CN116751803B - Application of VdCreC gene in verticillium dahliae growth, pathogenicity and carbon metabolism inhibition - Google Patents
Application of VdCreC gene in verticillium dahliae growth, pathogenicity and carbon metabolism inhibition Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention is suitable for the technical field of functional genes, and provides an application of VdCreC genes in the growth of verticillium dahliae. Provides the application of VdCreC genes in the pathogenicity of verticillium dahliae. Provides the application of VdCreC genes in the carbon metabolism inhibition of verticillium dahliae. A medicine for preventing and treating cotton diseases is disclosed, which contains the reagent for down-regulating VdCreC gene or protein expression. The invention proves that VdCreC not only plays an important role in inhibiting carbon metabolism, but also influences the growth and the pathogenicity of the verticillium dahliae, has important theoretical value for preventing and controlling the verticillium dahliae and developing a novel bactericide, and has important practical significance for promoting the safe production of cotton.
Description
Technical Field
The invention belongs to the technical field of functional genes, and particularly relates to application of VdCreC genes in verticillium dahliae growth, pathogenicity and carbon metabolism inhibition.
Background
The verticillium dahliae (Verticilliumdahliae) belongs to the fungus of the phylum Verticillium of the phylum Deuteromycotina, contains a plurality of degrading enzymes (Cazymes) for degrading plant cell wall components, including pectinases, cellulases, amylases and the like, and generates different carbohydrate hydrolases according to the polysaccharide components of the cell wall of a host through long-term co-evolution with the host, so that the functions of the verticillium dahliae are endowed with the characteristic of infecting plants by the verticillium dahliae, and the pathogenic capability of the verticillium dahliae on the plants is directly influenced by some genes for encoding the carbohydrate hydrolases. Carbon metabolism inhibition (Carbon Catabolite Repression, CCR) is used as a global regulatory factor to participate in regulating the expression of a plurality of carbohydrate hydrolase genes, carbon metabolism inhibition in fungi such as saccharomyces cerevisiae and aspergillus nidulans is involved in pathogenic force of pathogenic bacteria, and in addition, the carbon metabolism inhibition genes are involved in the energy metabolism, normal growth and development, carbohydrate metabolism and other cellular processes of the fungi, so that the pathogenicity of verticillium dahliae can be directly influenced when the carbohydrate hydrolase related genes are destroyed.
In order to study the function of the carbon metabolism inhibitor genes of Verticillium dahliae, 1 carbon metabolism inhibitor gene was found by searching the Verticillium dahliae genome database under the gene number VDAG-05549, which was designated VdCreC.
Disclosure of Invention
The embodiment of the invention aims to provide an application of VdCreC genes in the growth of verticillium dahliae and aims to solve the problems in the background technology.
The embodiment of the invention is realized in such a way, and the VdCreC gene is applied to the growth of the verticillium dahliae.
Preferably, the growth comprises growth rate, propagule yield.
Preferably, the propagules include microsclerotia and conidia.
It is a further object of embodiments of the present invention to provide the use of VdCreC genes in verticillium dahliae pathogenicity.
Preferably, the pathogenicity is expressed on one or several of the following indicators: index of disease, hyphal penetration ability, and host colonization ability.
It is yet another object of an embodiment of the present invention to provide the use of VdCreC genes in the inhibition of carbon metabolism in verticillium dahliae.
Preferably, the VdCreC gene is involved in glucose-induced inhibition of carbon metabolism by Verticillium dahliae.
It is yet another object of an embodiment of the present invention to provide a drug for controlling cotton diseases, comprising a reagent for down-regulating VdCreC gene or protein expression.
