CN113897377B - Application of ethylene forming enzyme gene VdEFE in verticillium dahliae growth and development, pathogenicity and ethylene synthesis - Google Patents

Abstract

The invention provides an application of an ethylene forming enzyme gene VdEFE in verticillium dahliae growth and development, pathogenicity and ethylene synthesis, belonging to the technical field of functional genes. The invention researches the functions of VdEFE genes, obtains VdEFE gene knockouts, complements and overexpression bodies by utilizing the homologous recombination principle, and determines the function of VdEFE genes in the ethylene synthesis process and the relationship between VdEFE genes and EFE ethylene synthesis paths by determining the content of ethylene and succinic acid; the effect of VdEFE genes in the colony growth speed, the microsclerotium yield, the conidium yield and the pathogenicity of the verticillium dahliae is clarified through biological trait measurement and pathogenicity identification, the effect of ethylene in the interaction process of verticillium dahliae and cotton is deduced, a theoretical basis is provided for verticillium dahliae control, and a foundation is laid for further analysis of the interaction mechanism of verticillium dahliae and host plants.

Description

Application of ethylene forming enzyme gene VdEFE in verticillium dahliae growth and development, pathogenicity and ethylene synthesis

Technical Field

The invention belongs to the technical field of functional genes, and particularly relates to application of an ethylene forming enzyme gene VdEFE in verticillium dahliae growth and development, pathogenicity and ethylene synthesis.

Background

In 1984, verticillium dahliae (VerticilliumdahliaeKleb) was first reported to have the ability to produce ethylene during growth and interaction with hosts (fukuda et al, 1984). In 2016, zhang Ting et al found that both strong and weak pathogenic strains of Verticillium dahliae produced ethylene, and that the strong pathogenic strains produced significantly higher levels of ethylene than the weak pathogenic strains (Zhang Ting et al 2016). During the course of gene function studies, it was also found that some genes are linked to the production of ethylene. Gene deletion mutants of VGB and VdPKAC1 in verticillium dahliae can lead not only to reduced pathogenicity of pathogenic bacteria, reduced microsclerotium, but also to reduced amounts of ethylene biosynthesis (tzima et al, 2012; tzima et al, 2010). In 2017, zhangTing et al found that verticillium dahliae melanin synthesis-related gene VdPKS1, and that after the gene knockout, verticillium dahliae pathogenicity was reduced, melanin was not produced, and gene expression of VGB and VdPKAC1 was reduced, and further experiments found that after VdPKS knockout, ethylene biomass synthesis was reduced (zhangetal, 2017). In 2012, the Guo Huishan group found that VdNLP family genes encode necrosis and ethylene-inducing proteins, of which VdNLP and VdNLP encoded proteins also have exciton activity, which can activate the ethylene biosynthetic pathway and the SA, JA-dependent defensive signaling pathway (santhane, 2013; zhouetal, 2012). In 2019, tsolakidou found VdACCd gene encoding ACC transaminase (ACCDEAMINASE), through which the ethylene synthesis precursor ACC can be digested, the ACC content in the gene deletion mutant is increased, the virulence is reduced, and the difference in the production of ethylene is not obvious, and ACC is proposed to act as a signal molecule, participating in the interaction of verticillium dahliae with plants (tsolakidaou et al, 2019). However, it is not clear whether the ethylene forming enzyme gene VdEFE in Verticillium dahliae is involved in the pathway of ethylene synthesis.

Disclosure of Invention

In view of the above, the present invention aims to provide a new application of ethylene forming enzyme gene VdEFE, in particular VdEFE in verticillium dahliae growth, pathogenicity and ethylene synthesis.

The invention provides an application of an ethylene forming enzyme gene VdEFE in the biosynthesis of verticillium dahliae.

Preferably, the biosynthesis comprises ethylene synthesis and succinic acid synthesis.

The invention provides an application of an ethylene forming enzyme gene VdEFE in down regulating the expression quantity of genes related to the ethylene formation of verticillium dahliae.

The invention provides an application of an ethylene forming enzyme gene VdEFE in maintaining the growth and development of verticillium dahliae.

Preferably, the growth comprises growth rate and propagule yield.

Preferably, the propagules include microsclerotia and conidia.

Preferably, the nucleotide sequence of the ethylene forming enzyme gene VdEFE is shown in SEQ ID NO. 1.

The invention provides an application of an ethylene forming enzyme gene VdEFE in the biosynthesis of verticillium dahliae. To determine whether VdEFE gene knockout affects the ability of verticillium dahliae to produce ethylene, the ethylene content of the VdEFE gene knockout mutant was measured with wild type V592 as a control, and the results indicate that the ethylene produced by knockout delta vdefe is significantly lower than wild type V592, only 49.01% of V592. The recovery of ethylene content of the complement EC-vdefe was not significantly different from V592, whereas the overexpression of mutant OE-vdefe produced significantly higher ethylene than that of wild-type V592 and knockout Δ vdefe, which were 1.73 and 3.53 times that of V592 and vdefe, respectively. By adding the sole substrate of the EFE pathway, 2-oxoglutarate (OXO), in the medium, the results indicate that OXO enables all strains to produce more ethylene, and that the ethylene content of wild-type V592 is significantly higher than that of knockout delta vdefe under the induction of OXO. The VdEFE gene is described as being associated with ethylene formation and is a key gene in the EFE pathway. The succinic acid content of knockout delta vdefe was not significantly different from wild-type V592 and complement EC-vdefe, whereas the succinic acid content of over-expression OE-vdefe was significantly higher than that of wild-type V592 and other mutants, approximately 1.5 times that of wild-type V592, indicating that VdEFE gene was associated with Verticillium dahlia succinic acid formation and VdEFE gene was an ethylene forming enzyme gene.

