The ralstonia solanacearum and the phage are cultured at 30 ℃, and the Escherichia coli DH5 alpha, the Escherichia coli carrying the plasmid and the recombinant plasmid are cultured at 37 ℃. The working concentration of ampicillin was 100. mu.g/mL.

TABLE 1 test strains, plasmids and phages

2. Common culture medium and reagent

LB culture medium: 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast extract and 15g/L of agar powder.

NA medium: 3g/L of beef extract, 5g/L of peptone, 0.5g/L of yeast extract, 10g/L of glucose, 15g/L of agar powder and 7.0 of pH.

TTC medium: hydrolyzed casein 1g/L, peptone 10g/L, glycerol 5mL/L, agar powder 32g/L, before use every 100mL medium with 1% TTC 500. mu.L.

Cellulase detection plate: 1.0g/L of carboxymethyl ethyl cellulose, 3.8g/L of sodium phosphate, 8.0g/L of agarose and 7.0 of pH.

Ordinary bacterial solid medium: 3g/L of beef extract, 3g/L of yeast extract, 3g/L of peptone, 0.25g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate, 0.5g/L of potassium dihydrogen phosphate, 15g/L of sucrose and 18g/L of agar powder.

Common bacterial semi-solid medium: 3g/L of beef extract, 3g/L of yeast extract, 3g/L of peptone, 0.25g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate, 0.5g/L of potassium dihydrogen phosphate, 15g/L of sucrose and 8g/L of agar powder.

Example 1 bioinformatic analysis of trp574 Gene (orf30)

In the early stage of the invention, a transcription regulatory factor orf30 is found by performing genome sequencing on a lytic phage P574, and the protein sequence of the gene is compared by using Blastp of NCBI; the physicochemical properties of the proteins were analyzed using the ProtParam tool (https:// web. expasy. org/ProtParam /); protein domain analysis was performed using a SMART server (http:// SMART. embl-heidelberg. de /); protein modeling was performed using SWISS-MODEL (https:// www.swissmodel.expasy.org /).

The Blastp result of NCBI shows that the orf30 gene belongs to XRE family, the alignment result shows that the gene has 69% homology with the transcription regulatory factor of Burkholderia phage Bcepmigl belonging to XRE family, four protein sequences with the highest scores in the alignment result are downloaded, multi-sequence alignment is carried out by using ClustalX software, and the alignment result is shown in figure 1 after being subjected to coloring treatment by Boxshade (https:// embnet.visual-it.ch/software/BOX _ form.html); the physicochemical properties of the protein show that the protein has 184 amino acids, about 20.235KD, isoelectric point about 9.20, Instability Index (II) of 39.63, belongs to stable protein, fat index 81.20, total average hydrophilicity of-0.368, and belongs to hydrophilic protein. The SMART server analyzes the structural functional domain of the protein and finds that a Cro/CI type HTH structure exists at 1-51 positions of the protein, and the structure generally comprises 50-60 spiral-turn-spiral (HTH) structural domains combined with DNA. The protein was constructed in the SWISS-MODEL MODEL as shown in FIG. 2.

Example 2 cloning of trp574 Gene (orf30) and construction of recombinant plasmid

1. Method of producing a composite material

(1) Extracting phage DNA by using a general phage DNA extraction kit (cargo number: KG005-1) of Guangzhou Noocrystal biotechnology limited, and designing an amplification primer orf30-F/orf30-R (shown in Table 2) of a target gene orf30 according to P574 genome information; phage DNA is used as a template, orf30-F/orf30-R is used as a primer, and Novozan is utilized

Amplifying the target gene orf30 by the Master Mix, wherein the amplification system is

Master Mix 25. mu.L, Primer 11. mu.L, Primer 21. mu.L, template DNA 1. mu.L (<200ng), complement ddH ₂ O to 50. mu.L. The PCR amplification conditions were: 3min at 95 ℃; 35 cycles of 95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 40 s; 72 ℃ for 10min, 4 ℃ and infinity. And (3) after the PCR product is subjected to electrophoresis, cutting and recovering the target band in gel electrophoresis.

