CN112029789B - Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage - Google Patents
Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage Download PDFInfo
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Abstract
The invention discloses application of a phage trp574 gene in reducing the resistance of ralstonia solanacearum to phage, and the research of the invention shows that the phage trp574 gene can eliminate the resistance of ralstonia solanacearum to phage and change the resistance into sensitivity; by transferring the bacteriophage exogenous gene trp574 into a ralstonia solanacearum strain, a transgenic ralstonia solanacearum strain with reduced bacteriophage resistance can be obtained, so that the using and preventing effects of the bacteriophage are improved, and the bacterial wilt prevention and control are realized.
Description
Technical Field
The invention relates to the technical field of plant disease control, and in particular relates to application of a phage trp574 gene in reducing resistance of ralstonia solanacearum to phages.
Background
Laurella solanacearum (Ralstonia solanacearum, Ralstonia solanacearum for short) is a gram-negative plant pathogenic bacterium, is widely distributed in tropical, subtropical and temperate regions, can infect more than 50 families and more than 450 plants, and is a pathogenic bacterium causing bacterial wilt of crops. The bacteria invade from the roots of plants, colonize first in the root-cortex intercellular spaces and the like, and then rapidly proliferate and widely spread in ducts and adjacent tissues, thereby causing blockage and destruction of vascular systems, eventually leading to withering and death of plants. This devastating soil-borne disease is known as crop bacterial wilt, and the economic losses due to this disease are hundreds of millions each year worldwide. At present, the control method for the bacterial wilt of crops mainly comprises chemical control, biological control, planting disease-resistant varieties, crop rotation and the like, and although the measures play a certain control role in the bacterial wilt, the diseases 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 the obvious advantages of strong sterilization specificity, no harm to human and livestock, continuous self-proliferation by taking pathogenic bacteria as hosts and the like, and has great development potential as an effective component of a novel biological pesticide. The utilization of the phage for preventing and treating the crop bacterial wilt has important practical significance. Filamentous bacteriophage
And
the invasion of ralstonia solanacearum can reduce the toxicity of ralstonia solanacearum, and the physiology and biochemistry of ralstonia solanacearum are changed, so that the filamentous bacteriophage can be used for killing the ralstonia solanacearum
The infected Ralstonia solanacearum is inoculated on the root of the tomato, then wild type Ralstonia solanacearum is inoculated, and the fluorescence detection method finds that the toxicity of the wild type Ralstonia solanacearum is inhibited, the whole tomato incidence rate is reduced (Addy H S, Askora A, Kawasaki T, et al
filamentous phages[J]Phytopathology,2012,102(5): 469-477). Fujiwara et al (2011) use of bacterial wilt bacteriophage to biologically control bacterial wilt, demonstrating that bacteriophage has some effect in bacterial wilt control (Fujiwara A, Fujisawa M, Hamasaki R, et al.Biocontrol of Ralstonia solanaceum by means of viral bacteriophages [ J ]Applied and Environmental Microbiology,2011,77(12): 4155-.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of a phage trp574 gene in reducing resistance of ralstonia solanacearum to phages.
Another object of the present invention is to provide the use of the gene trp574 of bacteriophage for producing a strain of Ralstonia solanacearum which is sensitive to bacteriophage.
It is a further object of the present invention to provide a method for reducing resistance of ralstonia solanacearum to bacteriophage.
The above purpose of the invention is realized by the following technical scheme:
according to the invention, a transcription regulatory factor orf30 is discovered by performing genome sequencing on a lytic phage P574, then a primer is designed according to a phage 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 present inventors have found that the trp574(orf30) gene can impair resistance of Ralstonia solanacearum, which originally exhibits resistance to a bacteriophage, to the bacteriophage, even if the resistance becomes sensitive. By transferring the phage exogenous gene trp574 into a ralstonia solanacearum strain, a transgenic ralstonia solanacearum strain with reduced phage resistance can be obtained, so that the use and control effects of the phage are improved; meanwhile, the results of the contemporaneous researches of the inventor also show that the ralstonia solanacearum transformed with trp574(orf30) gene almost loses pathogenicity, can obtain ralstonia solanacearum without pathogenicity and with reduced resistance to bacteriophage, and is better used for controlling bacterial wilt by matching with wide host bacteriophage.
Thus, the present invention claims the following applications of the trp574 gene:
SEQ ID NO: 1 to reduce the resistance of ralstonia solanacearum to the bacteriophage.
