CN118240041A - Cephalosporium camellia gene CcCp1 and application thereof - Google Patents
Cephalosporium camellia gene CcCp1 and application thereof Download PDFInfo
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a gene CcCp of a thorn-spore-forming fungus of camellia and application thereof, wherein the deficiency of CcCp gene leads to the reduction of the pathogenicity of the thorn-spore-forming fungus of camellia to host plants, ccCp gene and CcCp protein can be used as targets for the development of anti-thorn-spore-forming fungus medicaments, and compounds capable of inhibiting the expression, modification and positioning of the gene and the protein thereof are screened, so that the occurrence of thorn-spore-forming fungus of camellia can be effectively controlled, and the medicaments contain genetic engineering vectors for blocking or inhibiting the expression of the thorn-spore-forming fungus CcCp1 gene of the thorn-forming fungus of camellia are used for reducing the pathogenicity of the thorn-forming fungus of camellia or reducing the spore yield of the thorn-forming fungus of camellia or inducing the disease resistance of camellia sinensis.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a trametes camellia thorn spore gene CcCp to control hypha expansion rate, spore yield and pathogenicity of trametes camellia thorn.
Background
Anthrax was first isolated and observed in 1790 and classified as plexus anthrax (Vermicularia); fungi with bristle characteristics on the conidial of 1831 were separated and an independent genus, anthrax, was established (ColletolrichumCorda). Some researchers separate anthracnose pathogenic bacteria from tea trees in different tea producing areas in China, and after identification, 6 anthracnose strains such as C.camelliae, C.fructicola, C.gloeospori, C.siame nse, C.henanense and C.jiangxiense are found, and the strain C.camelliae is widely distributed in each large tea producing area in China and has a certain harm to most varieties of tea trees in China, so that the C.camelliae can be presumed to be a main dominant species of the tea trees endangered in China.
Plant anthracnose mainly infects leaves of plants. In the early stage of plant infection anthracnose, only the leaf edge can be affected by the anthracnose; with the development of disease, the disease portion gradually spreads to the middle part of the leaf or most of the leaf, and usually causes the complete leaf to die. In the early stage of anthracnose, only some elliptic or round reddish brown small spots are displayed on the leaves of plants, and until the later stage of anthracnose development, the spots become dark brown round spots with the diameter of about 1-4 mm, and the edges of the spots have yellow vignetting and are dark brown or dark green. By the later stage of anthracnose development, the lesions are thoroughly changed from dark brown to black brown and some small black spots in the shape of a wheel appear, which indicates that the pathogenic bacteria grow out of the conidiophore disc. When the disease spots are too much and dense, the plant leaves can be withered and fall off. Anthracnose can occur on the leaves of plants and can also infect the tender stems of the plants. When disease occurs on the stem, a plurality of circular and oval disease spots are usually generated. When anthrax infects tender tips of plants, the lesions generally appear as oval ulcerated spots.
Plant pathogenic fungi secrete large amounts of proteins that are involved in various aspects of parasitic and disease progression, such as transmission through vectors, attachment to plant organ surfaces, expression of symptoms, and induction of defensive responses. Among these proteins are various types of enzymes, receptors and cysteine-rich secreted proteins. This large group of cysteine-rich proteins is highly diverse and is classified by Templeton et al based on the number of cysteines, molecular weight and role in the pathogenic process. Currently, 7 non-catalytic fungal protein families have been identified based on their primary sequences, including certo-platanin (CP) family (PF 07249), class I hydrophobin (PF 01185), class II hydrophobin (PF 06766), kinetin (PF 00964), pc F family (PF 09461) and NIP-1 family (PF 08995). Among them, CP proteins are a class of proteins unique to fungi.