(2) plasmid extraction of pBBR1MCS4 plasmid extraction according to Axygen plasmid miniextract kit instruction, BamHI and Hind III double digestion, Novozam

II One Step Cloning Kit ligation and heat shock transformation to DH5 α. The plated plates were cultured in an inverted state at 37 ℃ for 24 hours until single colonies appeared, and colony PCR was performed using MCS-F/MCS-R primers (shown in Table 2) in the following PCR system: template 5. mu.L, 10 XBuffer 1. mu.L, dNTP 0.8. mu.L, MSC-F0.4. mu.L, MSC-R0.4. mu.L, Taq enzyme 0.08. mu.L, ddH ₂ O2.32. mu.L, 10. mu.L in total. The PCR procedure was: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 40 s; 72 ℃ for 5min, 16 ℃. Liquid culture is carried out on the bacterial strain containing the target band, plasmids are extracted and enzyme digestion verification is carried out by using BamHI and Hind III, and meanwhile, the recombinant plasmids are handed over to the Yinxie substrate for sequencing and identification. The obtained recombinant plasmid was named pBBR-orf 30.

TABLE 2 orf30-F/orf30-R and MCS-F/MCS-R primer sequences

2. As a result, the

(1) Designing a primer according to a phage genome sequence, carrying out PCR amplification to obtain a target fragment of about 688bp (figure 3A), and sequencing the target fragment to find that the orf30 sequence is 555bp, the nucleotide sequence of the orf30 sequence is shown as SEQ ID NO. 1, and the coded amino acid sequence of the orf30 sequence is shown as SEQ ID NO. 2; the BALST alignment search found that orf30 has 82% homology with Ralstonia phase GP4, and the protein search results showed that it has a conserved sequence of transcription regulatory factor (transcription regulator), and the amino acid sequence homology was not higher than 70%. Therefore, rf30 was named as trp574 gene, where T stands for Transcriptional regulator, R stands for Ralstonia (Ralstonia), and P574 stands for this gene from bacteriophage P574.

(2) The recombinant plasmid pBBR-orf30 obtained by a one-step cloning mode from the orf30 target fragment has a detection fragment size of 977bp (figure 3B), the recombinant plasmid enzyme digestion result shows two bands which are close to the predicted 646bp (figure 3E), and the sequencing result is consistent with the prediction result, so that the success of the recombinant plasmid construction is proved.

Example 3 preparation of transgenic trp574 gene (orf30) Ralstonia solanacearum Tb1546 and Tb15

1. Preparation and electrotransformation of ralstonia solanacearum competent cells

Preparation of ralstonia solanacearum Tb15 electric shock competence and electric shock transformation method refer to the method of Lavie et al (2002).

(1) Respectively inoculating ralstonia solanacearum Tb1546 and Tb15 in 10mL NA culture medium for overnight culture, placing the bacterial liquid on ice for 15min, subpackaging in precooled 1.5mL centrifuge tubes, centrifuging at 4 ℃ and 6000rpm for 5min, and discarding the supernatant.

(2) 1mL of 10% glycerol was added for resuspension, and the mixture was centrifuged at 6000rpm at 4 ℃ for 5min, and the supernatant was discarded.

(3) And (3) repeating the step (2) twice.

(4) Add 10% glycerol 100 u L heavy suspension obtained Ralstonia solanacearum competence.

(5) 50 mu L of prepared Tb1546 and Tb15 were taken respectively and competent for ralstonia solanacearum in 0.1cm electric shock cup, and about 2 mu g of recombinant plasmid pBBR-orf30 was added, and the voltage was set to 1.4kv in manual mode for electric shock transformation. Meanwhile, about 2 mu g of pBBR1MCS4 plasmid is taken to carry out empty vector electric shock transformation in ralstonia solanacearum competence. After transformation, 800. mu.L of NA liquid culture medium was added immediately for resuscitation for 12h, plates were coated on the NA culture medium containing Amp antibiotics, transformants orf30-Tb1546, pBBR-Tb1546, orf30-Tb15 and pBBR-Tb15 were selected by PCR, and PCR primers and conditions were constructed as in example 2.

