CN115747244B - Banana wilt No. 4 race pathogenicity gene FOXG_01465 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a banana vascular wilt No. 4 race pathogenic gene FOXG_01465 and application thereof, wherein the gene is derived from banana vascular wilt No. 4 race (Fusarium oxysporum f.sp.cubic surgical race 4). The FOXG-01465 gene deletion mutant strain is obtained by a gene knockout technology, and further discloses that the gene participates in regulating and controlling the hypha growth, spore production and pathogenicity of banana wilt No. 4 race, thereby providing theoretical and practical significance for elucidating the pathogenesis of banana wilt No. 4 race, grasping the banana wilt prevention and control strategy and disease-resistant breeding, and having important development value.

Description

Banana wilt No. 4 race pathogenicity geneFOXG_01465And applications thereof

Technical Field

The invention belongs to the technical field of molecular biology, and more particularly relates to a banana wilt No. 4 race pathogenicity geneFOXG_01465And applications thereof.

Background

Banana wilt is an endemic disease of a soil-borne fungus caused by Fusarium oxysporum Guba specialization (Fusarium oxysporum f. Sp. Cube) infection, which can cause necrosis of vascular bundles of banana plants, subsequent wilt yellowing and even death, and seriously threatens the development of the banana industry worldwide. The banana wilt No. 4 race is the physiological race of fusarium oxysporum with the strongest infection capability and toxicity, and almost all banana varieties can be infected, however, no chemical reagent and disease-resistant varieties capable of effectively preventing and treating the fungal diseases are found up to the present.

In the whole genome sequence of the presently published banana wilt No. 4 race, about 43% of the genes still have not been analyzed yet, and the appearance of a large number of unknown functional genes limits the understanding of scientists on the pathogenesis of the banana wilt No. 4 race, so that the research and development of the plant disease control method and disease resistant variety are affected. The functional mechanism of pathogenicity of the gene with unknown function in banana wilt No. 4 race is clarified, and theoretical and practical significance is provided for clarifying the pathogenesis of the plant pathogenic bacteria and developing disease-resistant control strategies and disease-resistant breeding.

Disclosure of Invention

The present invention is based on the above problems occurring in the prior art, and first provides a geneFOXG_01465Is used in the application of (a).

The aim of the invention is achieved by the following technical scheme:

geneFOXG_01465The application of the polypeptide in reducing the pathogenicity of banana fusarium wilt bacteria has the coded amino acid sequence shown in SEQ ID NO: 1.

The invention provides a banana wilt No. 4 small-species pathogenicity geneFOXG_01465To address the need for the disease target gene. The coding protein of the gene consists of 736 amino acids, and the sequence of the coding protein is shown as SEQ ID NO: 1.

The function of the gene has not been studied before. First pass construction of the inventionFOXG_01465The gene deletion mutant verifies the related functions from three aspects of hypha growth, spore production and pathogenicity of the mutant, and confirms the functional mechanism of pathogenicity of the mutant in banana wilt No. 4 race, which shows that the gene can be used for preventing and controlling banana wilt pathogenA target of bacteria.

Thus, the present invention also provides genesFOXG_01465The application of the method in reducing the spore yield of banana fusarium wilt bacteria.

The invention also provides genesFOXG_01465The application of the composition in reducing the mycelium morphology of banana fusarium wilt bacteria.

The invention also provides genesFOXG_01465The application of the composition in preventing and controlling banana vascular wilt caused by banana vascular wilt is characterized in that the prevention and control are carried out by down regulating genesFOXG_01465Is realized by expression of (a).

The invention also provides genesFOXG_01465Use as a target for a medicament for controlling banana vascular wilt No. 4 race.

The invention also provides genesFOXG_01465The expressed substances of (2) are used for preparing the medicines for preventing and treating banana vascular wilt.

