CN115747244A - Banana wilt disease No. 4 microspecies pathogenic gene FOXG _01465 and application thereof - Google Patents
Banana wilt disease No. 4 microspecies pathogenic gene FOXG _01465 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a banana vascular wilt disease number 4 microspecies pathogenic gene FOXG _01465 and application thereof, wherein the gene is derived from banana vascular wilt disease number 4 microspecies (Fusarium oxysporum f.sp. The FOXG _01465 gene deletion mutant strain is obtained by a gene knockout technology, and the gene is further disclosed to participate in regulating and controlling the hypha growth, spore production and pathogenicity of the banana vascular wilt disease No. 4 microspecies, so that the invention provides theoretical and practical significance for clarifying the pathogenic mechanism of the banana vascular wilt disease No. 4 microspecies, mastering the control strategy of the banana vascular wilt disease and breeding for disease resistance, and has important development value.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a banana vascular wilt disease No. 4 race pathogenic gene FOXG _01465 and application thereof.
Background
Banana vascular wilt is a epidemic disease of soil-borne fungi caused by infection of Fusarium oxysporum cubeba specialization (Fusarium oxysporum f.sp.cubense), can cause vascular bundle necrosis of banana plants and then withering and yellowing and even death, and seriously threatens the development of global banana industry. The fusarium oxysporum f.sp.4 is a physiological race of fusarium oxysporum with the strongest infection capability and toxicity, and can infect almost all banana varieties, however, no chemical reagent and disease-resistant variety capable of effectively preventing and treating the fungal diseases are found up to now.
In the whole genome sequence of the currently published banana vascular wilt disease No. 4 microspecies, about 43 percent of the functions of the genes are still not analyzed, and the occurrence of a large number of unknown functional genes limits scientists to understand the pathogenic mechanism of the banana vascular wilt disease No. 4 microspecies, and influences the research and development of plant disease control methods and disease-resistant varieties. The pathogenic function mechanism of the gene with unknown function in the banana vascular wilt 4 microspecies is determined, and theoretical and practical significance is provided for clarifying the pathogenic mechanism of the plant pathogenic bacteria, developing disease-resistant prevention strategies and disease-resistant breeding.
Disclosure of Invention
The present invention is based on the above problems of the prior art and provides the application of the gene FOXG _01465.
The purpose of the invention is realized by the following technical scheme:
the gene FOXG _01465 is applied to reducing pathogenicity of banana fusarium oxysporum, and the coded amino acid sequence is shown as SEQ ID NO:2, respectively.
The invention provides a banana vascular wilt disease No. 4 race pathogenic gene FOXG _01465, which is used for meeting the requirements of a disease target gene. The open reading frame of the gene has the length of 3296bp, the full-length sequence is shown as SEQ ID NO:1, the coded protein consists of 736 amino acids, and the sequence is shown as SEQ ID:2, respectively.
The function of this gene has not been studied previously. According to the invention, the FOXG _01465 gene deletion mutant strain is constructed for the first time, and the related functions of the mutant strain are verified from three aspects of hypha growth, spore production and pathogenicity of the mutant strain, so that the function mechanism of the pathogenicity of the mutant strain in the banana vascular wilt 4 microspecies is clear, and the gene can be used as a target for preventing and controlling banana vascular wilt pathogenic fungi.
Therefore, the invention also provides application of the gene FOXG _01465 in reducing spore yield of fusarium oxysporum f.sp.cubense.
The invention also provides application of the gene FOXG _01465 in reducing the hypha morphology of banana vascular wilt.
The invention also provides application of the gene FOXG _01465 in preventing and treating banana wilt caused by banana fusarium wilt, which is characterized in that the prevention and the treatment are realized by down-regulating the expression of the gene FOXG _01465.
The invention also provides application of the gene FOXG _01465 as a target of a medicine for preventing and treating banana vascular wilt disease No. 4 race.
Preferably, the nucleotide sequence of the gene FOXG _01465 is shown as SEQ ID NO:1 is shown.
The invention also provides application of the substance expressed by the gene FOXG _01465 in preparing medicines for preventing and treating banana vascular wilt.