The invention provides an application of an ethylene forming enzyme gene VdEFE in maintaining the growth and development of verticillium dahliae. Experiments prove that the growth rates of knockout bodies delta vdefe-1 and delta vdefe-2 are obviously reduced compared with the wild type. The growth rates of the complements EC-vdefe-1 and EC-vdefe-2 were restored, and the differences from the wild type were not obvious, which suggests that the knockdown of the VdEFE gene affects the growth rate of Verticillium dahliae. Microsclerotium yield assay, wild type V592 produced a significant amount of black microsclerotium, whereas knockout Δ Vdefe-1 and Δ Vdefe-2 formed significantly less black microsclerotium than wild type V592; the number of microsclerotium formed by the complements ECvdefe-1 and ECvdefe-2 and the wild type V592 was significantly recovered, which suggests that the VdEFE gene has a certain effect on the formation of microsclerotium of Verticillium dahliae. The conidiophoresis measurement results show that compared with the wild type V592 and the complementary strains ECvdefe-1 and ECvdefe-2, the sporulation amounts of knockout bodies delta vdefe-1 and delta vdefe-2 are obviously reduced, which shows that VdEFE genes are closely related to the sporulation capacity of the Verticillium dahliae.

Drawings

FIG. 1 is a phylogenetic tree of VdEFE and other microorganisms constructed based on ethylene-forming enzyme amino acid sequences;

FIG. 2 is a schematic representation of VdEFE gene knockout, complementation and over-expression verification; fig. 2A: vdEFE knockout vector construction strategy schematic diagram; fig. 2B: detecting the knockout of VdEFE genes at the genome level; DL2000 is Marker, CK is negative control; fig. 2C: verifying the transcript level of VdEFE genes by using RT-qPCR; * Representing significant differences compared to V592 by Duncan test (P < 0.01); fig. 2D: determining the transcript level of the VdEFE gene over-expression using RT-qPCR, the different lower case letters indicating significant differences (P < 0.05) by Duncan test;

FIG. 3 is a graph showing the determination of ethylene content of VdEFE mutants under non-induction conditions and under substrate OXO induction conditions; wherein the different lowercase letters indicate significant differences (P < 0.05) as tested by Tukey in the chaste medium treatment. The different capital letters indicate significant differences (P < 0.05) in the Tukey test in the treatment of the Charles medium with addition of OXO; * Indicating that the addition OXO treatment versus the non-addition treatment has a significant difference (P < 0.01) at a probability level of 0.01; error bars represent standard error of 3 repeated calculations;

FIG. 4 shows the detection of the succinic acid content of VdEFE gene mutants; different lowercase letters indicate significant differences (P < 0.05) as tested by Tukey;

FIG. 5 is the transcription level of ethylene formation-related genes in wild-type strains V592 and VdEFE gene knockouts; wherein sum is significantly higher than wild-type strain V592 at probability levels of 0.05 (P < 0.05), 0.01 (P < 0.01), respectively;

FIG. 6 is a phenotypic characterization of wild type V592, vdEFE knockout and complementation strains;

FIG. 7 shows the formation of microsclerotia in VdEFE gene knockouts; wherein fig. 7A: microsclerotium formation in non-vegetative conditions for all strains; fig. 7B: the wet weight and dry weight of microsclerotia produced by all strains were tested for significance using the Dunnett method; * Significantly lower than wild-type strain V592 at a probability level of 0.01 (P < 0.01);

FIG. 8 shows spore production and germination of VdEFE gene mutants; wherein fig. 8A: vdEFE gene mutant spore germination rate measurement; fig. 8B: vdEFE knockout strain produced spore amount determination, and x were significantly lower than wild type strain V592 at probability levels of 0.05 (P < 0.05), 0.01 (P < 0.01), respectively; fig. 8C: morphology observation of the sporulation peduncles of VdEFE gene mutants;

FIG. 9 is a graph showing the effect of VdEFE gene knockouts, complements and overexpression on Verticillium dahliae virulence; wherein FIG. 9A shows symptoms of wild type strain V592, vdEFE gene knockout, complementation and overexpression of cotton inoculated; fig. 9B: index of disease after onset of cotton under non-induced conditions.

Detailed Description

The invention provides an application of an ethylene forming enzyme gene VdEFE in the biosynthesis of verticillium dahliae.

In the present invention, the biosynthesis preferably includes ethylene synthesis and succinic acid synthesis. Experiments prove that the ethylene forming enzyme gene VdEFE not only participates in ethylene synthesis, but also has an effect on the expression of genes related to ethylene synthesis. Deletion of the ethylene forming enzyme gene VdEFE can up-regulate the expression level of the related ethylene forming genes of verticillium dahliae, and it can be seen that the expression relationship between the ethylene forming enzyme gene VdEFE and other related ethylene forming genes has antagonism. Ethylene formation-related genes preferably include VdNLP2, vdPKS, vdPKAC1 and VGB genes.

The invention provides an application of an ethylene forming enzyme gene VdEFE in maintaining the growth and development of verticillium dahliae.

In the present invention, the growth preferably includes a growth rate and a propagule yield. Experiments prove that the deletion of the ethylene forming enzyme gene VdEFE reduces the growth speed of the verticillium dahliae, which proves that VdEFE has the effect of regulating the growth speed of the verticillium dahliae; and VdEFE gene knockout does not affect the colony morphology of the verticillium dahliae on the PDA culture medium. The propagules preferably include microsclerotia and conidia. Experiments show that knockout delta vdefe-1 and delta vdefe-2 form significantly less black microsclerotia than wild-type V592. The number of microsclerotia formed by the complements EC-vdefe-1 and EC-vdefe-2 and the wild type V592 is obviously recovered; vdEFE gene knockout has the effect of reducing formation of microsclerotia. In terms of spore yield, the knockout bodies delta vdefe-1 and delta vdefe-2 had spore yields of only 75.01% and 74.56% of wild type V592, and the complementation strains EC-vdefe-1 and EC-vdefe-2 recovered to wild type levels, which were 98.67% and 102.68% of the spore yields of V592, respectively, indicating that VdEFE gene was closely related to Verticillium dahliae spore production. Meanwhile, the conidiophore of the knockout mutant is obviously reduced, the rotabranch is hardly generated, and the morphology of the conidiophore of the complementation strain is not obviously different from that of the wild type, which indicates that the reduction of the sporulation quantity of the VdEFE gene knockout mutant can be related to the reduction of the sporulation quantity of the conidiophore. Furthermore, the germination rate of the knockout Δ vdefe conidia was significantly improved compared to wild-type V592 and complement EC-vdefe, while the germination rate of the overexpressing OE-vdefe was minimal in all strains, indicating that the VdEFE gene was associated with verticillium dahliae spore germination.