According to the embodiment of the invention, the expression of VdCreC genes in different times and different tissues is analyzed, and the result shows that the expression quantity of VdCreC genes in 12d and hypha is highest in culture of wild strain V592 in a PDA solid culture medium, a knockout carrier taking VdCreC as a target gene is constructed according to the homologous recombination principle, conidia of cotton verticillium V592 strain are transformed by an agrobacterium-mediated genetic transformation method (ATMT), and 2 VdCreC gene knockout mutants are obtained by screening; meanwhile, an over-expression vector taking VdCreC genes as targets is constructed, and 2 VdCreC over-expression body strains are obtained through screening. Compared with wild strain V592 and over-expressed strain, the VdCreC knockout strain has reduced aerial hypha, reduced growth rate, spore yield, spore germination rate and microscler yield, and reduced pathogenicity to cotton;
The carbon source utilization result shows that VdCreC promotes the utilization of glucose, sucrose, lactose, raffinose and pectin, inhibits the metabolism of xylan, does not influence the utilization of galactose, carries out a carbon metabolism inhibition test on a VdCreC gene knockout mutant, and after glucose is added into a culture medium taking cellulose and starch as carbon sources, the inhibition rate of the VdCreC knockout mutant on the utilization of the cellulose and the starch is obviously lower than that of a wild strain V592, thus indicating that VdCreC genes participate in the carbon metabolism inhibition caused by the glucose;
In conclusion, vdCreC genes influence the growth and the pathogenicity of verticillium dahliae and play an important role in inhibiting carbon metabolism.
Drawings
FIG. 1 is a colony morphology of the Verticillium dahliae delta VdCreC mutant, complementation strain and wild-type strain V592 provided in example 3;
FIG. 2 is a microscopic observation of microsclerotium formation of the Verticillium dahliae delta VdCreC mutant, the complementation strain and the wild-type strain V592 provided in example 4;
FIG. 3 shows the sporulation measurement results of the Verticillium dahliae delta VdCreC mutant, the complementation strain and the wild-type strain V592 provided in example 5;
FIG. 4 is a microscopic observation of the pedicel-producing strain of Verticillium dahliae Δ VdCreC mutant, complementation strain and wild-type strain V592 provided in example 5;
FIG. 5 shows the pathogenicity determination of Verticillium dahliae delta VdCreC mutant, complementation strain and wild strain V592 on cotton provided in example 6 (upper panel is pathogenicity plant morphology; lower panel is disease index result);
FIG. 6 is a graph showing the results of measurement of the penetration ability of the hyphae of Verticillium dahliae delta VdCreC mutant strain, complementation strain and wild-type strain V592 to cotton provided in example 7;
FIG. 7 shows the phenotype of the Δ VdCreC gene mutant provided in example 8, the inhibition of 9d on different carbon source media;
FIG. 8 is an amylase and cellulase activity assay for the delta VdCreC gene mutant strain provided in example 9.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the embodiment of the invention, the nucleotide sequence of the VdCreC gene is shown as SEQ ID NO. 1.
In order to determine whether VdCreC gene knockout affects formation of verticillium dahliae conidium, the invention constructs a verticillium dahliae mutant strain delta VdCreC for knocking out VdCreC genes on a wild type strain V592 as a basic strain, and carries out gene overexpression on the basis of the wild type strain V592 to obtain two over-expression strains OE-VdCreC-1 and OE-VdCreC-2, wherein the construction method of the verticillium dahliae mutant strain delta VdCreC for knocking out VdCreC genes is completed by utilizing a homologous recombination principle, and can obtain knocking out and over-expression mutants according to the prior art (WANG S,XING H,HUA C,GUO H S,ZHANG J.An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation(ATMT)-based gene-disruption method for Verticillium dahliae.Phytopathology,2016,106(6):645-652.);