Preferably, the nucleotide sequence of the gene FOXG _01465 is shown as SEQ ID NO:1 is shown.
Preferably, the substance comprises any one of:
(i) Small interfering RNA, dsRNA, shRNA, microRNA and antisense nucleic acid which take FOXG _01465 transcript as a target sequence and can inhibit the expression of FOXG _01465 gene expression product or gene transcription;
(ii) (ii) capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct of (i);
(iii) A construct selected from the group consisting of interfering molecules that contain the complementary sequence of FOXG _01465 and are capable of forming an inhibitory molecule on the expression of the FOXG _01465 gene expression product or on gene transcription after transfer into the body;
(iv) A cell, differentiated cell or construct thereof, wherein the FOXG _01465 gene sequence is suppressed or deleted.
More preferably, the substance is selected from the group consisting of knockout homology arm sequences 465u _HYand YG _465d, and mutant strain Δ FOXG _01465.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a banana vascular wilt disease number 4 microspecies pathogenic gene FOXG _01465 and application thereof, wherein the gene is derived from banana vascular wilt disease number 4 microspecies (Fusarium oxysporum f.sp. The FOXG _01465 gene deletion mutant strain is obtained by gene knockout technology, and the gene is further disclosed to participate in regulating and controlling hypha growth, spore production and pathogenicity of the banana vascular wilt disease No. 4 microspecies, so that the invention provides theoretical and practical significance for clarifying the pathogenic mechanism of the banana vascular wilt disease No. 4 microspecies, mastering the prevention and control strategy of banana vascular wilt disease and breeding for disease resistance, and has important development value.
Drawings
FIG. 1 is a flow chart of an experiment for constructing a FOXG _01465 gene deletion mutant strain;
FIG. 2 is a graph comparing the growth patterns of normal banana vascular wilt disease No. 4 race and gene FOXG _01465 deletion mutant strain on PDA agar plate;
FIG. 3 is a comparison of hyphae of normal banana vascular wilt disease No. 4 microspecies and hyphae after deletion of gene FOXG _01465 under the observation of a scanning electron microscope;
FIG. 4 is a graph showing the comparison of the spore yields of normal banana vascular wilt disease number 4 microspecies in YEPD medium and the spore yield after deletion of the gene FOXG _ 01465;
FIG. 5 is a comparison of the pathogenicity of normal banana wilt disease No. 4 microspecies and gene FOXG _01465 deletion mutant to banana plants.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed drawings and examples. In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1
This example 1 provides a banana vascular wilt disease number 4 race pathogenic gene FOXG _01465, which has a sequence shown in SEQ ID NO:1, and the protein sequence is shown as SEQ ID NO:2, respectively.
Example 2
The embodiment provides a knockout of a banana wilt disease pathogenic gene FOXG _01465 and a construction of a gene deletion mutant strain thereof, which comprises the following steps:
1. culture condition of banana vascular wilt No. 4 microspecies
The preparation method of the PDA culture medium comprises the following steps: peeling fresh potato 200g, boiling with distilled water for 20-30min, filtering to remove potato, adding glucose 20g and agar 15g into the filtrate, adding distilled water to desired volume of 1L, and autoclaving at 121 deg.C for 20min. The banana vascular wilt No. 4 microspecies are inoculated in a PDA culture medium and then are cultured in a constant temperature incubator at 28 ℃. The YEPD formula is as follows: 10g/L of yeast extract, 20g/L of peptone, 20g/L of glucose and 15g/L of agar. After the banana vascular wilt disease No. 4 microspecies are inoculated in a YEPD liquid culture medium, the culture is carried out by shaking under the conditions of 180rpm and 28 ℃.