The invention provides an application of ethylene forming enzyme gene VdEFE or ethylene in verticillium dahliae pathogenicity. Experiments prove that the disease condition of cotton seedlings inoculated with wild type, knocked-out body and complements is more serious, even the whole plant necrosis phenomenon occurs, the disease index reaches more than 95, the disease symptoms caused by cotton seedlings inoculated with over-expression bodies are obviously reduced, the disease index is only 55.21, and the effect index indicates that VdEFE gene over-expression leads to the reduction of the pathogenicity of verticillium dahliae. This suggests that VdEFE gene is involved in the pathogenic process of verticillium dahliae and that low concentration ethylene can enhance the pathogenicity of verticillium dahliae and high concentration ethylene inhibits the pathogenicity of verticillium dahliae.

The use of the ethylene forming enzyme gene VdEFE provided by the present invention in the growth and development of Verticillium dahliae, pathogenicity and ethylene synthesis will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.

Material reagent Source description

1 Test materials and primers

The Verticillium dahliae strong pathogenic lare strain V592 (see F Gao,Zhou B J,Li GY,et al.A Glutamic Acid-Rich Protein Identified in Verticillium dahliae from an Insertional Mutagenesis Affects Microsclerotial Formation and Pathogenicity[J].Plos One,2010,5.0.), which is separated from Xinjiang cotton in the laboratory and stored in an ultralow temperature refrigerator at-80 ℃ after being subjected to single spore purification), the test cotton variety is army cotton No. one (Gossypium hirsutum Linn.cv.Junmian No. 1), the T vector is pMD19-T vector (Takara), the knockout vector pGKO-HPT(Sheng W,Xing H,Hua C,et al.An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation(ATMT)-based gene disruptionmethod inVerticillium dahliae[J].Phytopathology,2016,106(6):645.) is a benefit of a Guo Huishan researchers of a Chinese sciences microorganism, the complementary vector and the over-expression vector p1300-Neo-oliC-Cas9-TtrpC, wherein the Kan resistance gene is used as a screening mark, the fungus promoter oliC and the terminator TtrpC are contained, the Escherichia coli DH5 alpha and the Agrobacterium EHA-105 are stored in the laboratory.

Primer design used in this chapter was performed using PRIMERPREMIER 6.0.0 and SnapGene, synthesized by general bioengineering (Anhui) Inc.

2 Conventional reagent, kit used and bioinformatics application software

The DNA extraction adopts a kit (BioFlux); gel recovery kit (OMEGA); common PCR was performed using 2X Taq PCR Mastermix from Beijing Tiangen Bio Inc.; homologous arm amplification was performed using I-5 ^TM XHigh-FIDELITY MASTER Mix High-fidelity DNA polymerase from New Biotechnology Co., ltd. Of Beijing qingke TsingKe; clonExpress II One Step Cloning Kit and ClonExpress MultiS One Step Cloning Kit used for In-fusion cloning were purchased from Vazyme company; restriction enzymes used in the experiments: pac I, xba I, bamH I and buffers were purchased from Takara. The conventional reagents are all of domestic analytical purity.

Sequencing results were analyzed by means of DANMAN and SnapGene. The amino acid sequence was subjected to treeing analysis using Mega 5.0. The conserved domains were predicted using InterPro (http:// www.ebi.ac.uk/InterPro /).

3 Medium and reagent preparation

Isolation of Verticillium dahliae V592 and VdEFE Gene mutants and determination of biological Properties Medium A Patato Dextrose Agar (PDA) solid medium was used; collecting verticillium dahliae spore suspension and mycelium, using Czapek liquid medium, using Luria-Bertani (LB) liquid medium for bacterial propagation, using IMAS liquid medium for ATMT conversion of verticillium dahliae conidium: IMAS to the medium was added 40mM acetosyringone.

The antibiotics and concentrations used for the experiments were as follows:

The concentration of cephalosporin (Cef) was 200. Mu.g/mL, of carbenicillin (Car) was 200. Mu.g/mL, of hygromycin (Hyg B) was 100. Mu.g/mL, of 5-fluorodeoxyuridine (F2 dU) was 20. Mu.g/mL, of geneticin (G418) was 50. Mu.g/mL, of timetin (Tim) was 200. Mu.g/mL, of ampicillin (Amp) was 50. Mu.g/mL, of kanamycin (Kan) was 100. Mu.g/mL, of rifampin (Rif) was 50. Mu.g/mL, of MES (2-N-Marin ethanesulfonic acid) was 40mmol/L, and of AS (acetosyringone) was 200mmol/L. The above antibiotics were all purchased from Shanghai Biotechnology services Inc.

Example 1

1. Construction method of over-expression vector, knockout vector and complementary vector

1.1 Cultivation of Verticillium dahliae and extraction of genomic nucleic acids

Cultivation of Verticillium dahliae V592: 200mL of the bacterial liquid is sucked by a pipette from the Verticillium dahliae conidium suspension stored at the temperature of minus 80 ℃ and evenly beaten on a PDA solid culture medium containing Kan, the mycelium is cultured in darkness for 5 days at the temperature of 22 ℃, and the mycelium which grows newly is transferred to a new PDA (containing Kan) plate and cultured in darkness for 5 days at the temperature of 22 ℃.

DNA extraction: referring to the method of Song et al (2019) (Song et al, 2019), the tissues and cells of V592 are thoroughly disrupted by cryogenic grinding. Genomic DNA was extracted with reference to DNA extraction kit instructions.

RNA extraction: using enzyme-free centrifuge tube and suction head, sterilizing the mortar and pestle in 121 deg.C autoclave for 2 times after DEPC water treatment, and oven drying the residual water, and pre-cooling the tool with small amount of liquid nitrogen or in 4 deg.C refrigerator before use. Trizol Reagent, chloroform, absolute ethanol, isopropanol and 75% ethanol used in the experiment should be used after precooling in a refrigerator at 4 ℃. Extracting total RNA of the verticillium dahliae V592 by using a Trizol (QIAGEN) method. The total RNA of Verticillium dahliae V592 was reverse transcribed into cDNA according to the procedure of the reverse transcription kit (TaKaRa) instructions.