Preferably, the substance comprises any one of the following:

(i) To be used forFOXG_01465Transcripts are target sequences and can inhibitFOXG_01465Small interference RNA, dsRNA, shRNA of gene expression product expression or gene transcription, micrornas, antisense nucleic acids;

(ii) Capable of expressing or forming the small interference RNA, dsRNA, shRNA, microrna, antisense nucleic acid construct of (i);

(iii) Is selected from the group consisting ofFOXG_01465Complementary sequences and capable of forming an inhibition upon transfer into the bodyFOXG_01465A construct of an interfering molecule for expression of a gene expression product or transcription of a gene;

(iv) Suppression or knockoutFOXG_01465Cells after the gene sequence, differentiated cells or constructs.

More preferably, the substance is selected from the group consisting of knockout homology arm sequences 4635 u_hy and yg_465d, and mutant strain Δfoxg_01465.

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

the invention provides a banana vascular wilt No. 4 race pathogenic geneFOXG_01465And uses thereof, the gene is derived from banana wilt No. 4 race (Fusarium oxysporum f, spse temporal distance 4). The invention is obtained by gene knockout technologyFOXG_01465The gene deletion mutant strain further reveals that the gene participates in regulating and controlling the hypha growth, spore production and pathogenicity of banana wilt No. 4 race, provides theoretical and practical significance for elucidating the pathogenesis of banana wilt No. 4 race, grasping the banana wilt control strategy and disease-resistant breeding, and has important development value.

Drawings

FIG. 1 is a schematic diagram of a conventional gas turbineFOXG_01465An experimental flow chart of the construction of the gene deletion mutant strain;

FIG. 2 shows normal banana vascular wilt No. 4 race and geneFOXG_01465Growth morphology of deletion mutants on PDA agar plates;

FIG. 3 shows the hyphae and deleted genes of normal banana vascular wilt No. 4 raceFOXG_01465A contrast diagram of the mycelium after the mycelium is observed by a scanning electron microscope;

FIG. 4 shows spore yield and deletion gene of normal banana vascular wilt No. 4 race in YEPD mediumFOXG_01465Comparison of post spore yield;

FIG. 5 shows normal banana vascular wilt No. 4 race and geneFOXG_01465Comparison of pathogenicity of deletion mutants on banana plants.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.

Example 1

This example 1 provides a banana vascular wilt No. 4 minivariety pathogenic geneFOXG_01465The protein sequence coded by the gene is shown as SEQ ID NO: 1.

Example 2

This example provides a banana vascular wilt pathogenicity geneFOXG_01465The knockout of (2) and the construction of the gene deletion mutant thereof comprises the following steps:

1. culture conditions of banana vascular wilt No. 4 seed

The PDA culture medium preparation method comprises the following steps: peeling fresh potato 200-g, boiling with distilled water for 20-30 min, filtering to remove potato, adding 20-g glucose and 15-g agar into the filtrate, adding distilled water to volume 1L, and autoclaving at 121deg.C for 20 min. The banana wilt No. 4 seed is inoculated in PDA culture medium and cultured in constant temperature incubator at 28 deg.c. The YEPD formula is: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, agar 15 g/L. The banana vascular wilt No. 4 seed is inoculated into YEPD liquid culture medium and then cultured in shaking mode at 180 rpm and 28 ℃.

2. GeneFOXG_01465Acquisition of deletion mutants

(1) Preparation of knockout boxes: the knockout cassette for the target gene was constructed using split-marker PCR-based techniques. Obtained by PCR amplification using primers 465uf and 465ur (Table 1)FOXG_01465The 5 'flanking sequence 465u (250-500 bp) of the gene (Genbank: XM_ 018378304.1), and primers 465df and 465dr (Table 1) amplified the 3' flanking 465d (250-500 bp) of the gene. For the screening marker hygromycin phosphotransferase (HYG) gene (Genbank: HQ 412578.1), the "HY" and "YG" fragments were amplified from the full-length HYG gene using HphF/HYR and YGFHphR primers (Table 1), respectively, with overlapping sequences of about 400 bp. Primers 465ur and 465df, each containing a sequence reverse complementary to the HphF and HphR primers, respectively, at the 5' end. The sequences 465u and HY, YG and 465d were fused by overlap extension PCR to obtain the respective 4635 u_HY and YG_465d sequences (as shown in SEQ ID NO:2 and SEQ ID NO:3, the overlapping fragment had 315 bp). Finally, fragments of the respective total amounts 5-10 ug of the fragments of 463 u_HY and YG_465d were prepared by cleaning and recovering the template 463 u_HY and the primers 465uf and HYR, and the template YG_465d and the primers YGFand 465dr by PCR. PCR conditions: pre-denaturation at 98℃for 2 min, 30 cycles of denaturation at 98℃30 s, annealing at 60℃30 s, extension at 72℃30 s/kb, and final extension at 72℃for 2 min.