In the embodiment of the invention, delta VdCreC, an over-expression strain OE-VdCreC and a wild strain V592 are respectively taken as experimental objects, and the following indexes are respectively measured: 2 VdCreC knocked-out mutant strains and 2 overexpressed body strains are obtained through agrobacterium-mediated homologous recombination principles, the functions of VdCreC genes are explored in biological characters, pathogenicity identification and other aspects of VdCreC gene mutant strains and wild type strains, vdCreC genes are expressed in verticillium dahliae hyphae, microsclerotia and spores, but the expression quantity is highest in the microsclerotia, so that the fact that the verticillium dahliae VdCreC genes have tissue expression difference is demonstrated, the time expression difference analysis of the same shows that the VdCreC genes grow for 16d in a verticillium dahliae PDA culture medium, and the biological character measurement shows that VdCreC genes influence the colony morphology of verticillium dahliae, and the spore germination and microsclerotia formation are promoted;
The growth conditions of VdCreC mutants in different carbon sources indirectly reflect the expression conditions of the genes on the corresponding carbon source metabolic enzyme genes, and the fact that the VdCreC mutants have no difference from the wild type strains in the galactose culture medium, have obviously higher inhibition rates in glucose, sucrose, lactose, raffinose and pectin culture mediums than the wild type strains, and have obviously lower inhibition rates in xylan culture mediums than the wild type strains indicates that VdCreC genes do not affect the expression of galactose-related metabolic enzyme genes, positively regulate the gene expression of glucose, sucrose, lactose, raffinose and pectin metabolic enzyme-related genes, negatively regulate the expression of xylan metabolic enzyme-related genes, so that different inhibition rates are shown in different carbon source culture mediums;
the pathogenicity identification result shows that VdCreC genes influence pathogenicity of verticillium dahliae;
In conclusion, vdCreC genes influence the mycelium morphology, growth and development and pathogenicity of verticillium dahliae, and play an important role in inhibiting carbon metabolism;
According to the embodiment of the invention, by knocking out and functionally complementing VdCreC genes and researching the functions of the genes in the growth and development of the verticillium dahliae and the carbon metabolism inhibition, vdCreC genes play an important role in the growth and development of the verticillium dahliae, spore production, pathogenicity and other development aspects, and have an important role in identifying and regulating and controlling carbon metabolism inhibition pathways of the verticillium dahliae.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
Example 1, construction method of a Verticillium dahliae mutant delta VdCreC with VdCreC knocked out:
Designing primers according to upstream and downstream homology arms of VdCreC genes (the nucleotide sequence of which is shown as SEQ ID NO: 1), constructing a knockout vector, taking a wild strain V592 as an initial strain, constructing a mutant strain, and finally obtaining a verticillium dahliae mutant strain (delta VdCreC-1, delta VdCreC-2) from which VdCreC genes are knocked out;
the specific construction process is as follows:
(1) Amplification of homologous arms of the target genes:
designing 2 pairs of primers by taking the upstream and downstream of VdCreC genes as homology arms, amplifying the upstream and downstream homology arms of VdCreC genes by using I-5 TM X High-FIDELITY MASTER Mix High-fidelity DNA polymerase by taking the genome DNA of the deciduous strain V592 as a template, amplifying the upstream homology arms of VdCreC genes by using primers VdCreC-s-f and VdCreC-s-r, and amplifying the downstream homology arms of VdCreC genes by using primers VdCreC-x-f and VdCreC-x-r;
PCR reaction system: i-5 TM XHigh-FIDELITY MASTER Mix 25. Mu. L, vdCreC-s (x) -f/VdCreC-s (x) -r 1. Mu.L each of DNA was added to a 0.2mLPCR tube and the system was made up to 50. Mu.L with ddH 2 O and the PCR reaction procedure was: preheating 1min at 98 ℃, melting for 15s at 98 ℃, annealing for 15s at 60 ℃, extending for 15s at 72 ℃, maintaining for 5min at 72 ℃, finishing the reaction at 20 ℃ for 2min, performing 30 cycles from melting to extending, taking 3-5 mu L of PCR products, performing gel electrophoresis detection in 1% agarose gel electrophoresis added with Goldenview, and observing and photographing under an ultraviolet gel imager. Purifying the target gene fragment of the residual PCR product with correct bands according to the Gel Extraction Kit step of OMEGA biological company product and measuring the concentration;
Wherein the upstream and downstream homology arm primers:
VdCreC-s-f
CTTGCTGAGGTCTTAATTAAGGACAGACTCTAGAGGTCAATCC (shown as SEQ ID NO: 2)
VdCreC-s-r
AGTGCTGAGGCATTAATTAACGTCTCGATCATGGGCATGGCTC (shown as SEQ ID NO: 3)
VdCreC-x-f
CCCGCTGAGGACTTAATTAAGTTAAGTTGGGTTCCGACGAG (shown as SEQ ID NO: 4)
VdCreC-x-r
CTCGCTGAGGGTTTAATTAAAGGCCGTCATCAAGGAGTAGAGG (shown as SEQ ID NO: 5)