Successfully transforming the recombinant plasmid into ralstonia solanacearum Tb1546 and Tb15 by electric shock to obtain transgenic strains orf30-Tb1546, orf30-Tb 15; the empty plasmid was simultaneously shock-transferred into Tb1546 and Tb15 to obtain pBBR-Tb1546 and pBBR-Tb15 (FIGS. 3-C, D).

2. Colony morphology observation and growth curve determination

Culturing Tb1546, orf30-Tb1546, pBBR-Tb1546, Tb15, orf30-Tb15 and pBBR-Tb15 in NA liquid culture medium to obtain seed liquid with bacteria concentration of 1 × 10 ⁸ cfu/mL, dilution plating to TTC plate, observation colony morphology. Another seed liquid is taken to adjust the concentration of the bacteria to be about 1 multiplied by 10 ⁸ cfu/mL, in a 1: 100 respectively inoculating LB liquid culture medium and NA liquid culture medium, and measuring OD once every 2h ₆₀₀ The experiment was repeated 3 times.

The results show that the growth rate of orf30-Tb1546 and orf30-Tb15 in LB liquid medium is significantly faster than that of wild bacteria, the growth rate is significantly increased at 12-14 h, and the growth is close to the late growth period after 24h (FIG. 4-A, B). In the NA liquid culture medium, the orf30-Tb1546 and orf30-Tb15 strains transferred with orf30 genes have slightly higher speed in the logarithmic growth phase than that of wild bacteria, but no significant difference exists. In the later growth stage, the concentration difference between orf30-Tb1546 and the wild strain is not significant (FIG. 4C), but the concentration of orf30-Tb15 is significantly lower than that of the wild strain (FIG. 4D).

The ralstonia solanacearum can form pink colonies on a TTC culture medium, a large amount of white extracellular polysaccharide is generated outside the pink colonies, and the stronger the pathogenicity is, the higher the extracellular polysaccharide content is. Tb1546, pBBR-Tb1546, Tb15 and pBBR-Tb15 were able to form smooth pink colonies and produce large amount of extracellular polysaccharide on TTC, while orf30-Tb1546 and orf30-Tb15 strains transformed with orf30 gene formed shriveled colonies with less extracellular polysaccharide content on TTC plates (FIG. 5).

3. Extracellular polysaccharide production assay

Extracellular polysaccharide extraction was performed by the method of Vojnov et al (1998) and improved. Culturing to OD ₆₀₀ Ralstonia solanacearum (1 mL) of 0.5 was cultured in 100mL of NA medium for 1 day, centrifuged at 10000rpm for 10min, and the cells were oven-dried and weighed. Adding 2 times of anhydrous ethanol into the supernatant, and addingAdding sodium chloride to the final concentration of 5%, stirring to dissolve, and standing at 4 deg.C. Centrifuging for 10min at 10000rpm for 24h, taking the precipitate, adding absolute ethanol to wash once to obtain crude exopolysaccharide, drying and weighing, calculating the exopolysaccharide yield per unit bacterial load, and repeating the experiment for 3 times. The yield (g) of extracellular polysaccharide per gram of dry bacteria is extracellular polysaccharide mass (g)/dry bacteria mass (g).

The results show that the extracellular polysaccharide yields of orf30-Tb1546 and orf30-Tb15 are significantly lower than those of the corresponding wild bacteria and unloaded wild bacteria (FIG. 6). The determination of the extracellular polysaccharide yield of the strain shows that the extracellular polysaccharide content of the ralstonia solanacearum carrying the orf30 gene is obviously lower than that of wild bacteria, and the result shows that the orf30 gene can inhibit the production of EPS.