1.2 Cloning of VdEFE Gene

The genome DNA of the strong pathogenic defoliation strain V592 is used as a template, and the primer pair VdEFE-F/VdEFE-R is used for amplifying the full length of VdEFE genes. The specific operation is as follows: PCR reaction System (20. Mu.L System): to the PCR tube, 10. Mu.L of 2X Taq PCR MasterMix, 0.5. Mu. L VdEFE-F, 0.5. Mu. L VdEFE-R, 1. Mu.L of template DNA was added, and 20. Mu.L was complemented with ddH ₂ O, and amplification was performed as follows: pre-denaturation at 94℃for 3min (1 cycle), denaturation at 94℃for 30s, annealing at 64℃for 30s, extension at 72℃for 90s, cycle to the extension stage for 30 times, constant temperature at 72℃for 10min, and retention at 20℃for 2min for termination of the reaction.

3-5 ML of PCR product is taken to be subjected to gel electrophoresis detection in a 1% agarose gel electrophoresis added with Goldenview, and is observed and photographed under an ultraviolet gel imager. The PCR products of the correct bands were selected and purified according to OMEGA Bio-company gel recovery kit, and the PCR purified products were ligated to the sequencing vector pMD 19-T. Through heat shock conversion, the recombinant vector is led into colibacillus DH5 alpha, positive clone is screened out by using a Maiconk culture medium, plasmid of the positive clone is obtained through an alkaline cracking method, and sequencing is carried out by using M13F/M13R as a primer, so as to obtain VdEFE gene full-length sequence.

And (3) purifying a PCR product: with reference to OMEGA biosystems gel recovery instructions, a slight modification is made: ① Adding a Binding Buffer equal to the PCR product; ② Mixing, and adding into an adsorption column, 700mL each time; ③ Placing the adsorption column in a centrifugal machine for 8,000r/min, and centrifuging at normal temperature for 30-60 s; ④ Discarding the waste liquid, adding 650mL SPWwashbuffer,8,000r/min, and centrifuging at normal temperature for 30-60 s; ⑤ Repeating the step ④, and centrifuging at the normal temperature of 12,000r/min for 2min; ⑥ Taking out the adsorption column, standing for 2min at room temperature, and transferring to a new sterile 1.5mL centrifuge tube; ⑦ Adding ElutionBuffer mL of the mixture into an adsorption column, and standing for 10min; ⑧ Centrifuging at normal temperature for 2min at 12,000 r/min; ⑨ And (5) measuring the concentration of the purified product.

Heat shock conversion: ① Taking out DH5 alpha competent cells stored at-80 ℃ and thawing the cells on ice for 10min; ② Adding 1mL of the connection product into competent cells, gently mixing, and ice-bathing for 20min; ③ Carrying out heat shock in a water bath at 42 ℃ for 1 min; ④ Rapidly placing the mixture on ice after heat shock, and carrying out ice bath for 3-5 min; ⑤ 500mL of LB liquid medium is added into the centrifuge tube; ⑥ Placing in a shaking table, shaking and culturing at 37deg.C and 200r/min for 1 hr; ⑦ Taking 100-200 mL of bacterial liquid, and uniformly beating on a flat plate containing antibiotics; ⑧ Placing the mixture into 37 ℃ for dark culture for 12 to 16 hours.

Alkaline cracking: ① Taking 1.5mL of bacterial liquid, placing the bacterial liquid into a 1.5mL centrifuge tube, centrifuging at the normal temperature of 12,000r/min for 2min, and discarding the supernatant; ② 200mL of P1 is added, and the sediment is resuspended; ③ 200mL of P2 is added, and the mixture is inverted and mixed evenly; ④ 200mL of P3 is added, and the mixture is turned upside down, so that flocculent precipitate appears; ⑤ Placing on ice, ice-bathing for 10min, and centrifuging at normal temperature of 12,000r/min for 10min; ⑥ Adding 600mL of supernatant into a new 1.5mL centrifuge tube, adding 500mL of isopropanol, mixing uniformly upside down, centrifuging at normal temperature of 12,000r/min for 10min, and discarding the supernatant; ⑦ Adding 650mL of 75% ethanol, washing the precipitate, centrifuging at normal temperature of 12,000r/min for 2-5 min, and removing all liquid; ⑧ 50mL of ddH ₂ O was added and stored at-20 ℃.

1.3 Obtaining of knockout vector

1.3.1 Amplification of homologous arms up and down of the VdEFE Gene

Using the full genome sequence of Verticillium dahliae published by NCBI, 2 pairs of primers were designed with about 1,000bp of each fragment on the upstream and downstream of VdEFE gene as homology arms (primer sequences are shown in Table 1). Genomic DNA of the Verticillium dahliae forced strain V592 was used as a template. The upstream and downstream homology arms of the VdEFE gene were amplified using I-5 ^TM Xhigh-FIDELITY MASTER Mix High-fidelity DNA polymerase, the upstream homology arm of the VdEFE gene was amplified by about 1249bp using primer VdEFE-up-F/VdEFE-up-R, and the downstream homology arm of the VdEFE gene was amplified by about 990bp using primer VdEFE-dn-F/VdEFE-dn-R. The PCR product was purified to obtain a desired gene fragment according to the procedure of purifying the PCR product of 1.2 and the concentration was measured.

1.3.2 Knockout vector linearization

Single cleavage was performed using Pac I restriction enzyme. The method comprises the following specific steps: mu.L of Pac I,10 mu L pGKO-HPT plasmid and 5 mu L Cutsmart were taken, 50 mu L of the plasmid was complemented with ddH ₂ O, the plasmid was digested for 12 to 16 hours in a water bath at 37 ℃, detected by gel electrophoresis, and the target gene fragment was purified and the concentration was determined according to the procedure of PCR product purification in 1.5.

1.3.3In-fusion cloning

The purified homology arm fragment and the linearization vector are connected according to the following system: taking 0.5 mu L of upstream homology arm fragment, 0.5 mu L of downstream homology arm fragment, 5 mu L of linearization vector, 1 mu L Exnase Multi,2 mu L CE Multi Buffer, supplementing 10 mu L with ddH ₂ O, connecting the connecting product with DH5 alpha competent heat shock for 30min in a water bath kettle at 37 ℃, screening on a LB+Kan flat plate, selecting positive clone, extracting plasmid, and obtaining knockout vector pGKO-HPT: vdEFE.