(2) The knockout vector used in the experiment is pGKO-HPT, and the vector plasmid is subjected to linearization digestion by using PacI, and the system is as follows: 5. Mu.L of PacI, 35. Mu.L of vector plasmid, 5. Mu.L of 1X Cutsmart and ddH 2 O are complemented to 50. Mu.L, the mixture is subjected to constant temperature enzyme digestion for 10 to 12 hours in a water bath at 37 ℃,3 to 5. Mu.L of PCR products are taken next day and subjected to gel electrophoresis detection in a 1% agarose gel electrophoresis added into Goldenview, the mixture is observed under an ultraviolet gel imager to detect enzyme digestion strips, the target fragments are carefully cut off, the linearized vector fragments are purified by the Gel Extraction Kit steps of OMEGA biological company, the purified vector fragments are split-packed and the concentration thereof is measured after gel electrophoresis verification again, and the split-packed vector fragments are stored in a refrigerator at-20 ℃ for standby, so that repeated freeze thawing is avoided;
(3) In-fusion cloning: connecting an upstream and downstream homology arm of a target gene obtained after amplification and purification and a carrier fragment obtained after linearization and purification by using In-fusion enzyme, wherein the system is as follows: 3 mu L of the purified products of the homologous arm genes at the upstream and downstream of the target gene, 1 mu L of the purified products of the homologous arm genes at the 5 multiplied by CE Multis Buffer and Exnase Multis respectively, 2 mu L of the linearized vector, placing the above solution in a water bath kettle at 37 ℃ for connecting for 30min, placing the solution on ice for subsequent experiments or temporarily storing in a refrigerator at-20 ℃, transforming escherichia coli by heat shock, and extracting recombinant plasmids;
(4) Agrobacterium-mediated genetic transformation: electric shock transformation of agrobacterium: taking out recombinant plasmid and agrobacterium competent cells from-20deg.C refrigerator and-80deg.C refrigerator respectively, and rapidly thawing on ice; taking out 1 mu L of recombinant plasmid, adding the recombinant plasmid into the agrobacterium competent cells, and standing on ice for 10min; wiping off the water on the wall of the sterilized and precooled electric shock cup, adding the mixed solution, and then placing the electric shock into an electric shock instrument for instantaneous electric shock; adding 500 mu L of LB liquid medium without any antibiotics, blowing and mixing by a pipettor, sucking the mixed liquid in a electric shock cup, placing in a 1.5mL centrifuge tube, and shaking and culturing at 200rpm in a shaking table at 28 ℃ for 45min; instantaneously centrifuging, discarding 500 mu L of supernatant, fully mixing the rest bacterial liquid by a pipettor, uniformly coating the bacterial liquid on an LB solid culture medium containing Kan and Rif antibiotics in an ultra-clean bench, placing the LB solid culture medium in a constant temperature incubator at 28 ℃ for dark culture for 2-3 d, screening positive transformants by PCR, shaking a colony with correct PCR to obtain the agrobacterium bacterial liquid, taking 100 mu L of the agrobacterium bacterial liquid into a 10mL IMAS (Kan-containing) liquid culture medium, shaking and culturing at 200rpm in a shaking table at 28 ℃ until the OD600 value is about 0.5, thawing the collected verticillium dahliae conidium on ice in advance, and uniformly mixing the verticillium dahliae conidium and agrobacterium according to the proportion of 1:1; uniformly coating 200 mu L of the mixed solution on IMAS solid culture medium paved with sterilized bacteria, repeating for 3 times, wherein only a plate coated with the conidium of the verticillium dahliae is used as a positive control, and only a plate coated with the bacterial solution of the agrobacterium recombinant plasmid is used as a negative control; after 48h, the filter paper is removed from the IMAS solid culture medium and placed on a PDA (containing HygB +Cef+Car+F2dU) plate, the strain is dark cultured for 5-7 d at 26 ℃, a single fungus colony is picked on an ultra-clean workbench, streaked and inoculated on the PDA (containing Cef+ HygB) culture medium, dark culture is carried out for about 10d at 26 ℃, the growth condition of the fungus in the plate is always observed, only homologous recombinant knockout transformants grow on the resistant plate, false positive transformants do not grow, the positive transformants grow out, single spore separation is carried out on the positive transformants, and subsequent experiments are carried out, so that two knockout transformants are finally obtained.