(2) Preparation of protoplasts

Washing PDA plate of 7 d banana wilt No. 4 seed with sterile water, scraping the surface of mycelium plate with sterile glass rod, collecting spores in the plate, preparing spore suspension for inoculation, adjusting concentration to 1×10 with sterile water ⁸ Each mL was then inoculated into YEPD medium, incubated at 20℃and 180 rpm for 12-16 h, germinated mycelia were collected by centrifugation, and the mycelia were washed at least 3 times with 1.2M potassium chloride (sterile) solution. 20 The mL protoplast formation solution was prepared by filtration through 0.8M magnesium sulfate, 1% (w/v) muramidase, 0.1% (w/v) crashase, and 0.2 μm sterile filtration membranes. The germinated mycelia were transferred to a protoplast formation solution, treated at 30℃and 100 rpm for 2-4. 4 h, and the protoplasts were observed under a microscope to account for at least 50% of the same field of view. The protoplasts were collected by filtration with absorbent cotton having a thickness of 2 cm, collected by centrifugation at 2500 rpm at 4℃and washed at least 3 times with 1.2M potassium chloride, and finally with STC solution (1.2M sorbitol, 10 mM Tris-HCl, pH8.0 and 50 mM CaCl) ₂ ) Protoplasts are washed and enriched. Split charging 200 μl to 1.5 mL EP tube, adding DMSO at final concentration 7%, quick freezing with liquid nitrogen, and storing at-80 ℃.

(3) Transformation and screening of protoplasts

200. Mu.L of protoplast solution was added to the upstream and downstream target gene knockout homology arms 4635 u_HY and YG_465d, and after ice bath for 25 min, PTC solution (40% w/v PEG4000,1 XSC) was added thereto, and the mixture was left at room temperature for 25 min. Transfer protoplast solution to 50 mL YEPD agar medium at 50-60 ℃, pour into 14 cm plate Petri-dish, make plate. When hyphae appeared on the plates, YEPD 50 mL agar medium +200. Mu.g/mL hygromycin was poured into the upper layers of the plates and single colonies were selected as double plates.

(4) Screening of mutants and isolation of mutant monospores

Single colony mycelium fragments are picked into 50 mu L of sterile water, the mixture is vibrated and evenly mixed, the mixture is heated in a metal bath at 100 ℃ for 10 min, the supernatant is obtained by centrifugation, and PCR is carried out by using primers 465u and HYR and primers YGFs and 465d in a PCR system of 1 mu L to 50 mu L, wherein the PCR conditions are as follows: pre-denaturation at 98℃for 2 min, 30 cycles of denaturation at 98℃30 s, annealing at 60℃30 s, extension at 72℃30 s/kb, and final extension at 72℃for 2 min. And (5) observing the target fragment by electrophoresis. The correct mutant was determined based on the single colony PCR identification.

Mutant hyphal fragments were picked with the tip of the toothpick onto PDA plates containing hygromycin at 200. Mu.g/mL. Collecting spores after 7 days, mixing with PDA culture medium to make plate, and picking up monospore to form colony to obtain gene FOXG_01465Deletion mutants.

Example 3

The present example provides genesFOXG_01465Is involved in the regulation and control of the pathogenicity of No. 4 race of banana vascular wilt.

1. GeneFOXG_01465Analysis of Effect of deletion on growth of No. 4 microspecies mycelium of Banana wilt

Banana wilt No. 4 minispecies and genes FOXG_01465The deletion mutants were cultured on PDA agar medium, respectively, and cultured in a constant temperature incubator at 28℃and after 7. 7 d, the growth of hyphae on PDA agar plates was observed.