4. Cellulase detection

And (5) reversing the plate, solidifying and airing the cellulase detection plate, and punching the plate by using a 7mm puncher for later use. Culturing wild type strain, recombinant strain and unloaded strain in 10mL NA liquid to OD ₆₀₀ After centrifugation at 6000rpm for 5min, 50. mu.L of each supernatant was added to each well and incubated in an incubator for 24 h. Dyeing with 0.1% Congo red solution for 20min, eluting with 1mol/L sodium chloride solution for 15min, washing with water once, measuring the diameter of the transparent ring, and repeating the experiment for 3 times.

The result shows that the orf30-Tb1546 cellulase activity is obviously less than that of wild bacteria, and the pathogenicity of the cellulase is reduced. The Tb15, orf30-Tb15 and pBBR-Tb15 cellulase have no significant difference in activity, and probably the Tb15 cellulase has low activity per se, so orf30 has no significant effect on the Tb15 cellulase (FIG. 7).

5. Competitive growth

Liquid culture of Tb1546 and orf30-Tb1546 to obtain seed liquid, adjusting the concentration of bacteria to about 1 × 10 ⁸ cfu/mL as Tb 1546: orf30-Tb 1546: LB is 1: 1: 100 inoculating LB liquid culture medium for shaking culture, sampling every 4h and coating the plate on a TTC plate, counting the number of different colony forms on the plate, and repeating the experiment for 3 times.

The results show that the proportion of Tb1546 is significantly less than orf30-Tb1546 (FIG. 8) with increasing culture time during the culture process, indicating that orf30 gene can influence the growth rate of bacteria.

6. Determination of pathogenicity

The tobacco K326 was cultivated in pot plants until the seedling stage, and 20 plants were used as a group. Tb1546 and orf30-Tb1546 were cultured in NA until logarithmic growth phase, and the concentration of the culture was adjusted to about 1X 10 ⁸ cfu/mL, 1mL is extracted by an injector to stab and inoculate the tobacco stems in the seedling stage of the potted plant, cotton is covered on the wound, and redundant bacterial liquid is injected into the cotton. And within 3d after inoculation, carrying out moisture preservation treatment on the cotton every 12 h. Meanwhile, another batch of potted seedling stage tobacco is taken and irrigated and inoculated by adopting an irrigation method. Firstly, the root of the tobacco is scratched by a cutter, and the concentration of the bacteria is about 10 ⁸ The bacterial liquid of cfu/mL is diluted by 25 times, and each plant is irrigated by 500mL to ensure that the soil in the potted plant is thoroughly irrigated.

The number of disease progression is recorded every 2d from the onset of wild flora, and the disease index is counted. The disease progression refers to tobacco pest grading standard (GB/T23222 and 2008):

level 0: the whole plant is disease-free;

level 1: occasionally, the stem has chlorotic streak; or withering of leaves below the diseased side 1/2;

and 3, stage: 1/2 with black streak but no higher than stem height; or withering of diseased side 1/2 to 2/3 leaves;

and 5, stage: stem black streaks exceed the stem height 1/2, but do not reach the top of the stem; or withering leaves above the diseased side 2/3;

and 7, stage: the black streak of the stem reaches the top of the stem; or withering all the leaves of the diseased plant;

and 9, stage: the diseased plants die basically.

The disease index formula is: disease index (Σ (each level representative × plant) ÷ highest level representative × total plant × 100.

The result shows that the infection effect of pathogenic bacteria can be more obviously displayed by the needle punching method. After onset, the index of the tobacco bacterial wilt disease treated by the needle punching method increases significantly faster than that treated by the irrigation method (fig. 9). The needling inoculation result shows that the control group inoculated with Tb1546 is attacked 22d later, the experimental group inoculated with orf30-Tb1546 is attacked, and the attack rate is stable after 34 d. At this time, the difference between the disease indexes of the control group and the experimental group is more than 50. The irrigation experiment group does not attack the disease at all, and the final disease index of the control group reaches more than 40. The results show that the pathogenicity of orf30-Tb1546 to tobacco is obviously reduced, and orf30 obviously influences the pathogenicity of Tb 1546.