SEQ ID NO: 2 in the application of the protein coded by the gene trp574 of the bacteriophage shown in the figure, in reducing the resistance of ralstonia solanacearum to the bacteriophage.
SEQ ID NO: 1 in the preparation of bacterial strain ralstonia solanacearum sensitive to bacteriophage.
Specifically, a recombinant expression vector containing a phage trp574 gene is transformed into ralstonia solanacearum to obtain a transgenic ralstonia solanacearum strain with reduced phage resistance.
Preferably, the recombinant expression vector is pBBR1MCS 4.
Preferably, the ralstonia solanacearum is wild-type ralstonia solanacearum.
Preferably, the ralstonia solanacearum is Tb15 or Tb 1546.
According to the application, the invention also provides a method for reducing the resistance of ralstonia solanacearum to the bacteriophage, which is to transfer the trp574 gene of the bacteriophage into the ralstonia solanacearum to obtain a transgenic ralstonia solanacearum strain with reduced resistance to the bacteriophage, so that the resistance of the ralstonia solanacearum to the bacteriophage is reduced.
Preferably, the conversion is an electrical conversion.
The invention also claims a transgenic ralstonia solanacearum strain with reduced resistance to bacteriophage, which is obtained by the method.
Compared with the prior art, the invention has the following beneficial effects:
the research of the invention shows that the gene trp574 of the bacteriophage can eliminate the resistance of the ralstonia solanacearum to the bacteriophage, so that the resistance is changed into sensitivity; by transferring the bacteriophage exogenous gene trp574 into a ralstonia solanacearum strain, a transgenic ralstonia solanacearum strain with reduced bacteriophage resistance can be obtained, so that the using and preventing effects of the bacteriophage are improved, and the bacterial wilt prevention and control are realized.
Drawings
FIG. 1 shows the results of orf30 gene cloning and enzyme digestion verification. M1 is DL2000 Marker, 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, 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. 2 is a plaque of 6 phages on Tb1546, orf30-Tb1546 and pBBR-Tb1546 plates. A1-A4 are plaque forms of Tb1546 No. 1, No. 4, No. 7 and No. 10d, respectively; B1-B4 are plaque forms on 1 st, 4 th, 7 th and 10 th days of orf30-Tb1546 respectively; C1-C4 are plaque forms of pBBR-Tb1546 on days 1, 4, 7 and 10, respectively. 1, 2, 3, 4, 5 and 6 in each figure respectively represent P1556-1, P1556-2, P7-1, P1521, P1553 and P574.
FIG. 3 shows the variation of the sensitivity of Tb15, orf30-Tb15 and pBBR-Tb15 to 6 phages.
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 medium: 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast extract and 15g/L of agar powder.
NA culture 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 cloning of trp574 Gene (orf30) and construction of recombinant plasmid
1. Method of producing a composite material
(1) In the early stage of the invention, a transcriptional regulator is discovered by carrying out genome sequencing on lytic phage P574 Section factor orf30, using general phage DNA extraction kit (cat # KG005-1) of Noocrea Biotechnology, Guangzhou to extract phage DNA, and designing amplification primers orf30-F/orf30-R of gene orf30 of interest (as shown in Table 2) according to P574 genome information; phage DNA is used as a template, orf30-F/orf30-R is used as a primer, and Novozan 2 is used as a template
The Master Mix is used for amplifying a target gene orf30, and an amplification system is 2
Master Mix 25. mu.L, Primer 11. mu.L, Primer 21. mu.L, template DNA 1. mu.L (<200ng), complement ddH2O 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 was performed according to the Axygen plasmid miniprep kit, and after double digestion with BamHI and Hind III, Novozam was used
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 2O2.32. 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. Results
(1) Designing a primer according to a phage genome sequence, carrying out PCR amplification to obtain a target fragment of about 688bp (shown in figure 1A), sequencing the target fragment, and finding 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 an orf30 target fragment has a detection fragment size of 977bp (figure 1B), the recombinant plasmid enzyme digestion result shows two bands which are close to the predicted 646bp (figure 1E), and the sequencing result is consistent with the prediction result, so that the success of the construction of the recombinant plasmid is proved.
Example 2 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 shock competence and 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 the pseudomonas solanacearum competence. After transformation, 800. mu.L of NA liquid medium was added immediately for resuscitation for 12h, plates were coated on the NA 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 1.
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 shocked into Tb1546 and Tb15 to obtain pBBR-Tb1546 and pBBR-Tb15 (FIGS. 1-C, D).