1.4 Complementation vector and over-expression vector acquisition

The method is as follows, 1.2, taking the genome DNA of the strong pathogenic deciduous strain V592 as a template, amplifying VdEFE full length gene (the primer sequence is shown in table 2-1) by using I-5 ^TM X High-FIDELITY MASTER Mix High-fidelity DNA polymerase and primer VdEFEcds-F/VdEFEcds-R, and purifying PCR product by using a purification kit; the complementary vector is subjected to double digestion by using restriction enzymes Xba I and BamH I, the purified homologous arm fragments are connected with a linearization vector by ClonExpress II One Step Cloning Kit, the connection product is subjected to DH5 alpha competent heat shock conversion, positive clones are selected on a LB+Kan plate, plasmids are extracted, and the complementary vector and the over-expression vector p1300-Neo-oliC-VdEFE-TtrPC are obtained.

TABLE 1 test primers

1.5 Electric shock transformation of Agrobacterium

① Taking out the agrobacteria EHA-105 competent cells preserved at the temperature of minus 80 ℃ and thawing on ice for 10min; ② Adding 1 μl of recombinant plasmid, ice-bathing for 20min, and pre-cooling the electric shock cup on ice; ③ Adding the mixture into a precooled electric shock cup, wiping the outer wall of the electric shock cup, and shocking for 5ms at 1800V; ④ Adding 500mL of LB non-antibiotic liquid culture medium into the electric shock cup, uniformly mixing, and then transferring to a new 1.5mL centrifuge tube; ⑤ Shake culturing at 28 deg.c for 1 hr/min and 150-200 r/min; ⑥ Sucking 200 mu L of bacterial liquid, and coating the bacterial liquid on a solid LB+Kan+Rif plate; ⑦ Culturing in dark at 28 deg.c for 2-3 d; ⑧ The positive transformants were screened, added to 5. Mu.L of LB+Kan+Rif liquid medium, and shake cultured at 28℃for 16h at 150 r/min.

1.6 Agrobacterium-mediated genetic transformation of Verticillium dahliae conidia

1.6.1 Collection of Verticillium dahliae conidia

Several fresh pieces (PDA medium inoculated with strain) with a diameter of 5mm were inoculated in Czapek-Dox liquid medium at 26℃at 200r/min and cultured in darkness for 3d. Filtering the bacterial liquid with 4 layers of sterilized gauze into a 50mL centrifuge tube, centrifuging at 8000r/min and 4 ℃ for 10min, discarding the supernatant, re-suspending with 20mL of ultrapure water for 3 times, centrifuging, discarding the supernatant; spores were resuspended in 10% glycerol and adjusted to a concentration of 1X 10 ⁸ CFU/mL, dispensed into 2mL EP tubes, snap frozen with liquid nitrogen and stored in a-80℃freezer.

1.6.2 Agrobacterium-mediated genetic transformation of Verticillium dahliae conidium (ATMT transformation)

① 200 Mu L of agrobacteria liquid after electric shock is inoculated into 10mL IMAS liquid culture medium; ② Culturing at 28deg.C for 12h at 150r/min under shaking to OD ₆₀₀ of about 0.5; ③ Putting the collected conidia of the verticillium dahliae on ice in advance for thawing, and uniformly mixing the conidia of the verticillium dahliae and agrobacterium according to the ratio of 1:1; ④ Culturing the mixed bacterial liquid at 28 ℃ for 10min under shaking at 200 r/min; ⑤ Uniformly coating 200 mu L of bacterial liquid on IMAS solid culture medium with filter paper, and culturing in dark at 22 ℃ for 2d; ⑥ Removing filter paper, spreading on PDA+Car+Cef+5F2U+HyB solid medium (knockout carrier) or PDA+Car+Cef+ Timetin +G418 solid medium (complementary carrier), and culturing at 22deg.C in dark for 7d; ⑦ White spots on the plates are picked, streaked on a PDA+Car+Cef+5F2U+HyB solid medium (knockout carrier) or a PDA+Car+Cef+G418 solid medium (complementary carrier), and placed at 22 ℃ for 7d of dark culture; ⑧ Picking mycelium or microsclerotium of the transformant, streaking on a solid culture medium of PDA+Kan, and culturing in darkness at 22 ℃ for 7d; ⑨ The obtained transformants were screened.

1.7 Selection of VdEFE knockout and obtaining of overexpression

Extracting the DNA of each transformant according to the method of 1.2, and amplifying the target gene in the transformant by using primers VdEFE-F and VdEFE-R (primer sequences are shown in Table 1), and detecting whether it is successfully knocked out or complemented; meanwhile, primers HPH-F and HPH-R are used for amplifying hygromycin resistance genes, and whether the hygromycin resistance genes replace target genes in the knocked-out transformant is detected. The total RNA of wild-type V592 and mutant strains was extracted according to the method of 1.1 and reverse transcribed into cDNA, and the target gene was analyzed by real-time fluorescent quantitative PCR using primers VdEFE-q-F and VdEFE-q-R to detect the expression of the target gene at the transcription level, and the reference gene used was beta-tubulin (DQ 266153) gene. Relative quantification was performed using the 2 ^-△△Ct method and data statistical analysis was performed using SPSS.

2. Results

2.1 Cloning and sequence analysis of Verticillium dahliae VdEFE Gene

The full length of VdEFE gene obtained by amplification of primer pair VdEFE-F/VdEFE-R is 1134bp by taking Verticillium dahliae strong pathogenic strain V592 as a template, and sequence analysis shows that VdEFE gene contains 4 exons and 3 introns. Predicting the VdEFE gene conservation domain, wherein the VdEFE gene codes for the amino acid at 37-364, and an isopenicillin N synthase and dioxygenase domain (IsopenicillinN synthase andrelateddioxygenases, IPNS) belonging to the PcbC superfamily exists; there is a diox_n domain at positions 46-156 that is the highly conserved N-terminal region of 2-oxoglutarate/iron (II) -dependent dioxygenase active protein (HAGEL ANDFACCHINI, 2010); there is a glutarate/iron dependent dioxygenase domain (Oxoglutarate/iron-DEPENDENT DIOXYGENASE, fe (II) 2OG dioxygenase) at positions 199-313. In plant gene function studies, diox_n domain and Fe (II) 2OG dioxygenase domain were found to be key domains for ACC oxidase (ACO) to synthesize ethylene (Houben andVan De Poel, 2019). The above results therefore indicate that VdEFE gene may be associated with ethylene formation.