7. qRT-PCR gene expression and regulation analysis

(1) RNA extraction and reverse transcription

RNA extraction was performed with reference to the instructions of the Total RNA extraction kit for cultured cells/bacteria (Cat. No. DP430) of Tiangen Biochemical technology (Beijing) Ltd., and PrimeScript (PrimeScript) of Takara was used immediately ^TM RT Master Mix (Perfect Real Time) kit (goods number RR036A) carries out reverse transcription, and the reverse transcription system is as follows: RNase Free ddH ₂ O7. mu.L, DNA-free RNA 1. mu.L: (<500ng)，

RT Master Mix 2. mu.L, total 10. mu.L. Reverse transcription was performed on a PCR instrument with the program: 37 ℃ for 15min, 85 ℃ for 5s, and 4 ℃ infinity. The reverse transcription product was stored at-20 ℃ for the next qRT-PCR experiment.

(2) qRT-PCR relative quantitative analysis

Primers were designed according to the qRT-PCR primer design rules (Table 3).

The reverse transcription product was diluted (generally 10 to 100 times) and TB Green from Takara was used ^TM Premix Ex Taq ^TM II (Tli RNaseH plus) (product number RR820A) to prepare qRT-PCR system, the system is: ddH2O 8.5.5. mu.L, 2 XTB Green Premix Ex Taq II (Tli RNaseH Plus) 12.5. mu.L, Primer 11. mu.L, Primer 21. mu.L, template 2. mu.L, total 25. mu.L. The qRT-PCR instrument is a Bio-Rad CFX96 fluorescent quantitative PCR instrument, the reaction tube adopts 8 calandria (purchased from Behcet, Cathaya, Katsuga, Katsugaku, Kazuki, Katsugaku, and Katsugaku, including (Katsugaku) 8-Katsugaku, and a, including (Katsugaku, Ski: hold (Pre-denaturation) 95 ℃ for 30 s; 2Step PCR95 ℃ for 5s, 60 ℃ for 30s, 40 cycles; dissociation 95 ℃ for 10s, 65 ℃ for 5s, 95 ℃ for 5 s. Data mining 2 _-ΔΔ The Ct method (Livak and Schmittgen,2001) was performed by repeating the analysis of relative expression amounts 3 times using 16S as an internal reference gene, Ct as the number of PCR cycles at which the fluorescence reached the threshold, test as the experimental group, and con as the control group. Delta Ct _test Ct value of target gene of experimental group-Ct value of 16S gene of experimental group; delta Ct _con Control group target gene Ct value-control group 16S groupDue to the Ct value; Δ Δ Ct ═ Δ Ct _test -ΔCt _con (ii) a Relative expression level of target gene 2 _-ΔΔCt 。

TABLE 3 primers used for qRT-PCR

The qRT-PCR expression level of phc system, EPS, T3SS, flagella system, T6SS, cellulase and important regulatory genes in Tb1546 were analyzed. The expression levels of all these disease-causing genes were significantly reduced (FIG. 10). Indicating that orf30 can seriously interfere with the production of ralstonia solanacearum pathogenic factors, thereby reducing the virulence of ralstonia solanacearum.

The above results of the present invention show that the trp574(orf30) gene belongs to the XRE family of transcriptional regulatory factors, and that the ralstonia solanacearum strain transformed with orf30 almost loses pathogenicity. The growth speed of the transformed strain in LB is accelerated, the colony on the TTC plate is dry and flat, and is deep red, the yield of extracellular polysaccharide is reduced, and the activity of cellulase is reduced. The qRT-PCR result shows that orf30 can regulate and control a phc system and a plurality of two-component systems, so that pathogenicity of ralstonia solanacearum is influenced, and orf30 has comprehensive effects of obviously interfering growth, metabolism, pathogenicity and the like of the ralstonia solanacearum. the trp574(orf30) gene can be used for preparing a bacterial strain without pathogenicity, and has a larger application prospect in bacterial wilt prevention and control.

Sequence listing

<110> southern China university of agriculture

JI'AN COMPANY OF JIANGXI TOBACCO Co.