2. Acquisition of FOXG _01465 Gene deletion mutant
(1) Preparation of knockout cassettes: a knockout cassette for the target gene was constructed using split-marker PCR based technology. FOXG _01465 gene (Genbank: XM _ 018378304.1) 5 'flanking sequence 465u (250-500 bp) was obtained by PCR amplification using primers 465uf and 465ur (Table 1), and primers 465df and 465dr (Table 1) amplify 3' flanking 465d (250-500 bp) of the gene. For the selection marker hygromycin phosphotransferase (HYG) gene (Genbank: HQ 412578.1), "HY" and "YG" fragments were obtained from the full-length HYG gene by amplification using HphF/HYR and YGF/HphR primers (Table 1), respectively, with about 400bp of overlapping sequence. Primers 465ur and 465df, each having 5' sequences complementary to the HphF and HphR primers, respectively. The sequences of 465u _HYand YG _465d (shown in SEQ ID NO:3 and SEQ ID NO:4, the overlapping fragments have 315 bp) are obtained by fusion of the sequences 465u and HY and YG and 465d through overlapping extension PCR. Finally, the 465u _HYand primers 465uf and HYR, the YG _465d and primers YGF and 465dr are used and are cleaned and recovered by PCR to prepare 465u _HYand YG _465d fragments with the total amount of 5-10ug respectively. PCR conditions were as follows: pre-denaturation at 98 deg.C for 2min; in 30 cycles, denaturation is carried out for 98 ℃,30s, annealing is carried out for 60 ℃,30s, extension is carried out for 72 ℃, and the length of the extension is 30s/kb; final extension 72 ℃ for 2min.
TABLE 1
(2) Preparation of protoplasts
Washing PDA plate of 7d banana vascular wilt 4 # microspecies with sterile water, scraping the surface of hypha plate with sterile glass rod, collecting spores in the plate, preparing spore suspension for inoculation, adjusting concentration to 1 × 10 with sterile water 8 one/mL, followed by inoculation in YEPD medium, culturing at 20 deg.C and 180rpm for 12-16h, centrifuging to collect the germinating hyphae, and washing the mycelium at least 3 times with 1.2M potassium chloride (sterile) solution. 20mL of protoplast forming solution was prepared by filtration of 0.8M magnesium sulfate, 1% (w/v) lywallzyme, 0.1% (w/v) lypase, 0.2 μ M sterile filter. Transferring germinated mycelium into protoplast generating solution, treating at 30 deg.C and 100rpm for 2-4 hr, and observing under microscope to obtain protoplast in the same visual field at least 50%. Filtering with absorbent cotton having a thickness of 2cm, collecting protoplasts, centrifuging at 2500rpm at 4 deg.C, washing the protoplasts with 1.2M potassium chloride at least 3 times, and washing with STC solution (1.2M sorbitol, 10mM Tris-HCl, pH8.0 and 50mM CaCl) 2 ) Washing and enrichingAnd (4) collecting protoplasts. Subpackaging 200 μ L to 1.5mL EP tube, adding DMSO with final concentration of 7%, quick freezing with liquid nitrogen, and storing at-80 deg.C.
(3) Transformation and selection of protoplasts
200 μ L of protoplast solution was taken, and the homology arms 465u _HYand YG _465d for removing the target genes at upstream and downstream were added, and after ice-bath for 25min, PTC solution (40 w/v PEG4000,1 × STC) was added, and the mixture was left at room temperature for 25min. Transferring the protoplast solution to 50mL YEPD agar medium at 50-60 deg.C, pouring into 14cm plate culture dish, and making into plate. When hyphae appear on the plate, YEPD 50mL agar medium + 200. Mu.g/mL hygromycin is poured to the upper layer of the plate, and a double-layer plate is used for screening single colonies.
(4) Screening of mutants and isolation of mutant monospores
Picking single colony hypha fragments to 50 mu L of sterile water, shaking and uniformly mixing, heating for 10min in a metal bath at 100 ℃, centrifuging to obtain supernatant, sampling 1 mu L to 50 mu L of PCR system, and carrying out PCR by using primers 465u and HYR and primers YGF and 465d under the PCR conditions: performing pre-denaturation at 98 ℃ for 2min; in 30 cycles, denaturation is carried out for 98 ℃,30s, annealing is carried out for 60 ℃,30s, extension is carried out for 72 ℃, and the length of the extension is 30s/kb; final extension 72 ℃ for 2min. The target fragment was observed electrophoretically. And determining the correct mutant according to the single colony PCR identification result.