Disclosure of Invention
The invention provides a trametes camellia sinensis gene CcCp, the nucleotide of which is shown as SEQ ID NO. 1, and the coded CcCp protein has the amino acid sequence shown as SEQ ID NO: 2. The gene CcCp of the thorn-spore of the camellia is derived from the thorn-spore of the camellia, and the thorn-spore of the camellia participates in controlling the hypha expansion rate, the spore yield and the pathogenicity of the thallus. The invention also provides application of the thorn-basidiomycete gene CcCp, a knockout vector and a knockout mutant. As the deficiency of CcCp gene leads to the decrease of the pathogenicity of the camellia sinensis and the encoding protein of CcCp gene to host plants, the gene can be used as a target for developing anti-camellia sinensis medicament, and the compound which can inhibit the activity of the gene and the expression, modification and localization of the protein can be screened, so that the occurrence of the camellia sinensis and the like can be effectively controlled, and the medicament contains a genetic engineering vector which can block or inhibit the expression of the camellia sinensis and the gene CcCp of the camellia sinensis, such as a knockout vector, and is used for reducing the pathogenicity of the camellia sinensis and the spore yield of the camellia sinensis or inducing the disease resistance of the tea tree.
The invention aims to provide an application of a thorn-agaricus camellia CcCp gene or CcCp protein serving as a control target in preparation of an anti-thorn-agaricus camellia medicament.
Further, the medicament is used for any one or more of A to C: A. regulating and controlling the expansion rate of the mycelium of the colletotrichum camellia; B. regulating and controlling the spore yield of the camellia thorn spore bacteria; C. regulating and controlling the pathogenicity of the camellia thorn spore bacteria.
Furthermore, the increase of the gene activity of the thorn-agaricus camellia CcCp or the expression quantity of the CcCp protein promotes the pathogenicity of the thorn-agaricus camellia, and the loss of the gene activity of the thorn-agaricus camellia CcCp1 or the expression quantity of the CcCp protein reduces the pathogenicity of the thorn-agaricus camellia.
Furthermore, the regulation and control of the pathogenicity of the tea tree acanthosphaeria is realized by improving the oil metabolic pathway of the tea tree.
Further, the agent comprises CcUOX gene inhibitor or knock-out vector containing CcUOX gene.
Furthermore, the reduction of the pathogenicity of the colletotrichum camellia sinensis is achieved by improving the lipid metabolism pathway of the tea tree.
Further, the plant is a host plant of the colletotrichum camellia.
Further, the host plant is one of plants of camellia, camellia oleifera, camellia japonica and the like.
It is still another object of the present invention to provide an application of the gene CcCp of the genus Cephalosporium in improving disease resistance of plants of the genus Cephalosporium, using the Cephalosporium calcaratus with CcCp gene deletion to induce plants to produce immunity against Cephalosporium calcaratus.
Further, the plant is a host plant of the colletotrichum camellia.
Further, the host plant is one of plants of camellia, camellia oleifera, camellia japonica and the like.
The invention also aims to provide a method for preventing and controlling the camellia thorn fungus, and the expression vector is knocked out to block or inhibit the expression of the camellia thorn fungus CcCp gene, so that the biocontrol strain with reduced pathogenicity is obtained.
The CcCp gene provided by the invention has an important application that the expression of the gene and the protein product encoded by the gene can be used as important candidate target sites for designing and screening anti-camellia thorn disc spore medicaments. The invention proves that the deletion of CcCp gene leads to the obvious reduction of the pathogenicity of the thorn-spore camellia, the slow growth speed of hyphae and the reduction of spore production. Therefore, the compound which can prevent the gene expression and the expression, modification and localization of the protein can be screened, and the occurrence of the colletotrichum camellia can be effectively controlled, thereby being beneficial to developing a novel bactericide.
Drawings
FIG. 1 is a schematic diagram of a knockout strategy (gene replacement by homologous recombination) of the tea plant anthracnose CcCp gene; wherein CCA is a wild type strain, pXEH is a knockout vector, and delta CcCp1 is a CcCp1 gene deletion mutant.
FIG. 2 is a schematic representation of qPCR expression of CcCp gene deletion mutants.
FIG. 3 shows the hyphal extension rates of knock-out mutant Δ CcEpl and wild-type strain Colletotrichum camelliae; wherein Delta CcCp1-9, delta CcCp1-14 and Delta CcCp1-15 are knockout mutants and Colletotric hum camelliae is wild type.
FIG. 4 is a comparative analysis of the sporulation of knockout mutant Δ CcCp1 with wild-type strain Colletotrichum camelliae and complement Δ CcCp 1-C; wherein Delta CcCp1-9 and Delta CcCp1-14 are knockout mutants, CCA is wild type, delta CcCp1-C is complement.