<120> phage trp574 gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 555

<212> DNA

<213> Ralstonia solanacearum)

<400> 1

atggcacagc ggaaaatctc tctgcggcag ctcgctgaga agatgggcct tgcgcactca 60

gctctcagca tcaccttcag cggctcgcgg cgcatgcagt tggaggaggc cgcacagctc 120

tccaacatct tcggcgttcc catccacgag atcatcgaca acgccggcgt gtctgcccgg 180

ccgatcagca gcgcgcgcgt gtcggtcgtc ggcgccctgc gcggtgacgg ccacgtcgag 240

aagatcggcg gaaagcacac agagcgcacg tcggcgccgc ctgggtcgcc tgaaggcaca 300

gaggcgatcc agtcacgcac ggcggacacg cccctatcat ggatggacgg ctgggtcttt 360

ttcttcgtcc cgtccgacag catccatccc gatgcgatag gccgcctgtg ctacctgaaa 420

atccacgagg gcgagcacgt catagccgcg atcaagcgag gatacaggga aaacacctac 480

aacctctctg gccctcacac caaggagaac gcgcggatcg attgggctac gccgatcagg 540

tggacgcgca actag 555

<210> 2

<211> 184

<212> PRT

<213> Ralstonia solanacearum)

<400> 2

Met Ala Gln Arg Lys Ile Ser Leu Arg Gln Leu Ala Glu Lys Met Gly

1 5 10 15

Leu Ala His Ser Ala Leu Ser Ile Thr Phe Ser Gly Ser Arg Arg Met

20 25 30

Gln Leu Glu Glu Ala Ala Gln Leu Ser Asn Ile Phe Gly Val Pro Ile

35 40 45

His Glu Ile Ile Asp Asn Ala Gly Val Ser Ala Arg Pro Ile Ser Ser

50 55 60

Ala Arg Val Ser Val Val Gly Ala Leu Arg Gly Asp Gly His Val Glu

65 70 75 80

Lys Ile Gly Gly Lys His Thr Glu Arg Thr Ser Ala Pro Pro Gly Ser

85 90 95

Pro Glu Gly Thr Glu Ala Ile Gln Ser Arg Thr Ala Asp Thr Pro Leu

100 105 110

Ser Trp Met Asp Gly Trp Val Phe Phe Phe Val Pro Ser Asp Ser Ile

115 120 125

His Pro Asp Ala Ile Gly Arg Leu Cys Tyr Leu Lys Ile His Glu Gly

130 135 140

Glu His Val Ile Ala Ala Ile Lys Arg Gly Tyr Arg Glu Asn Thr Tyr

145 150 155 160

Asn Leu Ser Gly Pro His Thr Lys Glu Asn Ala Arg Ile Asp Trp Ala

165 170 175

Thr Pro Ile Arg Trp Thr Arg Asn

180

<210> 3

<211> 41

<212> DNA

<213> Ralstonia solanacearum)

<400> 3

gtcgacggta tcgataagct tgtggtgttg tatgcgaacc g 41

<210> 4

<211> 41

<212> DNA

<213> Ralstonia solanacearum)

<400> 4

cgctctagaa ctagtggatc cctcgtttct agttgcgcgt c 41

Claims

1. A phage trp574 gene is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. The protein encoded by the phage trp574 gene of claim 1, wherein the amino acid sequence is set forth in SEQ ID NO. 2.

3. A primer pair for amplification detection of the phage trp574 gene of claim 1, wherein the sequence of the primer pair is shown as SEQ ID NO. 3-4.

4. A recombinant expression vector comprising the phage trp574 gene of claim 1.

5. A host bacterium comprising the recombinant expression vector of claim 4.

6. The host bacterium of claim 5, wherein the host bacterium is Escherichia coli or Ralstonia solanacearum Tb15 or Tb 1546.

7. Use of the bacteriophage trp574 gene according to claim 1 for producing a pathologically inert ralston strain.

8. The use according to claim 7, wherein the recombinant expression vector containing the gene of bacteriophage trp574 is transformed into wild-type Ralstonia solanacearum.

9. The use of the bacteriophage trp574 gene according to claim 1 for controlling bacterial wilt.