Scraping hyphae from a PDA plate cultured at 28 ℃ for 7 d by using a sterile glass rod, immersing 2.5% glutaraldehyde at 4 ℃ overnight, pouring out a fixing solution, rinsing the sample three times with 0.1M phosphoric acid buffer solution with pH7.0 for 15 min each time, fixing the sample 1-2 h with 1% osmium acid solution, and rinsing the sample three times with 0.1M and pH7.0 phosphoric acid buffer solution for 15 min each time after taking out osmium acid waste liquid carefully; samples were dehydrated using gradient concentrations (30%, 50%,70%,80%,90% and 95%) of ethanol, each concentration for 15 min, and finally treated with 100% ethanol twice for 20 min each. The sample was treated with a mixture of ethanol and isoamyl acetate (V: v=1:1) for 30 min, then treated with pure isoamyl acetate, sample 1 h, critical point dried, and observed using a scanning electron microscope model HITACHI SU 8010.

As a result, as shown in FIG. 2, it was found that the gene was compared with banana vascular wilt No. 4 raceFOXG_01465Deletion mutants grew very slowly on PDA agar plates with thin hyphae. As observed by using a scanning electron microscope, in the graph 3, the original banana vascular wilt No. 4 mycelium with uniform thickness and few branches becomes uneven in thickness and obvious in branchesIncrease, indicating gene FOXG_01465Can influence the growth of hyphae of banana vascular wilt No. 4 race and change the form of the hyphae.

2. GeneFOXG_01465Analysis of the Effect of deletions on production of banana vascular wilt No. 4 microspores

Banana wilt No. 4 minispecies and genesFOXG_01465The deletion mutants were cultured in YEPD liquid medium, shaking culture was performed at 180 rpm and 28℃for 7 d, the fermentation broth was collected, and the fermentation broth was filtered with a sterile syringe containing absorbent cotton to obtain spore suspension, spores were observed under an optical microscope (40 Xobjective lens, 10 Xeyepiece lens), and the spore yield was calculated.

The results are shown in FIG. 4, genesFOXG_01465Comparison of deletion mutant strain with banana wilt No. 4 race shows that geneFOXG_01465The deletion mutant strain hardly produces spores, and the spore yield is caused by genesFOXG_01465Is significantly suppressed.

3. GeneFOXG_01465Analysis of Effect of deletions on pathogenicity of banana vascular wilt No. 4 race

No. 4 minispecies and genes of banana vascular wilt were scraped from PDA plates incubated at 28℃with 7 d using sterile glass rodsFOXG_01465Hypha 1 g of the deletion mutant was ground in 100 mL sterile water to prepare a bacterial suspension, then roots of banana seedlings (Musa sp. AAA Cavendish subgroup cv. Williams B6) with 4 leaves were immersed in the bacterial suspension for 3 h, then transferred to a plastic pot with 450 g sterile soil, irrigated with clear water every three days, and after 30 days the rootstock was transected and cut vertically to evaluate pathogenicity.

As a result, as shown in FIG. 5, banana vascular wilt No. 4 seed can cause yellowing of banana leaves and blackening of rootstock atrophy. And warp geneFOXG_01465Banana plants treated with deletion mutants did not show obvious banana wilt characteristics, thus inferring genesFOXG_01465 The deletion of (3) reduces the pathogenicity of the banana vascular wilt No. 4 race.

In summary, the present invention, in combination with the results of FIGS. 2-5, reveals the genes for the first timeFOXG_01465Function in banana vascular wilt No. 4 race and its role in pathogenesis and provideFOXG_01465Application ways and application technologies in developing small germplasm of No. 4 resistant to banana vascular wilt.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (2)

1. The application of the gene FOXG_01465 in reducing the pathogenicity of banana vascular wilt pathogen No. 4 race is characterized in that the encoded amino acid sequence is shown in SEQ ID NO: 1.

2. The application of the gene FOXG_01465 in reducing the spore yield of banana vascular wilt pathogen No. 4 race is characterized in that the encoded amino acid sequence is shown as SEQ ID NO: 1.