FIG. 5 is a pathogenicity comparison analysis of knockout mutant Δ CcCp1 with wild-type strain Colletotrichum camelliae and overexpressing mutant Δ CcCp 1-C; wherein Delta CcCp1 is a knockout mutant, colletot richum camelliae is a wild type, delta CcCp1-C is an over-expression mutant, and a host is tea leaves.
Detailed Description
The present invention is further defined in the following examples, from the description and examples that follow, one skilled in the art will be able to ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Examples 1, ccCp knockout and genetic complementation of the Gene
1) The knockout vector construction strategy is shown in fig. 1. First, the flanking sequences of the target gene DNA are amplified. Primers were designed using DNAMAN and synthesized by Shanghai Biotechnology Co., ltd.) using primers CcCp-UP-F: 5'-GGCGGCCTCGAGGGATGTTGTTTGCCTGCG-3' (SEQ ID NO: 3) and CcCp-UP-R: 5'-CGGGGTACCTTTGGCGGTTGTGGGTGGA-3' (SEQ ID NO: 4) an approximately 1kbp fragment upstream of the CcEpl gene was amplified using genomic DNA of the B.anthracis strain Colletotrichum camelliae as a template; primer CcCp-DN-F was used: 5'-GCTCTAG AACCACCACACGACCTCGG-3' (SEQ ID NO: 5) and CcCp-DN-R: 5'-ACG CGTCGACGACCCACATGCAGCTCCGA-3' (SEQ ID NO: 6), and a fragment of about 1kbp downstream of the CcEpl gene was amplified using the genomic DNA of the B.anthracis strain Colletotrichum camelliaeCCA as a template. Reaction system (25 μl): TAKARA PRIMERSTAR Max DNA Polymerase (2×) 12.5 μl; 1. Mu.L of each of the upstream and downstream primers (10. Mu.M); 1. Mu.L of template DNA; ddH 2 O9.5. Mu.L. Amplification procedure: (1) 98 ℃ for 10s; (2) 53℃for 15s; (3) 10s at 72℃the 1-3 steps were cycled 35 times. The PCR products were recovered and purified using TAKAR A gel recovery kit. The knockout vector was constructed by ligating the upstream and downstream fragments with pXEH vectors using XhoI+KpnI and XbaI+SalI, respectively.
SEQ ID NO. 1 nucleotide sequence: 417bp
ATGCAGTTCT CCAACCTTGT CACCATCCTC TCCTCCGTCG CCGCG GCCGC GGCCGTCTCC
GTCTCCTTCG ACACCGGCTA TGATGACGGT GCCCGCTCCC TCACT GCCGT CTCCTGCTCC
GACGGCGCCA ACGGCCTGAT CACCAAGTAC AACTGGCAAA CCC AGGGCAA CATCGCCCGC
TTCCCCTACA TCGGTGGTTC CGACGCCGTT GCCGGCTGGA ACTC CCCCAA CTGCGGCACC
TGCTGGCAGC TCACCTACAA CGGCAAGAGC ATCAACGTCC TCGC CATCGA CCACGCCGGC
TCCGGCTTCA ACCTCGCCCT CGGCGCCATG AACGACCTCA CCAA CGGCCA GGCCAACCAG
CTCGGCCGCA TCGACGCCCA GGCCACCCAG GTCGCCCTCA GCGC CTGCGG TCTGTAA
SEQ ID NO. 2 amino acid sequence:
MQFSNLVTIL SSVAAAAAVS VSFDTGYDDG ARSLTAVSCS DGANGL ITKY NWQTQGNIAR FPYIGGSDAV AGWNSPNCGT CWQLTYNGKS INV LAIDHAG SGFNLALGAM NDLTNGQANQ LGRIDAQATQ VALSACGL*
2) Transformation of anthracnose
A. Culturing of Agrobacterium tumefaciens AGL-1: a single colony of Agrobacterium tumefaciens strain AGL-1 containing binary vector CcCp1 was picked and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL rifampicin, and shake cultured for 36h to OD 600 = 0.8 or so. Preparing an IM liquid culture medium (100 mL:90mLddH 2 O;1mL of 50% glycerol; 1mL of 20% glucose ;4mL 1MMES;2mL M-Nbuffer;1mL Tr acelement;80μL K-buffer;250μL 20％NH4N03;100μL 1％CaCl2;1mL 0.01％FeSO4;200μL 19.2％As),), adding medicines except for FeS04 and As into 90mL ddH 2 O, uniformly mixing, taking 5mL of a shaking bacterial liquid into a 15-mL centrifuge tube, centrifuging at 4000 rpm for 5 minutes, discarding supernatant, adding 5mL of an IM culture medium (without FeSO4 and As), blowing down, centrifuging at 4000 rpm for 5 minutes, discarding supernatant, adding FeSO4 and As into the IM culture medium, taking 5mL of the IM culture medium, adding the 5mL into a 15-mL centrifuge tube, blowing down to an empty bottle, shaking at 28 ℃ for 4 hours, and placing the rest IM liquid culture medium into a 4 ℃ refrigerator, and packaging newspapers.