Example 2, construction method of Verticillium dahliae VdCreC Gene overexpression OE-VdCreC:
A Verticillium dahliae wild-type V592 is taken as an initial strain to obtain a Verticillium dahliae VdCreC gene over-expression body (OE-VdCreC), a vector p1300-Neo-oLiC-Cas9-TtrpC is used, and a 50 mu L XbaI and BamHI double enzyme digestion system is prepared by using XbaI and BamHI double enzyme tangential digestion: the target bands were recovered by overnight digestion at 37℃with p1300-Neo-oLiC-Cas9-TtrPC 20. Mu.L, xbaI 1. Mu.L, bamHI 1. Mu.L, 1X Mbuffer 2.5. Mu.L, ddH 2O2 5.5.5. Mu.L, and the next day. Using II One Step Cloning Kit construction of an overexpressed recombinant plasmid, wherein p1300-Neo-oLiC-Cas9-TtrPC and VdCreC genes are connected by ExnaseII enzyme, and a single-segment connection system of the p1300-NeO-LiC-Cas9-TtrPC is as follows: 5X CEIIbuffer. Mu.L, p1300-Neo-oLiC-Cas9-TtrPC 200ng, 80ng of the gene fragment of interest, 1. Mu.L of ExnaseII, 10. Mu.L of ddH 2 O, and ligation at 37℃for 30min;
The remaining methods are consistent with obtaining knockout mutants, but differ from the obtaining of knockout positive transformants in that the knockout mutants are obtained by mixing 1.0X10 6 conidia/mL of wild-type V592 of Verticillium dahliae with equal proportions of Agrobacterium containing knockout vectors, whereas the obtaining of overexpressing mutant positive transformants is obtained by mixing 1.0X10 6 conidia/mL of conidia of V592 with equal proportions of Agrobacterium containing overexpressing vectors; secondly, the first screening culture medium of the positive transformants of different complementary mutants of the resistance screening culture medium is PDA+ HygB +Cef+Tim+G418, and the second screening culture medium is PDA+Cef+G418.
Application of example 3, vdCreC genes in verticillium dahliae growth:
(1) Determination of colony growth Rate
Culturing with the VdCreC gene knocked-out verticillium mutant strain prepared in example 1, the VdCreC gene overexpression mutant prepared in example 2 and the wild strain V592 as experimental objects, inoculating the mycelium to the center of a PDA culture medium, culturing in darkness at 22 ℃, measuring the colony diameters of all strains on the 5 th day and the 9 th day after inoculation, and calculating the average growth rate of the colonies according to the following formula:
Average growth rate of colony = colony growth rate = (9 th d average colony growth diameter-5 th d average colony growth diameter)/4;
3 replicates were set for each strain and the colony morphology was recorded by photographing on day 15;
(2) Morphological observation of hyphae
Culturing different strains of Verticillium dahliae on PDA plate by streaking, obliquely inserting sterilized cover glass into streaking position, culturing at 22deg.C for 3d, taking out cover glass, and observing hypha growth condition under microscope;
as a result, as shown in FIG. 1, it can be seen from FIG. 1 that the wild-type strain V592 formed a large amount of black microsclerotia on the PDA plate with white aerial hyphae, but 2 VdCreC gene knockout strains produced less white aerial hyphae than the colony edge of the wild-type strain V592, indicating that VdCreC affected the growth phenotype of Verticillium dahlia.
Example 4 determination of microsclerotia:
The test subjects were cultured with the VdCreC gene knocked-out verticillium dahliae mutant strain prepared in example 1, the VdCreC gene overexpression mutant prepared in example 2, and the wild-type strain V592 to obtain a bacterial cake. Respectively taking about 10 bacterial cakes from each bacterial strain by using a puncher, inoculating the bacterial cakes into a Charles (containing kan) liquid culture medium, and shaking and culturing for 3-5 d in a shaking table at 26 ℃ at 200 r/min; collecting conidium by filtration; adjusting spore concentration to 1.0X10 6 CFU/mL, absorbing 100 μl, uniformly coating on MM plate (NaNO 32g、KH2PO41g、MgSO4·7H2 O0.5 g, KCl 0.5g, citric acid 10mg,ZnSO4·7H2O 10mg、FeSO4·7H2O 10mg,NH4Fe(SO4)2)·12H2O 2.6mg,CuSO4·7H2O 0.5mg,NnSO4·H2O 0.1mg,H3BO30.1mg,Na2MoO4·2H2O 0.1mg, glucose 2g,1.5 agar, adding distilled water to 1L.113 deg.C, autoclaving for 20 min), culturing at 22deg.C in dark for 15d, photographing, observing, scraping the culture on the glass paper, weighing, recording wet weight, standing at room temperature for 48 hr, air drying, weighing on balance, and recording dry weight data;
As a result, as shown in FIG. 2, it was revealed from FIG. 2 that 2 VdCreC gene knockouts were not significantly changed from the wild-type strain V592, all strains produced black microsclerotia, the microsclerotia yield of 2 VdCreC gene knockouts was significantly lower than that of the wild-type strain V592, and the microsclerotia yield of 2 VdCreC gene overexpression strains were not significantly different from that of the wild-type strain V592.