The fragment of the mutant hyphae was picked with the tip of a toothpick onto a PDA plate containing hygromycin at 200. Mu.g/mL. Collecting spores after 7 days, mixing the spores into a PDA culture medium to prepare a flat plate, and selecting single spores to form colonies to obtain the gene FOXG _01465 deletion mutant strain.
Example 3
The embodiment provides application of the gene FOXG _01465 in regulating and controlling pathogenicity of a banana vascular wilt No. 4 race.
1. Analysis of influence of gene FOXG _01465 deletion on growth of hyphae of banana vascular wilt No. 4 microspecies
Respectively culturing the banana wilt disease No. 4 microspecies and the gene FOXG _01465 deletion mutant strain on a PDA agar culture medium, placing the culture medium in a constant temperature incubator at 28 ℃ for culture, and observing the growth condition of hyphae on the PDA agar plate after 7 days.
Scraping hyphae from PDA plate cultured at 28 deg.C for 7d with sterile glass rod, soaking with 2.5% glutaraldehyde at 4 deg.C overnight, pouring off the fixative, rinsing the sample with 0.1M phosphate buffer (pH7.0) for 15min three times, fixing the sample with 1% osmic acid solution for 1-2h, and rinsing the sample with 0.1M phosphate buffer (pH7.0) for 15min three times after taking out osmic acid waste solution carefully; the samples were dehydrated using graded concentrations (30%, 50%,70%,80%,90% and 95%) of ethanol for 15min each, and finally twice with 100% ethanol for 20min each. The sample was treated with a mixture of ethanol and isoamyl acetate (V: V = 1) for 30min, then with pure isoamyl acetate for 1h, dried at the critical point and observed using a scanning electron microscope of HITACHI SU8010 type.
As a result, as shown in FIG. 2, it was found that the FOXG _01465 gene-deleted mutant strain grew very slowly on PDA agar plates and had thin hyphae, as compared with the banana vascular wilt disease No. 4 microspecies. Scanning electron microscope observation shows that hyphae of banana vascular wilt disease No. 4 microspecies with uniform thickness and few branches in the original position in FIG. 3 become uneven in thickness and have obviously increased branches, which shows that the gene FOXG _01465 can influence the hypha growth of banana vascular wilt disease No. 4 microspecies and change the hypha form.
2. Analysis of influence of gene FOXG _01465 deletion on production of banana vascular wilt number 4 microspore
Respectively culturing the banana wilt disease No. 4 microspecies and the gene FOXG _01465 deletion mutant strain in YEPD liquid culture medium, carrying out shake culture at 180rpm and 28 ℃ for 7d, collecting fermentation liquor, filtering by using a sterile syringe containing absorbent cotton to obtain spore suspension, observing spores under an optical microscope (40 Xobjective lens and 10 Xeyepiece lens), and calculating the yield of the spores.
As a result, as shown in fig. 4, it was found that the gene FOXG _ 01465-deleted mutant hardly produced spores when compared with banana wilt disease number 4 microspore, and the spore production was significantly suppressed by the deletion of gene FOXG _01465.
3. Analysis of influence of gene FOXG _01465 deletion on pathogenicity of banana wilt disease No. 4 race
1g of hyphae of a banana vascular wilt 4 # microspecies and a gene FOXG _01465 deletion mutant strain were scraped from a PDA plate cultured at 28 ℃ for 7 days by using a sterile glass rod, respectively, and the hyphae were ground in 100mL of sterile water to prepare a bacterial suspension, and then roots of banana seedlings (Musa sp.AAA Cavendsh subgroucv.Williams B6) with 4 leaves were immersed in the bacterial suspension for 3 hours, followed by transferring to plastic pots with 450g of sterile soil, pouring clear water every three days, and after 30 days, transverse cutting and vertical cutting of the rhizome were performed to evaluate pathogenicity.