B. Spore-producing culture of colletotrichum gloeosporioides: activating Colletotrichum camelliae strain, inoculating the bacterial cake on PDB culture medium (20% boiling and filtering potato, 2% glucose), shaking culturing at 28deg.C for 3-8 days, filtering supernatant with gauze, observing with microscope, and regulating spore concentration to 5×10 5/mL with hemocytometer.
C. Co-culturing agrobacterium tumefaciens and anthracnose germ conidium: mixing the agrobacteria liquid induced in the IM liquid culture medium with the equal volume of anthrax spores with the adjusted concentration, taking 100 mu L of the agrobacteria liquid, coating the agrobacteria liquid on the IM solid culture medium with the glass paper, coating 6 plates, sealing the plates, and culturing for 2 days at the temperature of 28 ℃ in a dark place. After the co-cultivation, the cellophane was transferred to PDA medium containing 100. Mu.g/mL hygromycin and cultivation was continued under the same conditions. After 4-7 days, the expanded colonies were picked up and subjected to secondary screening on screening media containing the same antibiotics.
D. Verification of transformants: transformants were screened by qPCR amplification using CcCp gene primers. The RNA level was not able to successfully perform transcription due to the CcCp gene deletion, and at this time, NO expression was detected using the CcCp gene qPCR primers 5'-GCAAACCCAGGGCAACAT-3' (SEQ ID NO: 7) and 5'-ATGGCGAGGACGTTGATG-3' (SEQ ID NO: 8), and the results are shown in FIG. 2, which illustrate that using the CcCp gene qPCR primer, the wild-type cDNA had an expression level and knockouts Δ CcCp1-9, Δ CcCp1-14, and Δ CcCp1-15 had NO expression level. From the transformants, 3 independent CcCp gene deletion mutants Δ CcCp1-9, Δ CcCp1-14, Δ CcCp1-15 were selected for subsequent functional analysis.
Effect of the gene CcCp on the extension rate of the mycelium of the colletotrichum camellia in example 2.
Activating Colletotrichum camelliae and delta CcCp1 strains, inoculating 0.6cm of bacterial cake to the right center of a 9cm PDA culture plate, and culturing in a dark culture box at 28 ℃. Colony diameters were measured every 24h until a colony was confluent throughout the plate, and the rate of hyphal extension of knock-out mutant Δ CcEpl and wild strain Colletotrichum CA MELLIAE is shown in fig. 3.
Example 3, ccCp influence of the Gene on the spore yield of Cephalosporium camellia.
Each strain was cultured under the same culture conditions until all strains were confluent, 5mL of sterile water was added to the plates, mycelia on the surface of the medium was scraped sufficiently with a cotton swab, the numbers of spores in the liquid were observed with a microscope after the mycelia in the liquid were filtered with three-layer glass paper, and the rates of extension of the mycelia of the knocked-out mutant Delta CcEpl and the wild-type strain Colletotrich um camelliae were as shown in FIG. 4.
Example 4, ccCp effect of gene on pathogenic aspect of disc fungus camellia sinensis was verified.