Example 5 to determine whether VdCreC gene knockdown affects the formation of verticillium dahliae conidia, a specific assay was as follows:
knockout mutants (strain obtained by constructing in example 1), overexpressing strains (strain obtained by constructing in example 2) and V592, each having a concentration of 1.0X10 6 CFU/mL, were inoculated into Czapek-Dox liquid medium, shake-cultured at 26℃under 200r/min conditions for 5d, spores were isolated, 100. Mu.L of spore suspension having a concentration of 1.0X10 6 CFU/mL was pipetted into Charles (kan-containing) medium, 3 biological replicates were set for each strain, shake culture was performed at 200r/min speed in a shaker at 26℃for 1mL of bacterial liquid was pipetted every 24h, and spore concentration was measured with a hemocytometer for 7d continuously and data were recorded;
As a result, as shown in FIGS. 3 and 4, it can be seen from FIG. 3 that the sporulation amounts of the 2 VdCreC gene knockouts were all significantly lower than that of the wild-type strain V592, and that the sporulation amounts of the 2 VdCreC overexpressing strains were significantly higher than that of the wild-type strain V592, starting from FIG. 5 d. The VdCreC gene is described to promote the spore production of verticillium dahliae; as can be seen from fig. 4, the mutant was observed under a microscope for the peduncles, and the Δ VdCreC mutant produced little peduncles in wheel branches compared to the V592 strain.
Example 6, in order to clarify the effect of VdCreC gene knockout on verticillium dahliae pathogenicity, pathogenicity of VdCreC gene knockout mutant on cotton was determined with wild-type strain V592 and over-expressed strain as controls, and the pathogenicity determination method was as follows:
The knock-out mutant, the over-expression strain and V592 constructed above are inoculated into a Czapek-Dox liquid culture medium at 26 ℃ for 5 days under shaking culture at 200r/min, after the 5 th true leaves of cotton seedlings grow out, 200mL of bacterial liquid with the concentration of 1.0X10 7 CFU/mL is inoculated into each pot of cotton by using a root dipping inoculation method, 3 water culture boxes (36 cotton seedlings in total) are repeated for each strain, the disease fingers are observed every day after inoculation, the disease classification standard is that the disease is recorded after the disease is developed every 3 days, and the disease classification standard is that: level 0: does not cause disease; stage 1: 1-2 pieces She Fabing; 2 stages: 1 leaf of true leaf; 3 stages: 2 pieces of true leaves are ill; 4 stages: 3 or more than 3 true leaves are diseased, and the disease index is calculated according to the following formula:
disease index= [ Σ number of plants at each stage×number of stages/(total number of plants×highest disease stage) ]×100;
the disease index is the repeated average value of 3 biological experiments;
as a result, as shown in FIG. 5, it can be seen from FIG. 5 that compared with the wild-type strain V592, the cotton inoculated with the VdCreC gene knockout strain exhibited symptoms of leaf yellowing, wilting and scorch after 3 days, and the disease symptoms at 25d were significantly lower than those of the wild-type strain, and the disease index of the VdCreC gene knockout strain was also significantly lower than those of the wild-type and overexpresser strains.
Example 7 to analyze whether VdCreC gene knockdown resulted in a decrease in verticillium dahliae virulence related to its ability to penetrate the host, cellophane penetration experiments were performed on each of the knockdown mutant strains against V592 as follows:
Spreading glass paper with the size basically consistent with that of the culture dish after high-temperature sterilization treatment on the poured MM basic culture medium, picking hypha by using a toothpick, inoculating the hypha to the center of the glass paper, respectively culturing for 3d, removing the glass paper, allowing strains to grow for 7d again, observing whether colonies can grow on the culture dish, and setting 3 repetitions for each strain;
The results are shown in FIG. 6, and it can be seen from FIG. 6 that VdCreC gene knockout mutant can penetrate the cellophane when inoculated with 3d, and the results show that VdCreC gene knockout does not result in loss of the penetrability of the Verticillium dahliae on the cellophane.