The amino acid sequence encoded by the VdEFE gene was aligned and analyzed for a VdEFE amino acid sequence having 99.47% similarity to homologous protein VdEFE in verticillium dahliae vdls.17 (v.dahliae vdls.17), a higher amino acid sequence similarity to homologous proteins in other fungi and bacteria, such as 92.99% similarity to verticillium gracile (v.alfalfaevams.102), 70.20% similarity to fusarium oxysporum (f.oxysporum), 60.19% similarity to anthrax graminis (Colletotrichum graminicola), 62.34% similarity to penicillium digitatum (p.digitatumpd 1), 61.54% similarity to aspergillus clavatus (Aspergillus clavatus), 52.46% similarity to alternaria (ALTERNARIA ALTERNATA), and 44.90% similarity to pseudomonas syringae (Pseudomonas syringae).

The result is shown in figure 1, fungi and bacteria form 2 large evolutionary branches respectively, the fungi are further divided into 4 branches, alternaria fungi form one evolutionary branch, penicillium fungi form one evolutionary branch, anthrax and Pyricularia oryzae and other fungi form one evolutionary branch, and Verticillium and Fusarium and other soil facsimile bacteria form one evolutionary branch, which indicates that VdEFE has the closest relation with the soil facsimile bacteria in evolution.

2.2VdEFE knockout and overexpression obtaining and identification

By using the principle of homologous recombination (figure 2A), 2 knockout body strains delta vdefe-1 and delta vdefe-2 obtained by carrying out ATMT genetic transformation on the knockout vector and the wild type V592 are extracted, and genome DNA is extracted for identifying homologous recombination transformants. From the DNA level, the mutant DNA was PCR amplified using 2 pairs of primers VdEFE-F/VdEFE-R and HPH-F/HPH-R, respectively, and the knockout mutant amplified only a 600 bp-sized HPH gene fragment, but not 1134 bp-sized VdEFE gene, compared to the wild-type strain, indicating successful substitution of the gene by the HPH gene (FIG. 2B). The complement strains EC-vdefe-1 and EC-vdefe-2 can amplify the 1134 bp-sized VdEFE gene, indicating the complementation success of the gene (FIG. 2B). Transcription level measurements of the knockout mutants and complements showed that the transcription levels of knockout mutants Δ vdefe-1 and Δ vdefe-2 were almost 0 and that the transcription levels of the complementation mutants ECvdefe-1 and ECvdefe-2 were restored to 95.30% and 95.95% of V592 (FIG. 2C). The over-expression vector and the wild type V592 are subjected to ATMT genetic transformation to obtain an over-expression body, and 2 over-expression mutants with higher expression level are selected and named OEvdefe-1 and OEvdefe-2 through RT-qPCR analysis (figure 2D). The above results demonstrate that VdEFE gene was successfully knocked out to obtain knock-out mutants Δ vdefe-1 and Δ vdefe-2, and the VdEFE gene was complementarily and overexpressed to obtain complements ECvdefe-1 and ECvdefe-2, and overexpressers OEvdefe-1 and OEvdefe-2.

Example 2

Ethylene content determination

To determine the ethylene yield of VdEFE gene mutants and wild type V592, 1mL of the conidium suspension at a concentration of 1.0X10: 10 ⁸ cfu/mL was beaten into 250mL injection glass flasks containing 100mL of Charles liquid medium, 26℃at 200r/min, and shake-cultured in the dark for 9d. Before ethylene measurement, the rubber plug of the injection glass bottle is opened for 5min in an ultra-clean workbench to remove the generated ethylene, and then the rubber plug is used for sealing; in the substrate induction experiment, the plug of the injection glass bottle was opened for 5min in an ultra clean bench, 20mM OXO was added, and then sealed with a rubber plug. After 3-4 hours of culture, the headspace air is collected by a syringe.

Ethylene standard gas was purchased from Shanghai Wei invasive standard gas analysis Co. Ethylene content in a 10mL air sample was measured using 7890B Agilent gas chromatograph equipped with flame ionization detector and chromatographic column HP-PLOT/Q (30 m 0.53mm 40 mm), nitrogen as carrier gas, inlet temperature 120 ℃, gas split ratio 4:1, column box temperature 100 ℃, detector temperature 160 ℃, total run time 10min, ethylene peak detected at 6-7 min. The test was repeated 3 times.

VdEFE mutant ethylene yield determination results

To determine whether VdEFE gene knockout affects the ability of verticillium dahliae to produce ethylene, the ethylene content of the VdEFE gene knockout mutant was measured with wild type V592 as a control, and the results indicate that the ethylene produced by knockout delta vdefe is significantly lower than wild type V592, only 49.01% of V592. The recovery of ethylene content of the complement EC-vdefe was not significantly different from V592, whereas the overexpression of mutant OE-vdefe produced significantly higher ethylene than that of wild-type V592 and knockout Δ vdefe, which were 1.73 and 3.53 times that of V592 and Δ vdefe, respectively. To further demonstrate that VdEFE gene is a key enzyme gene on the EFE pathway, the sole substrate of the EFE pathway, 2-oxoglutarate (OXO), was added to the medium. As a result, OXO was found to allow all strains to produce more ethylene, and the ethylene content of wild-type V592 was significantly higher than that of knockout Δ vdefe under OXO induction (fig. 3). The VdEFE gene is described as being associated with ethylene formation and is a key gene in the EFE pathway.