2. Phage sensitivity assay
Tb1546 is cultured by phages PTb1556-1, PTb1556-2, PTb7-1, PTb1521, PTb1553 and PTb574, the phage culture solution is centrifuged for 10min at 10000rpm in a centrifuge, and the supernatant is diluted to 1 × 108cfu/mL for use. Liquid culture of Tb1546, orf30-Tb1546 and pBBR-Tb1546 to a bacterial concentration of 1X 108cfu/mL, uniformly mixing bacterial liquid of every 100 mu L and common bacterial semi-solid culture medium of 15mL respectively, pouring the mixture on a culture dish to prepare a detection plate, dripping phage of 2 mu L on the detection plate after blow-drying, carrying out inverted culture until the plaque sizes are recorded at 1 st, 4 th, 7 th and 10 th days, and repeating the experiment for 3 times. The liquid culture of Tb15, orf30-Tb15 and pBBR-Tb15 were carried out for phage sensitivity detection in the same manner as described above, and the results of phage sensitivity were recorded 24 hours after culture.
The results show that Tb1546 and orf30-Tb1546 have different sensitivities to different phages, and after 1d of culture, plaques formed on orf30-Tb1546 detection plates are all smaller than plaques on Tb1546, and are most significant with P1556-1; the plaques are increased to a certain extent along with the extension of the culture time, except for P1556-1, the sizes of plaques formed by other phages are obviously different from those of wild bacteria, and are most obvious with P7-1 and P1553 (figure 2); in the three strains of Tb15, orf30-Tb15 and pBBR-Tb15, orf30-Tb15 appeared sensitive to phages P7-1 and P1553, indicating that orf30 affected the anti-phage mechanism of Tb15 (FIG. 3).
The results show that the trp574(orf30) gene can weaken the resistance of ralstonia solanacearum which originally shows resistance to the bacteriophage as the bacteriophage, even if the resistance becomes sensitive, the application range and the control effect of the bacteriophage are improved, and the application prospect in bacterial wilt control is wide.
Sequence listing
<110> southern China university of agriculture
Application of <120> phage trp574 gene in reducing resistance of ralstonia solanacearum to phage
<141> 2020-08-03
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 555
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<400> 1
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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
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<211> 184
<212> PRT
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His Glu Ile Ile Asp Asn Ala Gly Val Ser Ala Arg Pro Ile Ser Ser
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Ala Arg Val Ser Val Val Gly Ala Leu Arg Gly Asp Gly His Val Glu
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Lys Ile Gly Gly Lys His Thr Glu Arg Thr Ser Ala Pro Pro Gly Ser
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Pro Glu Gly Thr Glu Ala Ile Gln Ser Arg Thr Ala Asp Thr Pro Leu
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Ser Trp Met Asp Gly Trp Val Phe Phe Phe Val Pro Ser Asp Ser Ile
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Claims (10)
1, SEQ ID NO: 1 to reduce the resistance of ralstonia solanacearum to the bacteriophage.
SEQ ID NO: 2 in the application of the protein coded by the gene trp574 of the bacteriophage shown in the figure, in reducing the resistance of ralstonia solanacearum to the bacteriophage.
3, SEQ ID NO: 1 in the preparation of bacterial strain ralstonia solanacearum sensitive to bacteriophage.
4. Use according to claim 1 or 3, characterized in that a recombinant expression vector containing the gene of bacteriophage trp574 is transformed into Ralstonia solanacearum.
5. The use according to claim 4, wherein the recombinant expression vector is pBBR1MCS4-trp 574.
6. The use according to claim 4, wherein said Ralstonia solanacearum is wild-type Ralstonia solanacearum.
7. The use according to claim 4, wherein the ralstonia solanacearum is Tb15 or Tb 1546.
8. A method for reducing resistance of Ralstonia solanacearum to bacteriophage, characterized in that bacteriophage trp574 gene is transformed into Ralstonia solanacearum.
9. The method of claim 8, wherein the conversion is to electrical conversion.
10. A ralstonia solanacearum strain transformed with trp574 gene having reduced resistance to phage produced by the method of claim 8 or 9.
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| CN112048514A (en) * | 2020-08-03 | 2020-12-08 | 华南农业大学 | Phage trp574 gene and application thereof |
| CN112210503A (en) * | 2020-08-03 | 2021-01-12 | 华南农业大学 | Bacterial strain of non-pathogenic ralstonia solanacearum transformed with bacteriophage trp574 gene and preparation method and application thereof |
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