Example 8, verticillium dahliae produced 514 carbohydrate hydrolases, of which 152 were associated with pectin, 92 were associated with cellulose, 69 were associated with xylan and 61 were examined for the use of different carbon sources by the knock-out mutants of verticillium dahliae VdCreC, which indirectly reflected the regulation of the different carbon source hydrolases by the VdCreC gene, thus inoculating each VdCreC mutant strain and wild type strain V592 onto different carbon source media to observe the growth of the strain;
As shown in fig. 7, it can be seen from fig. 7 that 2 VdCreC knockout strains produced less black microsclerotia in glucose, xylan, pectin medium than the wild-type strain V592, and that the phenotype on the medium without additional carbon sources, galactose, lactose, raffinose, and xylan carbon sources was not significantly different from the wild-type strain V592, and the measurement of colony diameters showed that the inhibition ratio analysis: compared with the wild type strain V592, the inhibition rate of 2 VdCreC gene knockout strains in a culture medium which takes glucose, sucrose, lactose, raffinose and pectin as carbon sources is obviously increased, the inhibition rate of 2 VdCreC gene knockout strains in a culture medium which takes xylan as a carbon source is obviously reduced, and the inhibition rate in a culture medium which takes galactose as a carbon source is not obviously different from that of the wild type strain V592; compared with the wild type strain V592, under the condition of taking galactose as a carbon source respectively, the inhibition rate of the 2 VdCreC gene overexpression body strains has no obvious difference with the wild type strain; under the condition of taking xylan as a carbon source, the inhibition rate of 2 VdCreC gene overexpression body strains is obviously reduced, and under the condition of taking glucose, sucrose, lactose, raffinose and pectin as carbon sources, the inhibition rate of 2 VdCreC gene overexpression body strains is obviously increased, so that the VdCreC gene does not influence the expression of galactose related enzyme genes, positively regulates and controls the gene expression of glucose, sucrose, lactose, raffinose and pectin metabolism related enzymes, negatively regulates the expression of the xylan metabolism related enzymes, and therefore, different inhibition rates are shown in different carbon source culture mediums.
Example 9, primary task of carbon metabolism inhibition was to sensitively identify a preferred carbon source, thereby regulating other secondary carbon sources, inoculating VdCreC each mutant strain and wild-type strain to a medium using starch, starch+glucose, cellulose, cellulose+glucose as carbon sources, respectively staining with iodine solution and congo red, measuring the size of the transparent circle, and calculating the inhibition rate in the presence of glucose;
The results are shown in FIG. 8, and according to FIG. 8, the phenotype of each strain of VdCreC genes on each carbon source culture medium is not obviously different from that of the wild strain V592, under the condition of glucose, 2 over-expression strains of VdCreC genes and the wild strain V592 lose the utilization of starch and cellulose, no transparent circle is generated, namely, no decomposition of starch and cellulose exists, so that the inhibition effect of glucose on the utilization of starch and cellulose is demonstrated, and the inhibition rate reaches 100%; in the presence of glucose, 2 VdCreC gene knockout strains can still observe obvious transparent circles, which shows that VdCreC gene knockout releases the inhibition effect of glucose on starch and cellulose metabolism, but promotes the utilization of starch and cellulose, the inhibition rate of starch utilization reaches 6.37% and 8.77%, and the inhibition rate of cellulose utilization reaches 19.15% and 22.80%, respectively, and in conclusion, vdCreC genes participate in carbon metabolism inhibition response caused by glucose in Verticillium dahliae.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (2)
- The application of the VdCrec gene in reducing the pathogenicity of the verticillium dahliae is characterized in that VdCreC genes in the verticillium dahliae are knocked out, the pathogenicity of the verticillium dahliae is reduced, and the nucleotide sequence of the VdCreC genes is shown as SEQ ID NO. 1.
- 2. The application of the reagent for down regulating VdCreC gene expression or protein expression in preparing the medicine for preventing and treating cotton diseases is characterized in that the pathogenic bacteria of the cotton diseases are verticillium dahliae, and the nucleotide sequence of the VdCreC gene is shown as SEQ ID NO. 1.
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