Inoculating spore liquid: culturing each strain under the same culture condition to produce spores, collecting and concentrating spore liquid, diluting the spore liquid obtained by centrifugation to 1X 10 6 spore liquid/mL by using sterile water, fully and uniformly mixing, sucking 20 mu L spore liquid by using a pipette gun, dripping the spore liquid to two sides of the leaves of a host plant with the same batch size and good state, carrying out moisturizing culture on the inoculated host, observing the disease condition, and photographing and recording; inoculating a fungus cake: activating the strain on PDA, when the strain grows for 4d, using a puncher to punch out uniform bacterial cakes, reversely buckling the bacterial cakes on leaves of host plants, carrying out moisturizing culture on the inoculated hosts, observing the disease condition, and taking photos and recording. The differences in pathogenicity of wild type, knockdown and complement to the host plant were investigated by means of spore fluid and bacterial cake combination, and the results are shown in fig. 5: when CcCp genes are knocked out, the pathogenicity of the anthrax on host plants is greatly reduced, and the pathogenicity of complement is basically the same as that of wild type, which shows that CcCp1 participates in regulating and controlling the pathogenicity process of the anthrax on host plants, and is an essential key gene for maintaining virulence of the anthrax on host plants.
The embodiment shows that the gene CcCp or CcCp protein of the invention can be used as a control target in preparation of anti-thorn-film-forming medicines for controlling the hypha expansion rate, the spore yield and the pathogenicity of thorn film. The activity of the gene CcCp of the colletotrichum camellia or the expression quantity of the CcCp protein is increased to promote the pathogenicity of the colletotrichum camellia, and the loss of the activity of the gene CcCp of the colletotrichum camellia or the expression quantity of the CcCp protein reduces the pathogenicity of the colletotrichum camellia. The regulation and control of the pathogenicity of the tea tree acanthosphaeria is realized by improving the oil metabolic pathway of the tea tree. The medicament comprises CcUOX gene inhibitor or CcUOX gene knockout carrier.
The above examples are given for clarity of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. The nucleotide sequence of the gene CcCp of the colletotrichum camellia sinensis is shown as SEQ ID NO:1, the application of the gene CcCp of the colletotrichum camellia sinensis as a control target in the preparation of an anti-colletotrichum camellia sinensis medicament, wherein the medicament is used for any one or more of A to C: A. regulating and controlling the expansion rate of the mycelium of the colletotrichum camellia; B. regulating and controlling the spore yield of the camellia thorn spore bacteria; C. regulating and controlling the pathogenicity of the camellia thorn spore bacteria.
2. The amino acid sequence of the protein CcCp of the colletotrichum camellia sinensis is shown as SEQ ID NO:2, the application of the gene CcCp of the thorn-leaf gentian in preparing the anti-thorn-leaf gentian medicament for any one or more of A to C: A. regulating and controlling the expansion rate of the mycelium of the colletotrichum camellia; B. regulating and controlling the spore yield of the camellia thorn spore bacteria; C. regulating and controlling the pathogenicity of the camellia thorn spore bacteria.
3. Use according to claim 1 or 2, characterized in that: the activity of the gene CcCp of the colletotrichum camellia or the expression quantity of the CcCp protein is increased to promote the pathogenicity of the colletotrichum camellia, and the loss of the activity of the gene CcCp of the colletotrichum camellia or the expression quantity of the CcCp protein reduces the pathogenicity of the colletotrichum camellia.
4. Use according to claim 1 or 2, characterized in that: the regulation and control of the pathogenicity of the tea tree acanthosphaeria is realized by improving the oil metabolic pathway of the tea tree.
5. The use according to claim 1, wherein: the medicament comprises CcUOX gene inhibitor or CcUOX gene knockout carrier.
6. The application of the gene CcCp of the thorn-leaf camellia in improving the disease resistance of camellia plants uses the thorn-leaf camellia with CcCp gene deletion to induce the plants to generate immunity to the thorn-leaf camellia.
7. The use according to claim 6, wherein: the plant is a host plant of the colletotrichum camellia.
8. The use according to claim 6, wherein: the host plant is one of plants such as camellia tea, camellia oleifera, camellia japonica and the like.
9. A method for controlling the camellia d.spinosa, which is characterized in that: the expression of the gene CcCp of the colletotrichum camellia can be blocked or inhibited by using a CcUOX gene knockout vector, so that the biocontrol strain with reduced pathogenicity can be obtained.
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