As shown in FIG. 5, the banana vascular wilt disease No. 4 microspecies can cause yellowing of banana leaves and atrophy and blackening of rootstocks. And the banana plants treated by the FOXG _01465 gene deletion mutant strain do not show obvious banana wilt characteristics, so that the pathogenicity of No. 4 banana wilt race is weakened by deducing the gene FOXG _01465 gene deletion.
In conclusion, the invention combines the results of fig. 2-fig. 5, firstly reveals the function of the gene FOXG _01465 in the banana vascular wilt disease number 4 race and the role thereof in pathogenesis, and provides an application path and an application technology of the gene FOXG _01465 in the development of the banana vascular wilt disease resistance number 4 race germplasm.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The application of the gene FOXG _01465 in reducing pathogenicity of banana fusarium oxysporum is characterized in that the coded amino acid sequence is shown as SEQ ID NO:2, respectively.
2. The use of the gene FOXG _01465 as claimed in claim 1 for reducing the sporulation of Fusarium oxysporum f.sp.
3. The use of the gene FOXG _01465 as claimed in claim 1 for reducing the hyphal morphology of Fusarium oxysporum f.sp.
4. Use of the gene FOXG _01465 according to claim 1 for controlling banana wilt caused by banana fusarium wilt, characterized in that the control is achieved by down-regulating the expression of the gene FOXG _01465.
5. The use of the gene FOXG _01465 as claimed in claim 1 as a target for a medicament for the prevention and treatment of banana vascular wilt disease race No. 4.
6. The use according to any one of claims 2 to 5, wherein the FOXG _01465 gene has the nucleotide sequence as shown in SEQ ID NO:1 is shown.
7. Use of a substance down-regulating the expression of the gene FOXG _01465 as claimed in claim 1 in the preparation of a medicament for the prevention and treatment of banana wilt.
8. Use according to claim 7, wherein the substance comprises any of:
(i) Small interfering RNA, dsRNA, shRNA, microRNA and antisense nucleic acid which take FOXG _01465 transcript as a target sequence and can inhibit the expression of FOXG _01465 gene expression product or gene transcription;
(ii) (ii) capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct of (i);
(iii) A construct selected from the group consisting of interfering molecules that contain the complementary sequence of FOXG _01465 and are capable of forming an inhibitory molecule on the expression of the FOXG _01465 gene expression product or on gene transcription after transfer into the body;
(iv) A cell, differentiated cell or construct thereof, wherein the FOXG _01465 gene sequence is suppressed or deleted.
9. The use according to claim 8, wherein the substance is selected from the group consisting of knockout homology arm sequences 465u _HYand YG _465d, and mutant strain Δ FOXG _01465.
10. The use according to claim 9, wherein the nucleotide sequence of the gene FOXG _01465 is as set forth in SEQ ID NO:1 is shown.
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| CN202211581102.8A CN115747244B (en) | 2022-12-09 | 2022-12-09 | Banana wilt No. 4 race pathogenicity gene FOXG_01465 and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656116A (en) * | 2019-10-17 | 2020-01-07 | 华南农业大学 | Application of gene FoCWM in regulation and control of pathogenicity of banana vascular wilt |
| CN113174390A (en) * | 2021-03-05 | 2021-07-27 | 华南农业大学 | Application of banana fusarium oxysporum FoNpp1 gene in regulation and control of pathogenicity of banana fusarium oxysporum |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656116A (en) * | 2019-10-17 | 2020-01-07 | 华南农业大学 | Application of gene FoCWM in regulation and control of pathogenicity of banana vascular wilt |
| CN113174390A (en) * | 2021-03-05 | 2021-07-27 | 华南农业大学 | Application of banana fusarium oxysporum FoNpp1 gene in regulation and control of pathogenicity of banana fusarium oxysporum |
Non-Patent Citations (2)
| Title |
|---|
| MA, L.J., ET AL: "hypothetical protein FOXG_01465 [Fusarium oxysporum f. sp. lycopersici 4287]", pages 018234209, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/1070332734?sat=54&satkey=86235070> * |
| 陈石，等: "尖镰孢菌致病机理研究进展", 中国农学通报, vol. 27, no. 13, pages 74 - 78 * |
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