Example 3

Succinic acid content determination

Succinic acid detection was carried out with reference to national standard GB/T5009.157-2003, with some modifications. 1mL of the conidium suspension having a concentration of 1.0X10 ⁸ CFU/mL was beaten into a 250mL injection glass bottle containing 100mL of Charles liquid medium, and was shake-cultured in the dark at 26℃for 9d at 200 r/min. Spores were collected according to the above method, and the collected spores were crushed by an ultrasonic cytoclasis apparatus, and the cell sap was taken for measurement.

Succinic acid content measurement results

Ethylene forming enzymes are multifunctional enzymes that catalyze 2 reactions simultaneously, which in turn catalyze the formation of ethylene and the production of succinic acid (fukuda et al, 1992). To further determine whether VdEFE gene was an ethylene forming enzyme gene, wild type V592 and succinic acid of all mutants were tested by HPLC. As a result, it was found that the succinic acid content of the knockdown body vdefe was not significantly different from that of the wild-type V592 and the complement EC-vdefe, whereas the succinic acid content of the over-expression body OE-vdefe was significantly higher than that of the wild-type V592 and other mutants, about 1.5 times that of the wild-type V592 (FIG. 4). The VdEFE gene was described as being associated with the formation of verticillium dahliae succinic acid, and the VdEFE gene was an ethylene forming enzyme gene.

Example 4

Analysis of expression of ethylene formation-related Gene in mutants

Total RNA was extracted from all mutants and reverse transcribed into cDNA according to the method described in example 1. qPCR detection was performed using the primers of Table 2, the reference gene used was the beta-tubulin gene, and the ethylene-related gene used for the expression level measurement included ：VdNLP2(VDAG_01995.1),VdPKS 1(VDAG_00190),VdPKAC1(VDAG_06474.1),VGB(JQ665433.1),VdNLP1(VDAG_04701.1),VdACS(VDAG_05021.1),VdACCd(VDAG_10392).

TABLE 2 test primers

Results of expression level of ethylene Gene involved in formation of VdEFE Gene knockouts

To further determine whether VdEFE gene knockouts affected expression of other ethylene formation-related genes. Transcription levels of genes related to ethylene formation in wild-type strains V592, vdEFE knockout mutants and complements were detected using RT-qPCR. As a result, it was found that the relative expression amounts of VdNLP, vdPKS, vdPKAC and VGB genes in the 2 deletion mutants were all significantly higher than that of the wild-type V592 by 14.1-fold, 4.5-fold, 1.5-fold and 1.9-fold, respectively. The amount of the VdNLP gene expressed in the 2 deletion mutants was not significantly different from that of the wild-type V592 (fig. 5). The VdEFE genes are described as affecting the expression of VdNLP, vdPKS, vdPKAC1 and VGB genes.

Example 5

Determination of biological Properties of 1VdEFE mutant

The colony growth rate, the phenotype, the spore yield and the microsclerotia content of the VdEFE gene mutant are measured by referring to a measuring method (Wang Chunqiao, etc. 2019) of the phenotype characteristics of fungi such as Wang Chunqiao (2019). Besides the biological property measurement, the spore yield morphology and spore germination rate of VdEFE gene mutant are also measured.

1.1 Morphology observation of the spore-forming stems

On a solid culture medium of PDA+Kan, 5 aseptic cover slips are obliquely inserted at 45 degrees, mycelia of the strain are picked up by using aseptic toothpicks, and the mycelia are streaked on the junction of the slide and the culture medium, and are cultured in darkness for 5 days at 22 ℃. From 3d, 1-2 slides per day were taken and the morphology of the sporophores of the wild type and mutant was observed by electron microscopy (Olinbas).

1.2 Determination of spore germination Rate

To 40mL of Charles liquid medium, 400. Mu.L of spores at a concentration of 1X 10 ⁸ CFU/mL, 26℃and 200r/min were added, and the culture was performed with shaking for 12 hours, and the measurement was performed every 2 hours. The number of germinated spores in 100 spores was counted by an optical microscope, and the spore germination rate was measured.

1.3 Determination of pathogenicity

Referring to the method of peak and the like (2010) (Gao et al, 2010), a high-sensitivity cotton variety "army cotton No. 1" is selected, and a root dipping method without injury is adopted to detect the pathogenic condition. Uniformly spreading sterilized cotton seeds in sterile vermiculite, soaking the vermiculite in sterile water, and culturing to obtain cotton seedlings.

And (3) transferring the cotton seedlings to a water culture box, adding Hoagland's culture solution for culture, inoculating when the third true leaves of the cotton seedlings grow out, and inoculating with a conidium suspension with the concentration of 1X 10 ⁶ CFU/mL, wherein the bacterial liquid for each pot is 200mL, and the inoculation time is 2h. After 24h, the cotton seedlings were cultivated with one tenth of Hoagland's broth. After inoculation, the occurrence of diseases of cotton seedlings is observed every day, and the occurrence of diseases of cotton seedlings is referred to the grading standard of Song et al (2019). The disease index is calculated according to formula I.

Disease index = [ Σ number of patients at each stage x number of stages/(total number of patients x highest disease stage) ]x100

Each strain was repeated 3 times and the disease index was an average of 3 times.

To determine the effect of ethylene in the interaction of verticillium dahliae and cotton, cotton seedlings were treated with the agent, 200mL ACC or CDA at a concentration of 200mM was added to the hydroponic cassette, root soaked for 8h, and after 24h clear water was exchanged, inoculation experiments were performed.

Results

2VdEFE Gene knockout biological trait

2.1 Observation of growth phenotype and growth Rate of VdEFE Gene knockout Strain

To determine whether VdEFE gene knockouts affected the colony growth phenotype and growth rate of verticillium dahliae, 2 knockouts and V592 were inoculated on PDA medium respectively with wild type V592 strain as a control for culture, and the colony morphology was observed (fig. 6). The colony morphology of the knockout mutants vdefe Δ VdEFE-1 and vdefe Δ VdEFE-2 is not obviously different from that of the wild type V592 when 14d is cultured on the PDA culture medium, and the result shows that VdEFE gene knockout does not influence the colony morphology of the verticillium dahliae on the PDA culture medium.

The colony growth rate on PDA was measured and showed a significant decrease in the growth rate of knockouts Δ vdefe-1 and Δ vdefe-2 compared to the wild type. The growth rates of the complements EC-vdefe-1 and EC-vdefe-2 were restored with no significant differences from the wild type (as in Table 3). It was demonstrated that the knockout of VdEFE gene affects the growth rate of Verticillium dahliae.

TABLE 3 colony growth rate of VdEFE knockout mutants

2.2 Microsclerotia assay of VdEFE Gene knockout Strain

To determine whether VdEFE genes were involved in the generation of verticillium dahliae microsclerotium, equal amounts of conidia of wild type V592, knockout mutant, complement were cultured on cellophane-plated PDA solid medium. At 14d, wild-type V592 produced a large amount of black microsclerotia, while knockout Δ vdefe-1 and Δ vdefe-2 formed significantly less black microsclerotia than wild-type V592. The numbers of microsclerotia formed by the complements EC-vdefe-1 and EC-vdefe-2 with wild-type V592 were significantly restored (FIG. 7). The VdEFE gene has a certain influence on the formation of verticillium dahliae microsclerotium.

2.3 Sporulation measurements of VdEFE Gene knockout strains

To determine whether VdEFE gene knockouts affected the sporulation of Verticillium dahliae, sporulation results indicated that sporulation was significantly reduced for knockouts Vdefe delta vdefe-1 and Vdefe delta vdefe-2 compared to wild-type V592 and complementation strains EC-vdefe-1 and EC-vdefe-2. The spore yield of all strains increased faster 5d before inoculation. At 6d, spore production of all strains began to become slow. At 8d, the sporulation amounts of knockouts Δ vdefe-1 and Δ vdefe-2 were only 75.01% and 74.56% of the sporulation amounts of wild-type V592, with complementation strains EC-vdefe-1 and EC-vdefe-2 reverting to wild-type levels, 98.67% and 102.68% of the sporulation amounts of V592, respectively (FIG. 8B). The VdEFE gene is closely related to the spore production of Verticillium dahliae.

Microscopic observation of the sporophores produced by each strain revealed that the knockdown mutant showed significantly reduced sporophores compared to the V592 strain, almost no wheel-like branching, and no significant difference in the morphology of the conidiophores of the complementation strain from the wild type (fig. 8C). It was demonstrated that VdEFE gene knockout mutants may be associated with decreased sporulation.

2.4 Determination of spore germination Rate of VdEFE Gene knockout Strain

The germination rate of the conidia of each strain is observed, and the result shows that the conidia of all strains begin to germinate by shaking in a Charles liquid culture medium for 2 hours, and the maximum level is reached for 10 hours. After 6h of shaking culture, the knockdown Δ vdefe conidia germination rate was 42%, while the wild type V592 and the complement EC-vdefe conidia germination rates were 24%, 28%, respectively. After 10h, the germination rate of knockout Δ vdefe was 56%, that of wild type V592 was 54% and that of complement EC-vdefe was 58%. Whereas the germination rate of the over-expression OE-vdefeE was the lowest in all strains, the germination rate was only 14% after 6h of culture in Charles liquid medium. After 10h, the germination rate of OE-vdefe was 25% (FIG. 8A). The VdEFE gene was described as being involved in the germination of Verticillium dahliae spores.

2.5VdEFE identification of the pathogenicity of the knockout mutant

In order to determine the role of VdEFE genes in verticillium dahliae pathogenicity, pathogenicity of VdEFE gene knockouts on "army cotton number 1" was determined with wild type V592 as a control. As shown in fig. 9: after 10d inoculation, cotton seedlings inoculated with wild type, knocked-out body and complements begin to show leaf yellowing and wilting symptoms, and the symptoms of the inoculated overexpression body are not obvious; leaf yellowing and wilting symptoms begin to appear when the overexpression body is inoculated for 16 d. Shows that VdEFE gene over-expression has a delay effect on the onset of cotton verticillium; after 20d inoculation, the disease condition of cotton seedlings inoculated with wild type, knocked-out body and complemented body is more serious, even the whole plant necrosis phenomenon occurs, the disease index reaches more than 95, the disease symptoms caused by cotton seedlings inoculated with over-expression bodies are obviously relieved, the disease index is only 55.21 (figure 9A), and the result shows that VdEFE gene over-expression leads to the reduction of the pathogenicity of verticillium dahliae. From the disease index observation, the disease index of the knockdown body reaches 65.27 when inoculated for 12d, the disease indexes of the wild type and the complement are 43.51 and 24.17 respectively, and the disease index of the over-expression body reaches only 7.85 (figure 9B) at the lowest, which indicates that VdEFE gene knockdown accelerates the pathogenicity of the verticillium dahliae. The VdEFE gene is involved in the pathogenic process of Verticillium dahliae. The low concentration of ethylene can enhance the pathogenicity of the verticillium dahliae, and the high concentration of ethylene can inhibit the pathogenicity of the verticillium dahliae.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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Claims (4)

1. The application of the ethylene forming enzyme gene VdEFE in regulating and controlling the growth speed of the verticillium dahliae is characterized in that the deletion of the ethylene forming enzyme gene VdEFE reduces the growth speed of the verticillium dahliae; the nucleotide sequence of the ethylene forming enzyme gene VdEFE is shown as SEQ ID NO. 1.

2. The application of the ethylene forming enzyme gene VdEFE in regulating and controlling the formation of the microsclerotium of verticillium dahliae is characterized in that the knockout of the ethylene forming enzyme gene VdEFE has the effect of reducing the formation of the microsclerotium; the nucleotide sequence of the ethylene forming enzyme gene VdEFE is shown as SEQ ID NO. 1.

3. The application of the ethylene forming enzyme gene VdEFE in regulating and controlling the yield of the verticillium dahliae conidium is characterized in that the conidium yield is reduced after the ethylene forming enzyme gene VdEFE is knocked out; the nucleotide sequence of the ethylene forming enzyme gene VdEFE is shown as SEQ ID NO. 1.

4. The application of ethylene forming enzyme gene VdEFE in regulating and controlling verticillium dahliae pathogenicity, which is characterized in that the overexpression of ethylene forming enzyme gene VdEFE leads to the reduction of verticillium dahliae pathogenicity; the nucleotide sequence of the ethylene forming enzyme gene VdEFE is shown as SEQ ID NO. 1.